KR20010022917A - Methods and compositions for detection or quantification of nucleic acid species - Google Patents

Abstract

The present invention provides a method for detecting a target nucleic acid species using a probe immobilized on a substrate and a plurality of labeled probe arrays. The invention also relates to oligonucleotide probes attached to separate particles, wherein the particles can be divided into various sets based on physical properties. Different probes are attached to each set of separate particles, and identification of each probe is performed based on the physical properties of the separate particles. The present invention also relates to methods of using materials which destabilize the binding of complementary polynucleotide strands (reduce binding energy) or increase the binding stability between complementary polynucleotide strands (increase binding energy). The figure illustrates the apparatus used to make the probe array.

Description

METHODS AND COMPOSITIONS FOR DETECTION OR QUANTIFICATION OF NUCLEIC ACID SPECIES}

The speed of ordering four nucleotides in nucleic acid samples is a major obstacle to further developments in molecular biology, medicine, and biotechnology. A method of sequencing nucleic acids associated with separating nucleic acid molecules from a gel has been used since 1978. Another proven method of determining the order of nucleic acids is by hybrid (SBH).

A method of determining the order of nucleotides (e.g., determining the order of A, G, C, T, etc. in a sample), such as randomly terminating a dideoxy chain termination of a strand that is degraded or replicated at a particular nucleotide. It is common practice to make and carry out nucleic acid fragment mixtures labeled with one another. The resulting nucleic acid fragments in the range of 1 to 500 bp are then separated on the gel to create a band ladder, where adjacent samples differ by one nucleotide.

The SBH alignment method does not require single base dissociation, nucleic acid degradation, synthesis or imaging when isolated. Hybrid reactions, which are identified by mismatches of short oligonucleotide K bases, can be used to determine the list of K-mer oligonucleotides that are the makeup of the target nucleic acid. The sequence of the target nucleic acid can be assembled using uniquely overlapping oligonucleotides.

There are several ways to obtain sequences using hybrid reactions. In a process called SBH Format I, nucleic acid samples are arranged and labeled probes hybridize with the sample. Replication membranes with the same set of nucleic acids are used to arrange several probes side by side. The nucleic acid sample is arranged and hybridized to a nylon membrane or other suitable support. Each membrane array can be used multiple times. Format 1 is effective when processing multiple samples in batches.

In the case of SBH Format 2, the probes are arranged at positions corresponding to each sequence on the substrate, and the labeled nucleic acid sample fragments are hybridized to the arrayed probes. In such a case, the sequence information for the fragment can be obtained by performing a hybrid reaction simultaneously with all the arranged bases. To sequence other nucleic acid fragments, the same oligonucleotide sequence can be used again. Arrays are made by spotting or in situ probe synthesis.

In the case of Format 3 SBH, two sets of probes are used. In one embodiment, probe configurations having known positions are used, in other cases labeled sets are stored in several well plates. In this case, the target nucleic acid need not be labeled. If one attached probe and one labeled probe are both hybridized in the target nucleic acid, they are ligated by covalent bonds, creating a sensed sequence equal to the sum of the ligated probe lengths. This process can sequence longer nucleic acid fragments, such as the complete bacterial genome, etc., without nucleic acid subcloning in smaller fragments.

In the present invention, SBH is used to effectively identify and sequence one or more nucleic acid samples. This process is used in many fields, including nucleic acid diagnostics, arguments, and genetic mapping. It can also be used to identify mutations that are responsible for genetic disease or other features, to assess biological diversity, and to produce many different forms of data depending on nucleic acid sequences.

Summary of the Invention

The present invention provides a method for detecting a target nucleic acid species, which method provides a probe array immobilized on a substrate and a labeled probe, wherein each labeled probe corresponds to a first position of the target nucleic acid. The nucleic acid sequence of at least one probe selected to have a first nucleic acid and immobilized on a substrate is complementary to a second portion of the nucleic acid sequence of the target, and the second portion is adjacent to the first portion; Providing a nucleic acid targeted to the array under conditions such that the probe sequence can hybridize to the complementary sequence;

Introducing a labeled probe into the array;

Hybridizing the probe fixed to the substrate to the target nucleic acid;

Immobilizing labeled probes to adjacent hybridized probes in an array; And

Consisting of sensing labeled probes fixed to the probes in an array. According to a suitable method of the invention, the probe arrangement immobilized on the substrate consists of a complete set of probes. At least two of the probes immobilized on the substrate have a sequence overlapping the target nucleic acid sequence, and more suitably, at least two of the labeled probes are overlapping sequences of the target nucleic acid sequence. In addition, according to another aspect of the present invention, there is provided a method for detecting a target nucleic acid having a known sequence, the method comprising the following steps; A set of immobilized oligonucleotide probes attached to a solid substrate under conditions capable of hybridization is contacted with a nucleic acid sample, wherein the immobilized probe has the ability to specifically hybridize with different portions of the target nucleic acid sequence; Contacting the target nucleic acid with a set of labeled oligonucleotide probes in solution under conditions capable of a hybrid reaction, wherein the labeled probe can specifically hybridize with another portion of the target nucleic acid sequence adjacent to the immobilized probe; The probe immobilized to the labeled probe immediately adjacent the immobilized probe on the target sequence is covalently ligated (eg, using ligase); Remove any ligated labeled probes; Detecting whether there is a labeled probe attached to the fixed probe. The present invention also provides a method for determining the expression of partially or fully sequenced gene set components in a cell type, tissue or tissue mixture, wherein the method is directed to immobilized labeled probe pairs specific for the sequenced gene. Providing; Hybridizing the unlabeled nucleic acid sample and its corresponding labeled probe to one or more immobilized probes; Forming a covalent bond to the hybridized label probe and the immobilized probe acid; And detecting the labeled probe previously bound to a specific position in the array to determine if the sequenced gene is present. In a suitable embodiment of the invention, the target nucleic acid can confirm the presence of an infectious agent.

The present invention also provides an oligonucleotide probe arrangement, which comprises a nylon membrane; Multiple oligonucleotide probe subarrays arranged on nylon membranes; Wherein the subarray consists of a number of individual spots; Each spot consists of a plurality of oligonucleotide probes having the same sequence; On the nylon membrane, a plurality of hydrophobic barriers are placed between subarrays, preventing the mixing of adjacent subarrays due to the multiple hydrophobic barriers.

The present invention also provides a method of sequencing a repeating sequence, which sequence has a first terminus and a second terminus in a target nucleic acid, which method comprises: (a) a plurality of space oligonucleotides of various lengths; Wherein the space oligonucleotide consists of a repeating sequence; (b) providing a first oligonucleotide known to be adjacent to a first terminus of the repeat sequence; Providing a plurality of second oligonucleotides adjacent to a second end of the repeat sequence, wherein the plurality of second oligonucleotides are in a labeled state; (d) hybridize one of the plurality of oligonucleotides to the second oligonucleotide and the plurality of space oligonucleotides to the target nucleic acid; (e) ligation of the hybridized oligonucleotides; (f) separating the ligated oligonucleotides from the unligated oligonucleotides; And (g) detecting a label in the ligated oligonucleotide.

The present invention also provides a method of sequencing a branch point sequence having a first end and a second end in a target nucleic acid, the method providing a second oligonucleotide complementary to a first portion of the branch point sequence The first oligonucleotide extends at least one nucleotide from the first end of the branch point sequence; (b) providing a plurality of second oligonucleotides that are complementary and labeled to a second portion of the branching sequence, wherein the plurality of second oligonucleotides extend at least one nucleotide from the second end of the branching sequence Is complementary to a plurality of sequences generated in the branching sequence; (c) hybridize the first oligonucleotide and one of the plurality of second oligonucleotides to the target DNA; (d) ligation of the hybridized oligonucleotides; (e) separating the ligated oligonucleotides from the ligated oligonucleotides; (f) detecting the label in the ligated oligonucleotide.

The present invention also provides a method for identifying a sequence using a probe known to be negative for a target nucleic acid sequence. Targeted nucleic acid sequences can be identified by hybridizing the targeted nucleic acid sequences to "negative" probes to confirm that these probes do not form a perfect match pair with the targeted nucleic acid sequence.

The present invention also provides a method for analyzing nucleic acids using oligonucleotide probes complexed with various labels, which probes can be complexed in a hybrid reaction without losing information about the sequence (eg, various other probes). By different labeling, it is possible to identify hybridization of different probes to the target). In suitable embodiments, the label may be a radioisotope, fluorescent molecule or enzyme or electrophoretic material label, and the like. In a more suitable embodiment, differently labeled oligonucleotide probes can be used in Format III SBH, and multiple probes (if one probe is a fixed probe but two or more) can be ligated together.

Another embodiment of the present invention provides a method for detecting whether a target nucleic acid has a known sequence when compared to a homologous nucleic acid in a sample, when there is a very small amount of the target. In a suitable embodiment, the target nucleic acid is an allele present in a very small proportion in samples having nucleic acids from various sources. In another suitable embodiment, the nucleic acid of interest has a mutated sequence, which is present at a very low frequency in the nucleic acid sample.

In another embodiment of the present invention, a method of identifying the sequence of a target sequence using one pass gel sequencing is provided. Primers for one pass gels are derived from sequences obtained by SBH, and these primers are used in standard Sanger sequence reactions to provide gel sequence information for the target nucleic acid sequence. The sequence is then identified by comparing the sequence obtained by single pass gel sequencing with the sequence derived by SBH.

The invention also provides a method of finding branch points using single pass gel sequencing. Primers for reaction with a single gel pass sequence are identified by the Sfs terminus obtained after the SBH first step and these primers are used in a standard Sanger-sequencing reaction to provide gel sequence information through the secretion points of Sfs. Then, by comparing the Sanger-sequence results from the branch to the Sfs, arranging the Sfs, we can see the adjacent Sfs.

The invention also provides a method of preparing a sample comprising a nucleic acid targeted by PCR without the need to purify the PCR product prior to the SBH reaction. In Format I SBH, the unpurified PCR product can be used on the substrate without prior purification and the substrate is washed before introducing the labeled probe.

The invention also provides methods and apparatus for analyzing a target nucleic acid. The device consists of two nucleic acid sequences mixed at the desired time. In suitable embodiments, the nucleic acid in one of the arrays is labeled. In a more suitable embodiment, a substance is placed between the two arrays to prevent mixing of nucleic acids on the array due to the substance. In another suitable embodiment, the nucleic acid in one array is the target nucleic acid and the other in the array is an oligonucleotide probe. In another suitable embodiment, the nucleic acids present in both arrays are oligonucleotide probes. In another suitable embodiment, the nucleic acid in one array is an oligonucleotide probe and a target nucleic acid, while the nucleic acid in another array is an oligonucleotide probe. In another suitable embodiment, the nucleic acids in the two arrays are oligonucleotide probes and target nucleic acids.

One method of the present invention using the apparatus described above consists of the following steps;

Providing a nucleic acid arrangement immobilized on a substrate;

Providing a second array of nucleic acids under conditions capable of contact with a fixed array of nucleic acids, where one of the nucleic acid sequences is the target nucleic acid and the other array is an oligonucleotide probe;

Analyzing the hybrid reaction results. In a suitable embodiment, the immobilized arrangement becomes the target nucleic acid and the second arrangement becomes the labeled oligonucleotide probe. In a more suitable embodiment, material is placed between the two arrays to prevent mixing between the arrays until the material is removed.

The second method of the present invention described above is as follows;

Providing two arrays of nucleic acid probes,

Providing conditions such that these two arrangements are in contact with each other and with the target nucleic acid;

Ligation with probes adjacent to the target nucleic acid;

-Analysis of the results. In suitable embodiments, the probes in one array are fixed and the probes in the other array are labeled. In a more suitable embodiment, material is placed between the two arrays such that the two probes are not mixed until the material is removed.

The present invention also provides a substrate in which an oligonucleotide probe array is immobilized, wherein each probe is distinguished from adjacent probes due to physical barriers that impede the flow of the sample solution. In a suitable embodiment, the physical barrier is made of hydrophobic material.

The present invention also provides a method of making oligonucleotide probe arrays distinguished by physical barriers. In a suitable embodiment, a lattice is provided to the substrate using an ink-jet head that supplies a substance that reduces the reaction volume of the arrangement.

The present invention also provides a substrate to which an oligonucleotide is immobilized to form a three-dimensional array. Three-dimensional arrays are compounded with a lot of information (multiple levels or probes) in three-dimensional space in order to present the probe results (each level with a relatively low density of probes per cm 2).

The present invention also provides a substrate to which an oligonucleotide probe is immobilized, wherein the oligonucleotide probe has a space, the space of the oligonucleotide probe that binds to the information portion of the substrate and the oligonucleotide probe to provide sequence information. Increase the distance between parts). In a suitable embodiment, the space consists of a ribose sugar and a phosphate salt, where the phosphate forms esters with the ribose sugar through the 5 'and 3' hydroxyl groups of the ribose sugar and covalently binds with the ribose sugar to form a polymer.

In addition, the present invention provides a method for bringing cDNA clones into groups with similar or identical sequences, such that each clone is selected to sequence each population. In suitable embodiments, the pooling method is used to sequence multiple clones, which signal each clone to multiple oligonucleotide probes; Pool the clones into multiple groups by identifying clones that bind to similar probes at similar strength; Sequencing at least one clone in each group. In a more suitable embodiment, the plurality of probes consists of about 50 to 500 different probes. In another suitable embodiment the plurality of probes consists of about 300 different probes. In the most suitable embodiment, the plurality of clones is a plurality of cDNA clones.

The invention also relates to oligonucleotide probes complexed (covalently or non-covalently) to separate particles, wherein the particles can be divided into a number of sets based on their physical properties. In suitable embodiments, different probes are attached to each set of separate particles, and probe identification is made according to the physical properties of the separate particles. In another embodiment, the probe is identified according to the physical properties of the probe. Physical properties include any used to distinguish discrete particles, including size, fluorescence, radioactivity, electromagnetic charge, absorbance, etc., and can be attached to particles such as dyes, radionuclides or EMLs. Included labels are included. In suitable embodiments, the separate particles are separated by a flow cell system that senses the size, charge, fluorescence, and absorbance of the particles.

The invention also relates to methods of using probes conjugated to separate particles to analyze target nucleic acids. Such probes can be used in any of the methods described above, wherein the probes are identified using the physical properties of the discrete particles. Such a probe is used in a format III method. A "free" probe is identified by a label, and physical probes can be used to identify probes complexed with separate particles. In a suitable embodiment, the probe is used to sequence the target nucleic acid using SBH.

The present invention is a method of using a substance that destabilizes the binding of complementary polynucleotide strands (reduces binding energy) or increases the binding stability between complementary polynucleotide strands (increase binding energy). In a suitable embodiment, the substance is a trialkyl ammonium salt, sodium chloride, phosphate, borate, formamide, glycol, dimethylsulfoxide, dimethylformamide, urea, organic solvents such as guanidium, amino acid derivatives such as betaine, spermi Positively charged molecules that can neutralize the negative charges of polyamines or phosphate backbones such as dine, spermine, surfactants such as sodium dodecyl sulfate, sodium lauryl sulfate, small / large groove binding materials, positively charged polypeptides, Intercalating agents such as acridine or ethidium bromide, anthracene, and the like. In suitable embodiments, the material is used to increase or decrease the T _m of the complementary polynucleotide pair. In a more suitable embodiment, the material mixture is used to reduce or increase the T _m of the complementary polynucleotide. In the most suitable embodiment, the substance or mixture of substances can be used to distinguish completely embroidered from mismatched in complementary polynucleotides. In suitable embodiments, materials are added such that the binding energy of the AT bond pair is approximately equivalent to the binding energy of the GC bond pair. The binding energy of such complementary polynucleotides is increased by adding a substance that blocks or neutralizes the negative charge of phosphate groups in the polynucleotide framework.

This patent application is a continuation of US patent application 08 / 912,885 (August 15, 1997), US patent application 08 / 947,779 (October 9, 1997), US patent application 08 / 892,503 (July 14, 1997), and also the present application Is a serial application of US patent application 08 / 812,951, filed March 4, 1997, and a serial application of US application 08 / 784,747, filed January 16, 1997.

The present invention relates to a method and apparatus for performing nucleic acid analysis, in particular, a method and apparatus for nucleic acid analysis.

1 is a plan view of a material making up the probe array.

2 is a side view of the material making up the probe array.

3 is an enlarged side view of the dispensing unit of the device making the probe arrangement.

Description

1-cylinder, 2-arm, 3-dispenser, 6-reservoir, 7-supply line

8, 14-dispensing tip, 9-chamber, 10-sample line, 11-pressure line

12-body, 13-sample well

When analyzing many sets of samples simultaneously, Format 1 SBH is suitable. Thousands of samples can be arranged side by side on large arrays using thousands of independent hybrid reactions using small membranes. DNA is identified using 1-20 probes per reaction, and in some cases more than 1000 probes specific for each sample are used to identify mutations. To characterize the mutated DNA fragments, one must use a specific or selected specific probe for each mutation detected in the first course of the hybrid reaction.

DNA samples are prepared in small arrays separated by appropriate spaces and tested simultaneously with probes selected from a set of oligonucleotides arranged in multiple well plates. The small array consists of one or more samples. DNA samples in each array may include mutant sequences or individual sample sequences. Subsequent small arrays can be organized into larger arrays. Such larger sequences may include sequences of identical sub-species copies or different DNA fragments. The complete probe set includes a sufficient amount of probes to analyze predetermined accuracy DNA fragments, including an excessive amount of reading each base pair ("bp"), for example. These sets include more probes than are needed for one particular fragment, but may contain fewer probes than necessary to test thousands of DNA samples with different sequences.

DNA or allele identification and characteristic sequencing processes include the following steps;

1) selecting a subset probe from a complete set or representative probe set capable of hybridizing to each of a plurality of small arrays;

2) add a first probe side by side in each subarray on each array to be analyzed;

3) run the hybrid reaction and record the result of the hybrid reaction;

4) remove the probes already used;

5) repeat the hybrid reaction, record and remove remaining probes;

6) process the obtained results for final analysis or to determine hybrid additional probes;

7) perform additional hybridization on specific subarrays;

8) Process the complete data set and do a final analysis.

In this way, one type of minority nucleic acid (eg, DNA, RNA) can be quickly identified, sequenced, and analyzed side by side with many sample types in the form of subarrays using pre-synthesized probe sets (suitable length). do. The two methods can be combined to make a variety of processes that are effective in determining DNA identity, identifying DNA, and identifying mutations.

To identify known sequences, short probe sets are used instead of long unique probes. In this method, there are more probes to record, but a complete set of probes can be synthesized to include any form sequence. For example, the 6-mer complete set contains only 4,096 probes and the 7-mer complete set contains only 16,384 probes.

Two levels of hybridization can be performed to fully sequence DNA fragments. One method is to hybridize at least once with a sufficient set of probes for all bases. To this end, a specific set of probes is synthesized against a standard sample. Hybridization with this set of probes allows us to know where and where (differences) mutations exist in non-standard samples. In addition, such a probe set may include a "negative" probe to confirm the hybrid reaction result of the "positive" probe. To determine the substance of the change, additional specific probes can be hybridized to the sample. Such additional probe sets include both “positive” (mutated sequence) probes and “negative” probes to detect sequence changes by positive probes and to identify such changes using negative probes.

In another embodiment, all probes in the complete set can be obtained. With a complete set of probes, relatively small amounts of probes between samples can be obtained in a two-step process without unnecessary time consumption. The hybrid reaction process is a step of continuous probes, which first calculates the appropriate set of probes to be hybridized first, and then determines which probes should be further obtained from the complete set based on the results obtained next. Both sets of probes have "negative" probes to identify positive probes in the set. In addition, the obtained sequence can then be identified by hybridizing the sample with the "negative" probe set identified in the SBH results in a separate step.

When combining SBH sequences, special consideration is given to K-1 oligonucleotides that occur repeatedly in the analyzed DNA fragments, either for accidental or biological reasons. In the absence of additional information, a relatively small amount of DNA is completely assembled so that all base pairs are read multiple times.

In the case of combining relatively long fragments, an ambiguous case occurs due to recurrence in a positively-recorded probe set of K-1 sequences (eg, sequences shorter than probe length). This problem does not arise when determining mutated or similar sequences (eg, K-1 sequences are not identically repeated). Using the knowledge of one sequence as a template, the positive probes are arranged for the unknown sequence that best fits the template to accurately combine the sequences known to be similar (identify their presence in the database).

Sample arrangements can be used to avoid the continuous recording of many oligonucleotides on one sample or a small set of samples. Using this method, more probes can be recorded side by side by adjusting only one physical target. In a relatively short time, DNA subarrays of 1000 bp in length can be sequenced. If the sample is spotted into 50 small arrays, the array can repeat 10 probes, recording 500 probes. When screening for mutagenesis, use a sufficient amount of probe to curve each base three times. If a mutation is present, the base covered several times will be affected. Information about negative probe entities can be used to map mutations with two bases of accuracy. To find single base mutations created in this manner, an additional 15 probes can be used. Such probes can curve arbitrary base complexes for the two questionable locations (presumably not related to deletions and insertions). These probes are recorded in one procedure in 50 subarrays containing a given sample. When multiplexing with multiple label colors, two to six probes (each probe is labeled differently with different fluorescent colors) are used together to reduce the number of hybridization steps and shorten the sequencing process.

In more complex cases, there are two adjacent mutations or insertions. This should be handled with more probes. For example, if three bases are inserted, 64 probes should be used. In the most complex cases, a few stages of hybrid reactions should be approached, and new probe sets should be selected based on the results of existing hybrid reactions.

If the subarray to be analyzed contains tens or hundreds of samples of one type, it can be seen that some of them contained one or more changes (mutations, insertions or deletions). For each fragment that is mutated, a specific set of probes should be used. The total number of probes written into each sample can be millions. By writing replicas side by side, you can write millions of probes in a relatively small number of times. In addition, compatible probes can be collected. Since specific DNA fragments differ by approximately 75% of these constituent bases, a probe selected to assign specific DNA fragments to positive hybrid reactions can be assigned.

By using a larger set of longer probes, longer targets can be analyzed. These targets are collections of fragments, such as exon clone collections.

Specific hybrid recording methods can be used to determine if there is a mutation in the genomic fragments that are sequenced in the fold chromosome set. There are two variables; 1) one chromosomal sequence represents a known allele, the other sequence represents a new mutation, or 2) a new but different mutation on the two chromosomes. In these two cases, the scanning step designed to change the map provides a twofold maximum signal difference at the location of the mutation. In addition, this method can be used to determine whether each gene has an allele individually and whether each individual is homologous or heterologous to that gene.

By comparing the signals corresponding to the homology and release criteria, the double signal difference required in the first case can be effectively written. Using this method, it is possible to determine whether the hybrid reaction signal is relatively reduced for each particular probe in each given sample. Of particular interest, the hybridization effect is more than doubled for certain probes that hybridize to different nucleic acid fragments having the same perfectly matched target. In addition, different mutation sites affect one or more probes depending on the number of oligonucleotide probes. The signal for the two to four consecutive probes is reduced, indicating the mutation site more carefully. The results can be checked using a probe subset selected from one or more selected probes to provide a perfectly matched signal that is on average eight times stronger than signals generated in a double helix containing mismatches.

Using partitioned membranes, highly fluid experiments can be performed to test a relatively large number of samples giving a given sequence type, or a variety of samples with a relatively small number of samples. The number of samples that can be effectively handled ranges from 4 to 256. Small arrays with multiple dot ranges are created to fit the size and shape of standard multiwell plates used to store and label oligonucleotides. The size of small arrays can be adjusted to accommodate different sample numbers, or some standard small array sizes are available. Proceed to the same probe using additional arrays or membranes because they do not fit into one array for all sample types. In addition, the number of each subarray replica is adjusted to vary the time taken to complete the identification and sequencing process.

As used herein, the term "middle fragment" refers to 5 to 1000 bases in length, preferably 10 to 40bp in length.

In the case of Format 3, a first set of oligonucleotide probes with known sequences is immobilized on a solid support under conditions such that the probes can hybridize to nucleic acids having respective complementary sequences. A second set of labeled origonucleotide probes is provided in solution. Between these sets and within the set are probes of the same length and probes of different lengths. The nucleic acid to be sequenced or an intermediate fragment thereof is provided to a first set of double stranded probes (particularly the portion with the recA protein that allows hybridization under undenatured conditions) or to a single stranded probe, wherein the conditions Is a condition that can hybridize with varying degrees of complementarity (eg, a condition that can identify a hybrid reaction that is a complete match and a mismatch of one base). The nucleic acid or intermediate fragment thereof to be sequenced is provided to the second probe set in advance, or following or concurrently. Probes bound to adjacent sites on the target are bound together (eg, by overlapping actions or ligase or by means of causing chemical bond formation between adjacent probes). After allowing adjacent probes to bind, fragments and probes that are not immobilized to the surface by chemical bonding to the first probe set component are dissolved by removing the hybrid using a high temperature (up to 100 ° C.) wash solution. Probes bound to the second set can be sensed using means appropriate to the label used (eg, chemiluminescence, fluorescence, radioactivity, enzymes, density measurements, or calorimetric labels).

Here, in the case of forming a stable double helix by hydrogen bonding under certain conditions, the nucleic acid bases are referred to as "match" or complementary. For example, under conditions commonly used for hybrid reaction testing, adenine ("A") pairs with thymine ("T") but not with guanine ("G") or cytosine ("C"). Do not. Similarly, G pairs with C, but not with A or T. Other modified bases, such as inosine or universal bases ("M" bases, Nichols et al 1994) or methylated bases, which are capable of hydrogen bonding in a weak specificity, may also form stable double helices under certain conditions. Complementary to base. When each base in a probe hydrogen bonds to a base in a nucleic acid to be sequenced according to Watson and Crick's base pairing principle to create a double helix, the probe is said to be "completely complementary" or "completely paired" (optional). Without affecting the surrounding sequence, the double helix formed has a maximum binding energy for a particular probe). The terms “fully complementary” and “fully paired” also include probes having similar or modified nucleotides. "Fairly paired" for similar or modified nucleotides is determined according to the "fully paired principle" selected for analogues or modified nucleotides (e.g., maximum for a particular similar or modified nucleotide). Binding pairs with binding energy). According to the “principle” each base in a probe that does not form a binding pair is mismatched under certain hybrid conditions, ie it is called a “mismatch”.

The probe list is combined, where each probe is completely paired with the nucleic acid to be sequenced. The probes in these lists are arranged in the maximum overlapping manner and analyzed. The order is determined by comparing the first probe to other probes in the list, determining the 3 'end with the maximum sequence of bases identical to the base sequence at the 5' end of the second probe in the probe. The first probe and the second probe overlap, and the 5 'end of the second probe is compared against the 3' end of the remaining probe, and the 5 'end of the remaining probe and the 3' end of the first probe are compared. Repeat. This process is repeated until none of the probes on the list overlap the other probe. Alternatively, one or more probes are selected from the list of positive probes, and the overlapping probe sets ("sequence nuclei") are side by side. Each probe list for each sequence merging process is a list of all probes that are completely complementary to the nucleic acid or portion thereof to be sequenced.

The 5 'and 3' ends of the probe overlap to create a longer sequence band. This probe coalescing process is repeated until ambiguity appears due to branching points (probes are repeated in fragments), longer repeat sequences than probes or uncloned fragments. The sequence band between any two ambiguities is called a fragment or subclonal sequence (Sfs). Ambiguity occurs during sequence consolidation because of the possibility of overlapping probes with another probe, with longer probes missing in another overlapping part and longer ends of the probe pair missing in hybrids, competitive hybrids, or ambiguities. Ligation or signal pass gel analysis (provide a clear sequence of Sfs) can be used.

By using the above process, hybrid patterns for overlapping or non-overlapping probes or intermediate fragments or whole DNA molecules (eg chromosomes) using coalesced Sfs can be used to identify nucleic acid samples that can serve as symbols to identify nucleic acid samples. Related) can be obtained at the desired level.

Sequences and processes generally consist of the following steps;

a) contacting the nucleic acid fragment with the immobilized oligonucleotide probe sequence under conditions such that the nucleic acid fragment can form a primary complex with the immobilized probe having a complementary sequence;

b) probes labeled with the primary complex can hybridize, under effective conditions contacting the labeled set oligonucleotide with the primary complex, thus forming a second complex, wherein the fragments are combined with the immobilized probe and the labeled probe Can be hybridized;

c) removing from the second complex any unhybridized labeled probe adjacent the immobilized probe;

d) detecting the presence of adjacent and unlabeled probes by sensing the presence of a label; And

e) The nucleotide sequence of the fragment is determined by linking the fixed probe with the known sequence of the labeled probe.

To detect a true paired hybrid in practice (fragments and probes are hybridized to six of seven positions), to identify a complete pair and one base mismatch, or complete Hybrid reaction and wash conditions are selected to detect only hybrids that are well matched.

Appropriate hybrid reaction conditions can be determined by optimizing the process or using pilot studies. Such procedures and studies can be made and implemented by protocols used by those skilled in the art (Ausubel et al., Current Protocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989); Sambrook et al., Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press (1989); Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Cold Spring Harbor, New York (1982). For example, conditions of temperature, component concentration, hybrid and wash time, buffer components and their pH and ionic strength dynamics are varied.

In embodiments in which the labeled probes and the immobilized probes are not physically or chemically linked, the region may only be detected in a controlled washing step. Under these conditions, the binding affinity between adjacent probes is increased due to overlapping interactions between adjacent probes. Conditions may be varied to optimize the process described above.

In embodiments in which immobilized and labeled probes are ligated, they may be ligated using chemical ligation reagents (e.g., water soluble carbodiimide or cyanogen bromide) or a lease enzyme such as commercially available T ₄ DNA ligase. Can be. Wash conditions are selected to identify adjacent labeled and immobilized and non-adjacent labeled and immobilized probes that exhibit stability differences between adjacent and non-adjacent probes.

Oligonucleotide probes can be labeled with fluorescent dyes, chemiluminescent systems, radioactive labels (eg, ³⁵ S, ³ H, ³² P, ³³ P), or isotopes that can be detected with a spectrophotometer.

If the nucleic acid molecule having an unknown sequence is 45 or 50 bp or more, the molecule is fragmented to determine the sequence of the fragment. For fragmentation, restriction enzyme treatment and shear EH can be performed using NaOH. Fragments are separated according to size (electrophoresis) to obtain appropriate fragments 10 to 40 bp in length.

Oligonucleotides can be immobilized using a number of methods known to those skilled in the art, for example using reagents such as nucleoside phosphoramidite or nucleoside hydrogen phosphorate. Using laser phosphate deprotection through a phosphate group. Glass, nylon, silicon, fluorescent carbon support can be used.

Oligonucleotides are arranged and these arrays include all probes or subsets of a given length or include a probe set of a selected length. Hydrophobic separations are used to separate probes or probe subarrays. It can be used for a variety of uses (eg, mapping, subsequences, sequences of target moieties, mRNA sequences and large-scale sequencing for diagnostic purposes). Specific chips are designed to select a complex and array of probes on a substrate for use in a particular application.

For example, all of the five base oligonucleotide probes can be made into 1024 fixed probe arrays, each comprising 1024 separate probes. The probes in this embodiment are 5-mers for informational purposes (these may actually be longer probes). A second set of 1024 5-mer probes can be labeled and one of each labeled probe is provided in a fixed probe array with fragments to be sequenced. In this embodiment, 1024 arrays are composited into a large "superarray" or "superchip". If one of the immobilized and labeled probes is end-end hybrid with the nucleic acid fragment, the two probes are ligated to bind, and after removal of the unbound label, the 10-mers complementary to the sample fragment are known sequences. Detected due to the presence of a label in an array having a fixed probe of provided with a labeled probe of known sequence. The sequence of the sample fragment is simply the sequence of the immobilized probe that is continuous to the labeled probe sequence. In this way, 10,000 possible 10-mers are tested in a complex process that uses only 5-mers and requires tens of millions of efforts for oligonucleotide synthesis.

In a suitable embodiment, the substrate that will support the array of oligonucleotide probes is divided into sections so that each probe in the array is separated from adjacent probes by a physical barrier made of hydrophobic material. In suitable embodiments, the physical barrier has a width of 100 μm to 30 μm. In a more suitable embodiment, the distance from the center of each probe to any adjacent probe center is 325 μm. The probe array is "mass produced" using a stationary fixed substrate fixed to a rotating drum or plate using an ink-jet deposition apparatus, which may include, for example, a micro drop dosing head, a suitable robotic system, anrad. And ananorad gantry.

In another suitable embodiment, the oligonucleotide probe is immobilized in a three dimensional array. The three-dimensional array consists of a plurality of layers, each layer separated from the other layers and analyzed separately. Three-dimensional arrays take many forms, for example, the array is placed on a substrate having multiple depressions and probes located at different depths in the depression (each level is placed on a substrate having a similar depth in the depression) or arrangement Is placed at the bottom of the depression or at a peak that separates the depression or on a substrate having a depth of depression different from the located probe where the peak and depression are combined; Or placed on a substrate composed of multiple layers layered to create a three-dimensional array.

In such an arrangement, the probe includes a space that increases the distance between the surface of the substrate and the information portion of the probe. Spaces consist of at least two covalent atoms, such as carbon, silicon, sulfur, phosphoric acid, or sugar-phosphate groups, amino acids, proteins, nucleosides, nucleotides, sugars, carbohydrates, aromatic rings, hydrocarbon rings, linear and branched It consists of molecules that form at least two covalent bonds, such as chain hydrocarbons.

The nucleic acid sample to be sequenced is fragmented or treated to prevent the hybridization from interfering with the secondary structure (eg, using recA). Samples can be fragmented by treatment with a restriction enzyme such as Cvi JI, shear (ultrasound grinding), NaOH treatment, and the like. The resulting fragments are separated by gel electrophoresis and 10 bp to 40 bp long fragments are extracted from the gel. In suitable embodiments, "fragments" of nucleic acid samples are not ligated with other fragments in the pool. Such fragments can be obtained by treating fractionated nucleic acids with phosphatase (eg, calf visceral phosphatase). Alternatively, non-ligated fragments of sample nucleic acids can be obtained using random primers (eg, N ₅ -N ₉ , where N = AGT or C) in the Sanger-versus sequencing reaction of the sample nucleic acid. This creates a DNA fragment having a sequence complementary to the target nucleic acid and terminates at the dideoxy residue not ligated to the other fragment.

The reusable Fromat 3 SBH array is created by introducing a cleavable bond between the fixed and labeled probes, which is terminated when the bond is cleaved after Format 3 analysis. The labeled probe can be a ribonucleotide, which is used to bind a base to the labeled probe, which is removed by RNAse or uracil-DNA-glycosylation treatment or NaOH treatment. In addition, bonds formed by chemical ligation can be selectively cleaved.

Other variables include using modified oligonucleotides to increase specificity or effect, using a cycling hybrid to increase hybrid signal, e.g., following conditions selected for the first set of labeled probes, followed by a second set of labeled probes. What is necessary is just to perform a hybrid reaction in cycles under suitable conditions. Probe mixture (appropriately the same molar amount mixture) for each of the four nucleotide bases A, T, C, and G can be used to detect the shift of the reading frame.

Branch points cause ambiguity in the ordering of fragments. Sequence information is determined by SBH, but (i) long read length, single pass gel sequencing to divide complete gel sequencing cost; Or (ii) comparing the relevant sequences to order hybrid data with this ambiguity (branch point). Primers for single pass gel sequencing through branch points can be identified from the SBH sequence information or from the same vector sequence, which performs a contiguous sequence, standard Sanger-sequence reaction, at the vector insertion site in the sample nucleic acid. The sequence obtained in this single pass gel sequencing is compared to Sfs, which reads inside branches, to confirm the Sfs sequence. Alternatively, the Sfs sequence is determined by comparing the Sfs sequence with the related sequence, ordering the Sfs, and making the sequence closest to the related sequence. In addition, the number of side-by-side repeating nucleic acid fragments in the target fragment is determined using single pass gel sequencing. Side-by-side repeats occur rarely in the portion of the gene encoding the protein, so the gel-sequencing step is performed only when the non-coding portion is identified as being of particular interest (eg, if it is an important regulatory sequence).

The information obtained about the degree of hybrid reaction seen for about 200 oligonucleotide probes is a unique signal for each gene, which is used to classify cDNA from the library to determine if there are multiple copies of the same gene in the library. Such signals can identify and examine the same, similar or different cDNAs.

Methods of isolating, cloning and sequencing nucleic acids and nucleic acids are known in the art (Ausubel et al., Current Protocols in Molecular Biology, Vol. 1-2, John Wiley & Sons (1989); and Sambrook et al. , Molecular Cloning A Laboratory Manual, 2nd Ed., Vols. 1-3, Cold Springs Harbor Press (1989)).

SBH is a well developed technique that can be implemented in a variety of ways known in the art. In particular, we introduce techniques related to sequencing with hybrids described in the following references; United States Patent No. 5,202,231-1993 to Drmanac et. Drmanac et al., Genomics, 4, 114-128 (1989); Drmanac et al., Proceeding of the First Int'l. Conf. Electrophoresis Supercomputing Human Genome Cantor et al., Eds, World Scientific Pub. Co., Singapore, 47-59 (1991); Drmanac et al., Science, 260, 1649-1652 (1993); Lehrach et al., Genome Analysis: Genetic and Physical Mapping, 1, 39-81 (1990), Cold Res., 4691 (1986); Stevanovic et al., Gene, 79, 139 (1989); Panusku et al., Mol. Biol. Evol., 1, 607 (1990); Nizetic et al., Nucl. Acids Res., 19, 182 (1991); Drmanac et al., J. Biomol. Struct. Dyn., 5, 1085 (1991); Hoheisel et al., Mol. Gen., 4, 125-132 (1991); Strezoska et al., Proc. Nat'l. Acad. Sci. (USA), 88, 10089 (1991); Drmanac et al., Nucl. Acids. Res., 19, 5839 (199); and Drmanac et al., Int. J. Genome Res., 1, 59-79 (1992)).

The invention is illustrated by the following examples. With reference to the content described in the present invention, those skilled in the art will be able to make other embodiments and variations within the scope of the present invention. Therefore, the scope of the present invention is not limited to the following examples.

Example 1; Preparation of Probe Sets

Prepare two universal probe sets. The first set is a relatively short probe complete set, for example, 4096 (or 200 non-complementary) 6-mers or 16,384 (or 8,000 non-complementary) 7-mers. Completely non-complementary subsets of 8-mers or longer probes are inconvenient because they contain more than 32,000 probes.

The second set of probe sets is chosen because a small subset of probes is sufficient to read all bp in any sequence with at least one probe. For example, 12 out of 16 mer is sufficient. For sequencing double helix DNA, for the 7-mers, 8-mers, and 9-mers, the subset would be about 3000, 10,000, and 30,000 probes, respectively.

The probe set is selected to identify target nucleic acids having known sequences and to identify alleles or mutations of target nucleic acids having known sequences. Such probe sets include a sufficient amount of probes so that at least once all the nucleotide positions of the target nucleic acid can be read. Alleles or mutations can be identified when one binding of a "positive" probe is lost. The specific sequence of these alleles or mutations includes all possible nucleotide changes, and is determined by examining the target nucleic acid with a set of probes comprising a combination of these changes at these probe positions.

The probe set consists of 30 probes to a universal probe set (all probes of a specific length), more suitably the set consists of 100-500 probes, and in the most suitable embodiment, the probe comprises 300 probe sets do. In a suitable embodiment, the probe set is 6-9 nucleotides in length, using which the cDNA clones are grouped into populations with similar identical sequences, and a single clone is selected from each group for sequencing.

Probes are made using standard chemistry with one to three non-specific bases (eg, A, T, C mixed) or universal bases (eg, M base or inosine) at the ends. When using radiolabeling, it has an OH group at the 5 'end to attach a radiolabeled phosphate group. Or probes labeled with any suitable system such as fluorescent dyes and the like. Other types of probes such as Protein Nucleic Acids (PNA) or probes containing modified bases that can change the stability of the double helix can be used.

Probes are stored in multi-well plates with barcodes. For a few probes, 96 well plates can be used; For example, for 10,000 or more probes, it is desirable to store in 384 or 864-well plates. By stacking 5-50 plates, all probes can be stored. About 5ng of probe is enough to hybridize one DNA sample. Thus, about 50 mg per probe can be synthesized, thus analyzing 10 million samples. When each probe is used for the third sample each time, if each sample is 1000 bp in length, it is possible to sequence 30 billion bases (10 human genomes) with 5,000 probe sets.

Example 2; Probes with Modified Oligonucleotides

The modified oligonucleotides are introduced into the hybrid probes and placed under appropriate conditions. For example, the use of halogen pyrimidines at the C ⁵ -position affects the stacking of bases to improve the stability of the double helix. 2,6-diaminopurine is used to provide a third hydrogen bond in base pairing with thymine to thermally stabilize the DNA-double helix. With 2,5-diaminopurine, shorter oligomers can be used by increasing the stability of the double helix, providing more stringent conditions for annealing and suppressing background problems.

The modified triphosphate nucleotide synthesis is described in Hoheisel & Lehrach (1990).

Nichols et al. As described in (1994), non-identified base analogs or universal bases can be used. Design of this new analogue, 1- (2-deoxy-D-ribfuranosyl) -3-nitropyrrole (designated as "M") resulting from degeneracy of oligonucleotide probes and genetic code It is used as a primer to solve a problem or when only fragment peptide sequence data is available. Such analogs maximize stacking and minimize hydrogen-bonding interactions without disrupting the spatial interference of the DNA double helix.

M nucleoside analogs utilize non-protonous polar substituents linked to heteroaromatic rings to maximize stacking interactions and enhance the stacking interactions within and between strands to reduce the role of hydrogen bonding in base pair specificity. Nichols et al. (1994) preferred 3-nitropyrrole 2-deoxyribonucleoside because it is structurally and electronically similar to p-nitroaniline. This derivative is one of the minimum intercaltors of double stranded DNA.

Dimethyloxytrityl-protected nucleoside M phosphoramidites can be used to bind nucleotides used as primers for sequencing and polymerase chain chain reaction (PCR). Nichols et al. (1994) replaces the actual number of nucleotides with M without compromising primer specificity.

The unique property of M is that it is possible to make functional sequence probes, replacing continuous nucleosides. When sequencing with 3, 6, and 9 M, a readable sequencing ladder is provided and the product is amplified correctly when PCR is performed using primers containing three different M- ( Nichols et a., (1994).

The ability of oligonucleotides to comprise 3-nitropyrrole to function as a primer means that the double helix structure forms a complementary strand. Oligonucleotide pair d (5-C ₂ -T ₅ XT ₅ G ₂ -3), d (5-C ₂ A ₅ YA ₅ G2-3), where X and Y are A, C, G, T, M It is reported that the optical thermal profile obtained in Fig. 3 corresponds to the normal signal pattern that appears when the DNA double strand is transferred to a single strand. It has been reported that the Tm values of oligonucleotides containing XM base pairs where X is A, C, G, T and Y are M are all in the 3 ° C. range (Nichols et al., 1994).

Example 3; Probe Selection and Labeling

When creating subarrays, limit each probe array to a set of probes that are hybridized in each of the hybrid processes. For example, 384 probe sets are selected from the universal set, and 96 probes are run in each of the 4 procedures. The probe selected to hybridize in one process has a similar G + C content.

The probe selected for each procedure is transferred to a 96 well plate and then labeled by kinase or other labeling procedure (unless labeled before use, for example if it is not labeled with a stable fluorescent dye).

Based on the first round hybrid procedure, a new set of probes is assigned to each subarray for further processing. Some arrays are not used in some processes. For example, if only eight of the 64 patient samples show mutations, eight probes are first written to each mutation, all 64 probes are recorded in one procedure and 32 subarrays are not used. This unused subarray is then treated with a hybrid buffer to prevent drying of the filter.

The probe may be recovered from the storage plate in any conventional manner, such as, for example, a single channel pipetting device, a robot station such as Beckman Biomek 1000 (Beckman Instruments, Fullerton, California) or a Mega Two robot (Megamation, Lawrenceville, New Jersey). The robot station includes a data analysis program and a probe adjustment program. The results of these programs are entered into one or more robot stations.

The probes are collected one by one and added to the subarray covered with hybrid buffer. The recovered probe is preferably placed in a new plate to mix or label with hybrid buffer. Appropriate recovery methods access plates stored one by one and pipet (or transfer using metal pins) a sufficient amount of each selected probe from each plate to a particular well of the intermediate plate. Individual pipettes or pin arrays are used to accelerate the recovery process.

Example 4; Labeled Probe Preparation

For example, a Biosystem system is used to prepare oligonucleotide probes by automatic synthesis, routinely known in the art. Alternatively, the probe may be prepared using a stacking method using a porous Teflon wafer of Genosys Biotechnologies Inc.

For 100-200 μm or 100-400 μm spot arrays the oligonucleotide probes are radiolabeled (eg ³⁵ S, ³² P, ³³ P, suitably ³³ P); Or a non-radioactive isotope (Jacobsen et al., 1990) or a fluorophore (Brumbaugh et al., 1988). Such labeling is routine in the art and related implications are described in Sambrook et al. (1989) or Schubert et al. (1990), Murakami et al. (1991), Cate et al. (1991) et al.

Regarding radiolabeling, conventional methods can be end-labeled using T4 polynucleotide kinase or labeled with high specific activity using Klenow or T7 polymerase. This is explained in the following.

Synthetic oligonucleotides were synthesized without phosphate groups at the 5 terminus and delivered ^-32 p using the enzyme bacteriophage T4 polynucleotide kinase or delivered ^-33 p from [ ^-32 P] ATP or [ ^-33 P] ATP. It can be easily labeled. When the reaction is carried out effectively, the specific activity of such probes is as high as the specific activity of [ ^-32 P] ATP or [ ^-33 P] ATP itself. The reaction described below is designed to label 10 pmole oligonucleotides with very high specific activity. While keeping all component concentrations constant, the reaction size can be increased or decreased to facilitate different amounts of oligonucleotide labeling.

1.0 μl oligonucleotide (10 pmoles / μl); 2.0 μl 10 × bacteriophage T4 polynucleotide kinase buffer; 5.0 μl [ ⁻³² P] ATP or [ ^-33 P] ATP (sp. Act. 5000 Ci / mmole; 10 mCi / ml on aqueous solution) (10 pmoles); Prepare the reaction mixture with 11.4 μl water. 8 units (˜1 μl) of bacteriophage T4 polynucleotide are added to the reaction mixture and incubated at 37 ° C. for 45 minutes. The reaction is heated at 68 ° C. for 10 minutes to inactivate the bacteriophage T4 polynucleotide kinase.

Effective delivery of ³² P or ³³ P to oligonucleotides and their specific activity is determined. If the specific activity of the probe is acceptable, it is purified. If the specific activity is very low, an additional 8 unit enzyme is added and incubated at 37 ° C for an additional 30 minutes, then the reaction is heated at 68 ° C for 10 minutes to inactivate the enzyme.

Methods for purifying radiolabeled oligonucleotides include ethanol precipitation; Precipitation with cetylpyrididium bromide; chromatography with bio-gel P-60; Chromatography on a Sep-Pak C ₁₈ column; Polyacrylamide gel electrophoresis is used.

In order to synthesize DNA strands complementary to synthetic oligonucleotides, Klenow fragments of E. coli DNA polymerase I can be used to obtain highly specific probes. The short primer is hybridized to an oligonucleotide template, which sequence is the complement of the desired radiolabeled probe. The primer is then extended with a Klenow fragment of E. coli DNA polymerase I to bind [ ^-32 P] dNTPs or [ ^-33 P] dNTPs in the template-direction. After the reaction, electrophoresis through a polyacrylamide gel under denaturing and denaturing conditions to separate the mold and the product. In this way, oligonucleotide probes containing several radioactive atoms in an oligonucleotide molecule can be made.

To use this method, the calculated amount of [a-32P] dNTPs or [a-33P] dNTPs necessary to achieve the desired specific activity and sufficient to synthesize all template strands is mixed in a microfuge tube. Appropriate amount of primer and template DNA is added to the tube and 3 to 10 times greater primer than template.

Then add 0.1 volume of 10 x Klenow buffer and mix well. Klenow fragments of E. coli DNA polymerase I are added 2-4 units to 5 [mu] l of the reaction, mixed and incubated at 4 [deg.] C. for 2-3 hours. If desired, the reaction process is monitored by drawing off a small amount of reactant and measuring the radioactive rate that can precipitate with 10% trichloroacetic acid (TCA).

The reaction is diluted with the same amount of gel-drop buffer, heated to 80 ° C. for 3 minutes, and then the entire sample is added dropwise to the modified polyacrylamide gel. After electrophoresis, the gel is self-radiographed to localize the probe and remove it from the gel. Various methods for fluorescent probe labeling are used, Brumbaugh et al. (1988) describe the synthesis of fluorescently labeled primers. Deoxyuridine analogs with a 12 atom primary amine "linker arm" attached at the C-5 position were synthesized. Analogue synthesis methods induce 2-deoxyuridines through organometallic intermediates to give 5 (methyl propenyl) -2-deoxyuridine. Reaction with dimethoxytrityl-chloride gives the corresponding 5-dimethoxytrityl adduct. The methyl ester is hydrolyzed, activated and reacted with the appropriate monoacylated alkyl diamine. After purification, the resulting linker arm nucleosides are converted to nucleotide analogues suitable for chemical oligonucleotide synthesis.

The oligonucleotides are then made to contain one or more linker cancer bases using modified phosphoridite chemicals. To 25 μl 500 mM sodium bicarbonate (pH 9.4) add 20 μl 300 mM FITC dimethyl sulfoxide to a 50 nmol linker arm oligonucleotide solution. The mixture is stirred for 6 hours at room temperature. Eluate with 20 mM ammonium acetate (pH 6) from a 1 x 30 cm Sephadex G-25 column to separate from free FITC.

In general, fluorescence labeling of oligonucleotides at the 5 'end consists of two steps. First, during automated nucleic acid synthesis, an N-protected aminoalkyl phosphoramidite derivative is added at the 5 'end of the oligonucleotide. After removal of all protecting groups, the appropriate fluorescent dye NHS ester is bound overnight to the 5 'amino group and purified labeled oligonucleotides from excess dye using reverse phase HPLC or PAGE.

Schubert et al. (1990) describe phosphoramidite synthesis, which can produce fluorescently labeled oligonucleotides during automated DNA synthesis.

Murakami et al describe a method for preparing oligonucleotides labeled with fluorescents.

Cate et al. (1991) describe the use of oligonucleotide probes conjugated directly to alkaline phosphodases in combination with a direct chemiluminescent material (AMPPD) to detect probes.

Labeled probes should be purchased from a variety of commercially available products, including GENSET, rather than synthetic.

Other labels include ligands that specifically bind to labeled antibodies, chemiluminescent materials, enzymes and the like that can act as binding partner members specific for labeled ligands. Various labels can be used for the immunoassays used. Other labels include antigens, groups with specific activity, electrochemically detectable moieties, and the like.

In general, labeling nucleic acids using shear labeling (“EML”) is described in Xu et al., J. Chromatography 764: 95-102 (1997). Electrophores are compounds that are sensitively detected by an electron capture mass spectrometer (EC-MS). EMLs can be attached to probes using known chemicals that can reversibly modify nucleotides (eg, known nucleotide synthesis chemistry teaches various methods of attaching molecules to nucleotides with protecting groups). EMLs are detected using known electron trapping mass spectrometer devices (eg, commercially available from Finnigan Corporation). In addition, techniques used to detect EMLs include rapid electron impact mass spectrometry (Koster et al., Biomedical Environ. Mass Spec. 14: 111-116 (1987)); Plasma desorption mass spectrometer; Electrospray / ion spray (Fenn et al., J. Phys. Chem. 88: 4451-59 (1984), PCT Appln. No. WO 90/14148, Smith et al., Annal. Chem. 62: 882-89 (1990)); Matrix-supported riser desorption / ionization (Hillenkamp, et al., "Matrix Assisted UV-Laser Desorption / Ionization: A New Approach to Mass Spectrometry of Large Biomolecules," Biological Mass Spectrometry (Burlingame and McCloskey, eds.), Elsevier Science Publishers, Amsterdam, pp. 49-60, 1990); Huth-Fehre et al., "Matrix Assisted Laser Desorption Mass Spectrometry of Oligodeoxythymidylic Acids," Rapid Communications in Mass Spectrometry, 6: 209-13 (1992)).

In suitable embodiments, the EMLs are attached to the probe by covalent bonds with low sensitivity. The EML is released from the probe after hybridization with the target nucleic acid using a laser or other light source that emits the desired wavelength of light. The EML is then fed to GC-MS (Gas Chromatography-Mass Spectrometer) or other suitable device to confirm the mass.

Example 5; Prepare Sequencing Chips and Arrays

The basic embodiment uses a 6-mer attached to a 50 micron surface to provide a 3 x 3 mm size chip, which is combined to create a 20 x 20 cm arrangement. Another example is the use of 9-mer oligonucleotides attached to 10 × 10 microns to make 9-mer chips (5 × 5 mm in size). Using each chip 4000 unit, make a 30 x 30 cm arrangement. In the case of 4,000 to 16,000 oligochip arrays, the array is square. The plate or tube is packaged with the array as part of the sequencing kit.

The arrangements are separated from one another either physically or due to hydrophobic surfaces. One possible method of using hydrophobic strip separation is to use the Iso-Grid Microbiology System technology produced by QA Laboratories, Toronto, Canada.

Over the past decade, hydrophobic lattice membrane filters (HGMF) have been used for analytical food microbiology, which automatically count a large range of unique affinity and colony counts. One commercially available lattice is ISO-GRID ^™ from QA Laboratories, Toronto, Canada. It is a polysulfone polymer (Gelman Tuffryn HT-450, 0.45 μ pore size) cube (60 x 60 cm), including 1600 ( A black hydrophobic ink grid of 40 x 40 square rectangles is printed. HGMF is already inoculated with bacterial suspensions using vacuum filters and incubated on differential or selective media.

Since microbial growth is confined to lattice fins of known position and size on the membrane, HGMF functions closer to the MPN device than conventional plates or membrane filters. Peterkin et al. (1987) can use these HGMFs to grow and store genomic libraries when used with HGMF replicators. Such a device can replicate growth in each of the 1600 mu s of ISO-GRID, making many copies of the original HGMF (Peterkin et al., 1987).

Sharpe et al. (1989) also use ISO-GRID HGMF, automated HGMF counter (MI-100 Interpreter), RP-100 Replicator from QA Laboratories. Many techniques have been reported for maintaining and screening microbial cultures.

Peterkin and his colleagues have recently described how to screen DNA probes using hydrophobic lattice-membrane filters (Peterkin et al., 1989). The authors report how to effectively and efficiently colony hybrids directly on HGMFs. Previously, poor results were obtained because DNA was poorly bound to the epoxysulfone polymers printed with HGMFs. However, Peterkin et al. (1989), by treating cloned cultured HGMF with polyethyleneamine, polycationic, prior to contacting DNA, can improve the ability of DNA to bind to the membrane surface. This initial work uses cellular DNA attachment, which employs Format 3 SBH, a method described for purposes other than the present invention.

To quickly identify useful sequences, see Peterkin et al. (1989) uses plasmid DNA radiolabeled from various clones and tests its specificity for DNA in prepared HGMFs. In this way, recombinant plasmid DNA can be quickly screened by colony hybrid reactions for 100 organisms in HGMF replicates that can be easily and repeatedly made.

Small (2-3 mm) chips can perform thousands of reactions side by side. The solution of the present invention is to keep the chip and probe in the corresponding arrangement. As an example, a chip containing 250,000 9-mers is synthesized on a silicon wafer, in the form of an 8 x 8 mM plate (15 uM / oligonucleotide) arranged in an 8 x 12 Format (96 chips) with 1 mM grooves. , Pease et al., 1994). The probe may add one probe to one needle, using a multichannel pipette or pin arrangement. To write all 4000 6-mers, 42 chip arrays must be used, either different or one set chip array multiple times.

In this case, using the initial list of applications, F = 9; P = 6; F + P = 15. The chip has a probe of the formula BxNn, where x represents the number of specific base B; n represents the number of non-specific bases, and therefore x = 4 to 10; n = 1 to 4. In order to obtain more effective hybrid reactions and to avoid the effects of any support oligonucleotides, certain bases are surrounded by unspecified bases and thus can be represented by (N) nBx (N) m (FIG. 4).

In another embodiment of the chip, the substrate supporting the array of oligonucleotide probes is divided into sections so that each probe in the array is distinguished from adjacent probes by a physical barrier that becomes a hydrophobic material. In a suitable embodiment, the physical barrier has a width of 300 μm to 30 μm, and the distance from the center of the bad physical barrier to the center of the adjacent physical barrier is at least 325 μm.

In a suitable embodiment, the hydrophobic material is deposited on a substrate to create a barrier of the desired width using an ink-jet head coupled to a suitable robotic system. For example, in the case of a microdrop dosing head, this is intended for use with a desired hydrophobic solution or suspension (e.g., an oil-based material that forms a barrier after the solvent has been vaporized), which is incorporated into an Anrod Gantry system, Secured in a suitable receiving and dispensing system, a grid of hydrophobic material is provided to the desired substrate to form a plurality of wells on the substrate. After the lattice of hydrophobic material is formed, a different probe is spotted into each well (or a probe mixture is supplied to each well) using a robotic system similar to that used to make the lattice but adapted for use in probe suspensions or solutions. In one embodiment, the same robotic system is used to provide hydrophobic gratings and probes. In such embodiments, the dispensing system is primed for probe delivery to the hydrophobic lattice and then filled with solution.

Example 6; Preparation of Support Bound Oligonucleotides

Oligonucleotides, such as small nucleic acid fragments, can be prepared by synthesizing oligonucleotides directly using chemical means such as those made using conventional automated oligonucleotide synthesizers and the like.

In general, oligonucleotides are bound to the support through appropriate reactors. Such reactors are well known in the art and include amino (-NH ₂ ), hydroxyl (-OH), carboxyl (CO ₂ H) groups and the like. Oligonucleotides bound to the support are made using any method known to those of skill in the art using any suitable support such as glass, polystyrene, Teflon. One method is to accurately match oligonucleotides synthesized by standard synthesizers. Immobilization is passive adsorption (Inouye & Hondo, 1990); UV light (Nagata et al., 1985., 1985; Dahlen et al., 1987; Morriey & Collins, 1989); Or by covalent linkage of base-modified DNA (Keller et al., 1988; 1989), to which reference is attached.

Another strategy available is to use strong biotin-streptavidin interactions with the linker. For example, Broude et al. (1994) describe the use of biotinylated probes immobilized on streptavidin-coated magnetic beads, although these are double helix probes. Streptavidin-coated beads can be purchased from Dynal, Oslo. Of course, the same link chemistry can be used to coat any surface with streptavidin. Biotinylated probes can be purchased from a variety of sources, such as Operon Technologies (Alameda, Calif.).

Nunc Laboratories (Naperville, IL) also commercially available suitable materials. Nunc Laboratories has developed a method of covalently binding DNA to the surface of a microwell called Covalink NH. CovaLink NH is a polystyrene surface with a secondary amino group (> NH) that acts as a linking head for further covalent bonds. CovaLink Modules are available from Nunc Laboratories. DNA molecules can be bound exclusively to CovaLink at the 5 'end by phosphoramidate linkages to immobilize more than 1 pmol DNA (Rasmussen et al., 1991).

The use of CovaLink NH strips for covalent binding of DNA molecules at the 5 'end is described (Rasmussen et al., 1991). In this technique, phosphoramidate binding is used (Chu et al., 1983). This is advantageous because immobilization using only one covalent bond is preferred. Phosphoramidate binding binds DNA to the CovaLink NH secondary amino group located at the end of the space covalently bound through a 2 nm long spacer arm on the polystyrene surface. In order to link oligonucleotides to CovaLink NH via phosphoramidate linkages, the oligonucleotide ends must have a 5 ′ end phosphate group. Perhaps it is possible to covalently bind biotin to CovaLink and use streptavidin, which is used to bind the probe.

More specifically, the linkage method involves dissolving DNA in water (7.5 ng / μl), denaturing at 95 ° C. for 10 minutes, and cooling on ice for 10 minutes. 0.1 M 1-methylimidazole, pH 7.0 (1-MeIm ₇ ), stored on ice, is added to a final concentration of 10 mM 1-MeIm ₇ . The ss DNA solution is then placed in CovaLink NH strips (75 μl / well) stored on ice.

Carbodiimide 0.2 M 1-ethyl-3- (3-dimethylaminopropyl) -carbodiimide (EDC) dissolved in 10 mM 1-MeIm ₇ is made fresh and 25 μl is added to each well. The strip is incubated at 50 ° C. for 5 hours. After incubation, the strips are washed with Nunc-Immuno washes; The wells are washed three times and then allowed to absorb the wash solution for 5 minutes; Finally wash 3 times (the wash solution was heated to 50 ° C. with 0.4 N NaOH, 0.25% SDS).

Further suitable methods of using the invention are described in PCT patent application WO 90/03382 (Southern & Maskos). The method for preparing an oligonucleotide bound to a support is to attach a phosphodiester covalent linkage to an aliphatic hydroxy group possessed by the support and attach it to the nucleoside 3'-reagent through a phosphate group. The oligonucleotides are then synthesized on the supported nucleotides and the protecting group is removed from the synthetic oligonucleotides under appropriate conditions such that the oligonucleotides are not cleaved from the support. Suitable reagents include nucleoside phosphoramidite and nucleoside hydrogen phosphate.

To prepare a DNA probe array, an on-chip strategy of preparing a DNA probe is used. For example, addressable laser-activated photodesorption is used to chemically synthesize oligonucleotides directly on the glass surface (Fodor et al. (1991)). Probes are also described in Van Ness et al. Fix it to nylon support as described in (1991) or connect it to Teflon using the method of Duncan & Cavalier (1988).

Van Ness et al. In order to link oligonucleotides to the nylon support as described in (1991), the surface of the nylon is activated through alkylation and selective activation of cyanuric chloride with the 5'amine of the oligonucleotide.

One particular method for preparing oligonucleotides bound to supports is to use the photo-generated synthesis described in Pease et al., (1994). The inventors now use photolithographic techniques to create immobilized oligonucleotide probe (DNA chip) arrays. This method of using light to synthesize oligonucleotide probes at high density utilizes light labile 5′-protected N-acyl-deoxynucleoside phosphoramidites, surface linker chemicals, and various complex synthesis strategies. 256 spatially defined oligonucleotide matrices are made in this manner and then used for beneficial Format 3 sequencing as described herein.

Of course, one can readily purchase DNA chips, one of the photo-activated chips described above that are commercially available. In this case, Affymetrix of Santa Clara, CA 95051, and Beckman can be contacted.

In a suitable embodiment, the probes of the present invention include an information portion (a portion hybridized to a target nucleic acid to provide sequence information); A reactor attached to a substrate (solid support); Random positions (eg, any of the four bases can be seen to be bound at these positions), and the like. Suitable probes have the sequence 5 '-(T) _6- (N) _3- (B) ₅ , where T is thymine (binds to a solid support) and N = A, C, G, T (randomized position) ), B = five information positions (information parts) of the probe. In the most suitable embodiment, the probe is bound to the support and the space portion is found at the end of the probe or inside the probe or at 5 'of (N) ₃ . Spaces are atoms or sugar-phosphate groups, amino acids, peptides, nucleosides, nucleotides, sugars, carbohydrates, aromatic rings, hydrocarbon rings, linear and at least two covalent bonds such as carbon, silicon, oxygen, sulfur, phosphate, etc. It consists of molecules that make at least two covalent bonds, such as branched hydrocarbons.

Example 7; Nucleic Acid Fragment Preparation

The nucleic acid to be sequenced can be obtained from any suitable source such as cDNA, genomic DNA, chromosomal DNA, finely cut chromosomal bands, cosmids or YAC inserts, RNA including mRNA without any proliferation step. For example, Sambrook et al. (1989) describe three processes for separating high molecular weight DNA from mammalian cells (p. 9.14-9.23).

Targeted nucleic acid fragments can be prepared by cloning into M13, plasmid or lambda vectors, made directly from genomic DNA, or obtained from cDNA using PCR or other amplification methods. Samples are prepared or dispensed in multiple well plates. Approximately 100-1000 ng DNA is allowed to have a final volume of 2-500 ml. The target nucleic acid prepared by PCR is directly applied to the substrate for Format I SBH without purification. Once the target nucleic acid is immobilized on the substrate, the substrate is washed or annealed directly to the probe.

The nucleic acid is then used in methods known to those skilled in the art, such as Sambrook et al. (1989) into fragments using the restriction enzymes described in 9.24-9.28 or by methods such as sonication and NaOH treatment.

Schriefer et al. Low pressure shear as described in (1990) is also an appropriate method. In this method, the DNA sample passes through a small French pressure shock at various pressures, from low to medium pressure. The lever system can be used to adjust the pressure from low to medium. The results show that low-pressure shear can be substituted for DNA fragmentation using ultrasound and enzymes.

One suitable method of fragmenting DNA is to use an endonuclease, CviJI, which recognizes the two bases described in Fitzgerald et al (1992). The inventors describe a rapid method of fragmenting and fractionating DNA at a particular size, which is suitable for shotgun cloning and sequencing. We have found this method to be useful for making relatively small random DNA fragments that can be used in the sequencing techniques of the present invention.

The restriction endonuclease CviJI normally cleaves PuGCPy, a cognitive sequence between G and C, to produce blunt ends. Using atypical reaction conditions that modify the specificity of this enzyme (CviJI ^** ), DNA fragments are made semi-random from small molecule pUC19 (2688 base pairs). Fitzgerald et al. (1992) quantitatively assessed the randomness of this segmentation strategy by fractionating pUC19 treated with CviJI ^** sized fractions using rapid gel filtration and ligating directly to the lac Z minus M13 vector without end repair. According to the sequence analysis of 76 clones, CviJI ^** was added to the PuGCPy site, limiting pyGCPy and PuGCPu and accumulating new sequence data at a rate consistent with random fragmentation.

As reported in the literature, the advantages of this method when compared to the methods of sonication and agarose gel fractionation are that the amount of DNA required is smaller (0.2-0.5 μg is sufficient instead of 2-5 μg); The number of steps involved is lower (no ligation, end repair, chemical extraction, agarose gel electrophoresis and elution). This advantage can be used when preparing DNA for sequencing by Format 3.

In suitable embodiments, "fragments" of nucleic acid samples are not ligated with other fragments in the pool. Such fragments can be obtained by treating fractionated nucleic acids with phosphatase (eg, calf visceral phosphatase). Alternatively, non-ligated fragments of sample nucleic acids can be obtained using random primers (eg, N ₅ -N ₉ , where N = AGT or C) in the Sanger-versus sequencing reaction of the sample nucleic acid. This creates a DNA fragment having a sequence complementary to the target nucleic acid and terminates at the dideoxy residue not ligated to the other fragment.

Regardless of how the nucleic acid fragments are obtained or prepared, it is important to denature the DNA into single stranded fragments available for hybridization. DNA can be denatured by incubating the DNA solution at 80-90 ° C. for 2-5 minutes. The solution is then rapidly cooled to 2 ° C. and contacted with the chip to prevent DNA fragments from regenerating again.

Example 8; DNA Array Preparation

Dot the DNA sample on a support such as a nylon membrane to create an array. Spotting is carried out using metal pin arrays (having positions corresponding to well arrays in microtiter plates) to repeatedly transfer about 20 nl DNA solution onto the nylon membrane. With offset printing, the density of dots can be made higher than the density of wells. Depending on the label type used, 1 to 25 dots are accommodated within 1 mm 2. By avoiding spotting on some preselected number of rows and columns, you can create separate subsets (subarrays). Samples in one subarray can be the same genomic DNA fragment (or the same gene) from another individual or different overlapping genomic clones. Each subarray represents duplicate spotting of the same sample. As an example, the selected gene fragment can be amplified from 64 patients. For each patient the amplified gene fragments may be in one 96-well plate (all 96 wells contain the same sample). Prepare plates for each of 64 patients. All samples are spotted on one 8 × 12 cm membrane using a 96-pin device. The subarray contains 64 samples, one for each patient. In the case where the 96 sub arrays are the same, the dot range is 1 mm 2 and there is a 1 mm space between the sub arrays.

Another approach is to use membranes or plates (NUNC, Naperville, Illinois) that are partitioned using physical spaces, for example, plastic prices molded over the membranes, where the grating is a membrane type or hydrophobic strip that provides the bottom of a multiwell plate Similar to Fixed physical space is not suitable when the image is made by exposure to a flat in-storage screen or x-ray film.

Example 9; Hybrid reaction and recording process

Labeled probes are mixed with hybrid buffer and pipetted into subarrays using multichannel pipettes. To prevent mixing of the probes between the subarrays (in the absence of a physical barrier injected between the hydrophobic strips or membranes), the corresponding plastic, metal or ceramic grid is securely fixed to the membrane. Alternatively, the volume of the buffer is reduced to about 1 ml or less per mm 2. Probe concentrations and hybrid reaction conditions used are the same as previously described, except that the wash buffer is poured quickly over an array of subarrays to quickly dilute the probes to prevent cross-hybrid reactions. For the same reason, use a minimum concentration of probes and the hybrid start extends to the maximum level. For DNA detection and sequencing, knowledge of the "normal" sequence is used, and a continuous stacking interaction phenomenon is used to increase the signal. In addition to the labeled probes, an unlabeled probe is added that is back to back hybrid with the labeled in the hybrid reaction. The amount of hybrid is increased several times. Probes are linked by ligation. This method is important for unpacking the "compressing" parts of DNA.

In the case of radiolabeled probes, phosphate storage techniques can be used to obtain the filter phase appropriately. Record the fluorescent label using a CDC camera, a confocal camera, or others. To properly discard and coalesce data from other hybrid experiments, the actual signal is normalized based on the target amount at each dot. The difference in the amount of target DNA per dot is corrected by dividing the signal of each probe by the average signal for all probes. Standardized signals are classified 1-100 in comparison with data obtained from other experiments. In each subarray, several reference DNAs are used to determine the mean of the background signal in the sample that does not contain a fully paired target. In the case of samples with dual grades (multiple grades), homology criteria can be used to recognize dysplasia in the sample.

Example 10; Oligonucleotides and Hybrid Reactions

Oligonucleotides were purchased from Genosys Inc., Houston, Texas and synthesized using an Applied Biosystems 381A DNA synthesizer. Most of the probes used were not purified using HPLC or gel electrophoresis. For example, probes include a completely complementary target in interferon, a 921 bp Eco RI-Bgl II human B1-interferon fragment (Ohno and Tangiuchi, Proc. Natl. Acad. Sci. 74: 4370-4374 (1981)). The M13 clone was designed to have at least one target with one base mismatch in the M13 vector itself.

Terminal labeling of oligonucleotides is described in Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labortory Cold Spring Harbor, New York (1982), T4-polynucleotide kinase (5units Amersham), g ³² P -10 ml containing ATP (3.3 pM, 10 mCi Amersham 3000 Ci / mM), oligonucleotides (4 pM, 10 ng). Specific activity of the probe is 2.5-5 × 10 ⁹ cpm / nM.

Spot single stranded DNA (2-4 mL / 0.5 NaOH, 1.5 M NaCl) on a Gene Screen membrane moistened with the same solution, filter neutralized to 0.05 M Na ₂ HPO ₄ pH 6.5 and placed in an oven at 80 ° C. for 60 min. Afterwards, UV is emitted for 1 minute. The filter is then incubated in hybrid solution (0.5 M Na ₂ HPO ₄ pH 7.2), 7% sodium lauryl sarcosine for 5 minutes at room temperature, and placed on a plastic dish surface. A drop of hybrid solution (10 mL 0.5 M Na ₂ HPO ₄ pH 7.2, 7% sodium lauryl sarcosine sodium) with ³² P-terminal labeled oligomer probes at 4 nM concentration was placed on 1-6 dots per filter and then Cover the piece (approximately 1 × 1 cm) and incubate in a wet room at the indicated temperature for 3 hours. The filter is placed in a 3 × 5 6 × SSC wash solution three times for 5 minutes at 0 ° C. to remove the unhybridized probe, ending the hybrid reaction. The filter is then dried or further washed at the indicated time and temperature and taken radiophotor (phosphoimager (Molecular Dynamics, Sunnyvale, California)). For identification measurements, after taking a radiograph, the dots are cut out of the dried filter, placed in a liquid scintillation caltail, and countered. The cpm uncorrected ratio for the IF and M13 dots is D.

The conditions reported here allow for the hybridization of very short oligonucleotides but for the identification of mismatched oligonucleotides with paired oligonucleotides that are complementary to and bind to the target nucleic acid. Based on the degree of identification (D) between a completely complementary target in a hybrid and an incomplete complementary target with one mismatch, it is defined as a factor that affects the effective detection of a hybrid of a particular short sequence. In experimental tests, 28 probes of 6-8 nucleotides in length were dot blotted hybridized to two M13 clones or hybridized to model oligonucleotides bound to membrane filters. The principle driving the experimental process is as follows.

The oligonucleotide hybrid reaction, which filters bound target nucleic acids several nucleotides longer than the probe under conditions with excess probes, becomes a simulated primary response to target concentrations. This reaction is as follows.

S _t / S _o = e ^{-kh [OP] t}

In this case, S _t and S _o represent target sequence concentrations at t and t _o time. (OP) is the probe concentration and t represents the temperature. The rate phase of hybrid formation, k _h, is slightly increased in the range of 0 ° C. to 30 ° C. (Porschke and Eigen, J. Mol. Biol. 62: 361 (1974); Craig et al., J. Mol. Biol. 62: 383 (1971)). Hybrid melting is a first order reaction to hybrid concentration as shown below;

H _t / H _o = e ^-kmt

In this formula, H _t and H _o represent hybrid concentrations at t and t _o times, respectively; k _m is a hybrid melt rate constant with temperature and salt concentration [Ikuta et al., Nucl. Acids Res. 15: 797 (1987); Porsclike and Eigen, J. Mol. Biol. 62: 361 (1971); Craig et al., J. Mol. Biol. 62: 303 (1971). During the hybrid reaction, a process in which the strands are associated, a reverse melting or strand separation reaction occurs. Thus, the hybrid amount formed in time represents the pre-reaction and reverse-reaction results. Increasing the probe concentration or decreasing the temperature shifts the equilibrium towards hybrid formation, but the melt reaction prevails during the washing process in a significant amount of buffer and the reverse reaction hybrid is weak because there is no probe. These assay results indicate that short oligonucleotide hybrid conditions (SOH) that can work on probe concentration or temperature vary.

Define for D or identification in the following formula;

D = H _p (t _w ) / H _i (t _w )

H _p (t _w ) and H _i (t _w ) refer to the amount of hybrid that the fully complementary double helix and the incomplete complementary helix remain during the washing time (t _w ) for each equal amount. At a given temperature, identification D changes 10 times the length of the wash time and reaches a maximum when H _i = B (Equation 5).

Background B represents the lowest hybrid signal the system can detect. Since no further reduction in H _i was examined, D increases with continuous washing. A wash past t _w decreases H _p over B, which can be seen to decrease. For incomplete hybrids from equations 3 and 5 the optimal wash time t _w is as follows;

t _w = -ln (B / H _i (T ₀ )) / K _mi

Since H _p is washed for the same t _w , the formula can be combined to obtain the optimal identification function as follows;

D = e ^{ln (B / Hi (t0) km, p / km, i} XH _p (t ₀ ) / B

The change in D as a function of T is important because it selects the optimum wash temperature. It is possible to obtain the final equation by substituting the following Arhenius equation (K- = Ae ^{-Ea / RT} ) for the existing equation;

D = H _p ((t ₀ ) / BX (B / H _i (t ₀ )) ^{(Ap / Ai) e (E} a, i ^-E a, p ^{) / RT} ;

At this time, B is less than H _i (t ₀ ).

Since the active energy of a complete hybrid, E _{a, p} and the active energy of an incomplete hybrid, E _{a, i} can be the same or E _{a, i} can be less than E _{a, p} , D is independent of temperature or temperature Can decrease with increasing. This result suggests that it is not suitable for investigating stringent temperature conditions for good identification in SOH. In the case of washing at lower temperatures, the same or better identification can be made, but the washing temperature increases exponentially with decreasing temperature. If H _i (t ₀ ) is increased for H _p (t ₀ ), the identification decreases very strongly with T.

At lower temperatures, D depends more on the H _p (t ₀ ) / B ratio than on H _p (t ₀ ) / H _i (t ₀ ). This result indicates that a sufficient amount of H _p can be obtained in the hybrid reaction regardless of the identification obtained at this stage. Larger amounts of complete hybrids require more differential melting times to be effective. Similarly, using a larger amount of target nucleic acid can provide the necessary identification even when the difference between K _{m, p} and K _{m, i} is small.

Given a more complex situation than this simple model would accept, the results show that it is important to wash at low temperatures to identify hybrid cases of probes with many terminal mismatches in a given nucleic acid target. .

Using the theoretical principles described as a guide for experiments, using probes with 6 to 8 nucleotides in length can yield reliable hybrids. All experiments were performed using a suspended plastic sheet providing a filter-topped hybrid solution film. This process can reduce the amount of probe to the maximum, thereby reducing the labeling cost of the dot blot hybrid reaction. Using high concentrations of lauryl sarcosine sodium in place of lauryl sulfate sodium in the hybrid phosphate buffer can reduce the reaction temperature from room temperature to 12 ° C. Similarly, using 4-6 X SSC, 10% lauryl sarcosine sodium buffer, hybrid reactions can be performed at low temperatures such as 2 ° C. Surfactants in such buffers provide a background that can accommodate concentrations of labeled probes up to 40 nM. Stability to the columns of short oligonucleotides can be previously investigated in prototypes with 50% G + C, such as octamers with probe sequence TGCTCATG. Its transition enthalpy is similar to the more stable heptamer or enthalpy of 6 nucleotides in length (Bresslauer et al., Proc. Natl. Acad. Sci. USA 83: 3746 (1986)). The temperature variable T _d at the time when 50% of the hybrid melts in minutes is 18 ° C. The results show that for hybrids with 8bp rather than 11bp double helix, T _d is lower to 15 ° C [Wallace et al., Nucleic Acids Res. 6: 3543 (1979).

In addition to experimenting with model oligonucleotides, the M13 vector was chosen as a system to describe the hybrid reaction of short oligonucleotides. The main goal is to provide useful end-mismatch identification between similar targets that may be used in the various methods of the present invention. Oligonucleotide probes of the M13 model were chosen because they contain terminal mismatch bases in the M13 model itself. The vector, an M13 recombinant comprising a 921 bp human interferon gene insert, carries one complete paired target. Thus, IF has the same or more mismatch targets when compared to the M13 vector itself.

Cold conditions and dot blots can be used to obtain a hybrid sig- nal difference between a dot with a complete target and a mismatched target and a dot with only a mismatch target. This is true for 6-mer oligonucleotides and for 7 and 8-mer oligonucleotides hybridized to IF-M13 large nucleic acid pairs.

Hybrid reaction signals depend on the amount of target available in the filter that reacts with the probe. The difference in sine intensity does not reflect the varying amounts of nucleic acid in the two dots. Hybrid reactions with probes having the same number and the same kind of targets in both IF and M13 show that the dots have the same amount of DNA. The hybridization effect increases with hybrid length, so that in the case of a double helix having six nucleotides, the signal is best detected when the amount of oligonucleotide target bound to the filter is high. Because of their low molecular weight, larger numbers of oligonucleotide target molecules can bind to a given surface as compared to the larger molecules of nucleic acid that act as targets.

To measure the sensitivity of detecting unpurified DNA, various amounts of phage supernatant are spotted on the filter and hybridized with ³² P-labeled octamers. Even 50 million unpurified phages containing 0.5 ng DNA provide a detectable signal, indicating that the sensitivity of the short oligonucleotide hybrid reaction method is sufficient. The reaction time is short.

As mentioned in the theoretical section above, the equilibrium yield of the hybrid depends on the oil probe concentration and the reaction temperature. For example, for targets of the same amount as 4 nM octamers at 13 ° C., the signal levels are three times lower than for 40 nM probe concentrations and decrease 4.5 times as the hybrid reaction temperature is raised to 25 ° C.

The use of cold washes for maximum identification has been described. For visual clarity, 50 times DNA is placed in the M13 dot than the IF dot using a hybrid reaction with a vector specific probe. In this way, after hybrid reactions with real probes, the signal appears stronger when mismatched than when paired. The H _p / H _i ratio is 1: 4. If the wash was extended at 7 ° C., the signal strength would be reversed by 2: 1, without quantitative loss of the complete hybrid. In contrast, it is not possible to make any identification at 25 ° C., because the paired target signals went down to the background level after a two minute wash; At the same time, the signals of mismatched hybrids are still detectable. Compared with 7 ° C, the loss of identification at 13 ° C is large and can be clearly seen. When the mismatched hybrid signal is close to the background level, 90 minutes at 7 ° C. and 15 minutes at 13 ° C. are several times greater at 7 ° C. than 13 ° C., given the optimum cleaning time for each condition. Is obvious. To further illustrate this, in washing the same amount of starting hybrid at two temperatures, a higher maximum D was shown at lower temperatures in the change of discernment over time. These results confirm that D tends to change with the ratio and temperature of the two types of hybrids at the start of the washing step.

To demonstrate the general utility of short oligonucleotide hybrid reaction conditions, hybrid reactions of four heptamers, ten octamers, and up to twelve nucleotides in length in a simple M13 system were investigated. This includes two extreme GC contents, the 9mer GTTTTTTAA and the 8-mer GGCAGGCG. Although GC content and sequence are expected to affect the stability of short hybrids [Bresslauer et al., Proc. Natl. Acad. Sci. U.S.A. 83: 3746 (1986)], in order to obtain sufficient identification, low temperature short oligonucleotide conditions can be applied to all probes tested. Since the identification value obtained with a 13 nucleotide probe of length 20 is the best 20, it is easily acceptable to be reduced several times due to sequence changes.

The M13 system has the advantage of showing the effect of target DNA complexity on the level of identification. In two octamers with no mismatched targets or with five mismatched targets and only one GC pair is different, the observed identification values are 18.3 and 1.7.

To demonstrate the utility of this method, a probe of 8 nucleotides in length was tested on 51 plasmid DNA dot collections made from a library in a Bluescript vector. There is one probe specific for the Bluescript vector, not in M13, and the other two probes have a target that is an insert of a known sequence. Such a system allows the use of hybrid reaction negative or positive reference DNA with each probe. This probe sequence (CTCCCTTT) has a complementary target in the interferon insert. Hybrid reactions are sequence specific because the M13 dot is negative and the interferon insert in M13 or Bluescript is positive. Similarly, in the presence of an appropriate target in the clone, a probe that either detects the target sequence only in one of the 51 inserts or does not detect the target sequence in any of the inserts tested with the criteria to confirm the hybrid response. May occur.

The thermal stability curves for very short oligonucleotides of 6-8 nucleotides in length are at least 15 ° C lower than for hybrids of 11-12 nucleotides in length [Fig. 1 and Wallace et al., Nucleic Acids Res. 6: 3543-3557 (1979). However, hybridization at low temperatures and at a wide variety of oligonucleotide concentrations of 0.4-40 can detect complementary sequences at known or unknown nucleic acid targets. To determine fully unknown nucleic acid sequences, a full set comprising 65,535 8-mer probes can be used. Sufficient nucleic acid is present for this purpose in conventional biological samples such as dozens of [mu] l of M13 culture, bacterial culture or plasmid preparation from 10 ml of single colony bacteria or less than 1 ml of standard PCR reaction.

Short oligonucleotides of 6-10 nucleotides in length provide good identification. Having a single terminal mismatch reduces the stability relative to the longer probe than the longer probe in the case of hybrids. The results with octamer TGCTCATG wish this conclusion. In the case of targets with G / T terminal mismatches in the experiment, hybridization to mismatch type targets is most stable for all of the following types of oligonucleotides. This identification is greater than or equal to the internal G / T mismatch in the 19 base pair double helix [Ikuta et al., Nucl. Acids res. 15: 797 (1987). Using such identification properties, using the described hybrid reaction conditions for short oligonucleotide hybrid reactions, oligonucleotide targets can be determined with great accuracy. In contrast to the ease of identification detection between complete and incomplete hybrids, the problem with using very short oligonucleotides is preparing a sufficient amount of hybrid. Indeed, increasing the amount of DNA in the dots, increasing the probe concentration, or decreasing the hybrid reaction temperature can help to identify H _p and H _i . However, increasing the probe concentration further increases the background. In addition, the amount of target nucleic acid that can be actually used is limited. This problem can be solved by increasing the surfactant sarcosyl concentration, which provides an effective background for 4 nM probes. In addition, it may be improved by using a competition material or a hybrid reaction support material change for non-specifically binding the probe to the filter. For probes with E _{a below} 45 Kcal / mol (for many heptamers and mostly hexamers), modified oligonucleotides provide a more stable hybrid than an unmodified pair [Asseline, et al., Proc. Nat'l Acad. Sci. 81: 3297 (1984). The hybrid reaction conditions described for short oligonucleotide hybrid reactions at low temperature in the present invention provide good identification for all sequences and double helix hybrid offerings. Only the cost of achieving uniformity in hybrid reaction conditions for different sequences is increased at up to 24 hours of wash time depending on the sequence. In addition, the wash time can be further reduced by reducing the salt concentration.

Although one matched hybrid can be immediately identified among mismatched hybrids, in the case of short oligonucleotide hybrid reactions, the signal that occurs in the mismatched hybrid is due to an end mismatch. will be. This means that the size that can be effectively tested with a specific length of probe is limited.

The impact of sequence complexity on identification cannot be ignored. However, in the case of defining sequence information by short oligonucleotide hybrid reactions in the case of certain non-random sequences, the effect of complexity is greater, which can be solved by using probes that are appropriate for the target length ratio. The length ratio is chosen so that no particular sequence with end-mismatch can eliminate false inverse identification. According to the results, we recommend using probes with 6, 7, or 8 oligonucleotide lengths in target nucleic acid inserts shorter than 0.6, 2.5, and 10 kb, respectively.

Example 11; DNA sequencing

Subarrays allow for efficient sequencing of small sample sets arranged in replicated subarray forms; For example, 64 samples are arranged at 8 x 8 mm, and 16 x 24 subarrays are replicated on a 15 x 23 cm film with 1 mm width between subarrays. Several replica membranes can be made. For example, probes in 3072 7-mers universal sets are divided into 32 96-well plates and labeled using kinase. Four membranes are processed side by side during one hybrid process. Each film contains 384 probes. The hybrid reaction intensity is recorded and the following sequences are coalesced.

In the case where one sample subarray contains several unknowns, especially when similar samples are used, fewer probes are sufficient if they were selected based on the results of the previously recorded probes. For example, if the probe AAAAAAA is not positive, it is rare that any of the eight overlapping probes becomes positive. If AAAAAAA is positive, both probes are positive. In this case, the sequencing process consists of first hybridizing a minimum overlapping probe subset to positive fixation and successfully selecting the probes that identify the most likely for the fixation sequence, the size and shape in between. For this second phase, a pool of 2-10 probes is used, where the probe is chosen to be positive in one DNA sample that is different from the sample expected to be positive with other probes in the pool. do.

The sub-array method can effectively perform probe competition (overlapping probes) or probe cooperation (continuous probe stacking) in solving branching problems. After a hybrid reaction of a universal probe set, the sequence subsegments (SFs) that are recommended in the sequence merging program are determined. For further incorporation of SFs, EH from overlapping DNA fragment sequences, analogous sequences, single pass gel sequences should provide additional information from other hybrid reaction or restriction enzyme map data. Through a branch point, primers for a single pass gel sequence can be identified from SBH sequence information or other vector sequences known in the art, eg, sequences adjacent to the vector insertion site, standard Sanger-sequence reactions can be performed on the sample DNA. The sequence obtained in this single pass gel sequencing is compared to Sfs, which reads inside the branch point, to confirm the order of Sfs. In addition, single pass gel sequencing complexes with SBH to sequence the nucleic acid again.

Incorporate Sfs using competitive hybrid reactions and continuous stacking interactions. This approach has limited value for sequencing multiple samples by SBH, but labeled probes are applied to samples immobilized on the array when using the same array. Fortunately, analyzing a small number of samples using replica subarrays makes both methods more efficient. Each replica subarray was tested on one or more DNA samples using a probe pool similar to solving mutated sequences in different samples spotted in the same subarray.

In the case where there are about 100 split points in each of the 64 samples described in this embodiment, when 8 samples are analyzed in parallel in each subarray, at least 800 subarray probes can solve all the branch points. . This means that an additional 800 probes (25%) are used for the 3072 base probes. More suitably, two probes are used at one branch point. If the subarray is small, additional probes are used less. For example, if the subarray contains 16 samples, 200 additional probes are recorded (6%). Using a 7-mer probe (N _1-2 B ₇ N _1-2 ) and a competitive or cooperative branching solution, about 1000 bp fragments can be incorporated into about 4000 probes. In addition, when using 8-mer probes (NB ₈ N), 4 kb or longer fragments can be incorporated into 12,000 probes. For example, to reduce the number of branch points, use a probe with a gap, that is, NB ₄ NB ₃ N or NB ₄ NB ₄ N.

Example 12; DNA analysis by transient attachment to subarray probes and ligation of labeled probes

Oligonucleotide bases having 4 to 40 information lengths are synthesized by standard chemistry and stored in tubes or multiple well plates. Specific probe sets consisting of from 1 to 10,000 probes are synthesized in precipitation or in separate supports or synthesized in separate parts of large supports. In the last case, partial or subarrays are separated using physical or hydrophobic barriers. Probe arrays are synthesized in situ. Appropriately sized sample DNA hybridizes into one or more specific arrays. Many samples react in pools in the same array or independently of other subarrays within one support. Simultaneously or sequentially with the sample, one labeled probe or labeled probe pool is added to each subarray. Attached labeled probes hybridize back-bcak to complementary targets within the DNA to which they are ligated. Detect labels from probes to detect ligation.

This process is a variation of the described DNA analysis process, in which the DNA sample is not primarily attached to the support. It can be temporarily attached using a probe fixed to the support. In this case, no target DNA alignment process is necessary. Short immobilized probes and short labeled probes can also be combined to detect longer oligonucleotide sequences.

This process has some unique features. Basically, the temporary attachment of the target can be used again. After ligation occurs, the target is released and the label remains covalently attached to the support. This feature allows circulating detectable signal production for targets and small targets. Under appropriate conditions, the target does not need to be amplified, for example, a natural source DNA sample can be used directly for diagnostic and sequencing purposes. The target is released by cycling the temperature between the effective hybrid reaction and the effective melting of the double helix. More appropriately, do not cycle. The temperature and concentration of components causes the proportion of free and hybrid targets to equilibrate to about 50%: 50%. In such a case. Ligation products can be produced continuously. If the objectives are different, different equilibrium ratios are suitable.

The electric field can be used to enhance target use. At the beginning, providing horizontal pulses within each subarray allows for faster target classification. In this phase, the equilibrium is shifted towards hybrid formation and an unlabeled probe can be used. Following the target classification, an appropriate washing step (using a vertical electric field to limit the movement of the sample) is performed. To increase specificity, several processes, such as harvesting targets, can be introduced, such as identifiable hybrid melting, hybrid reactions, ligation and removal of unused targets. In the next step, a labeled probe is added and a vertical cell pulse is provided. Increasing the temperature achieves an optimal ratio for the free and hybrid targets. Vertical electric fields prevent the spread of classified targets.

Subarrays of immobilized and labeled probe sets (particularly selected or specially designed from universal probe sets) can be arranged in a variety of ways to effect effective flexible sequencing and diagnostic procedures. For example, when partially or completely sequencing a short fragment of the bacterial genome (100-500 bp), small array probes (5-30 bp in length) designed to fit the base of a known sequence can be used. When reacting with a pool of 10 labeled different probes per subarray, it is estimated to record only two bases linked by ligation into 10 subarrays with 10 probes each capable of checking 200 bases. Under conditions capable of identifying mismatches through hybridization, probes may be replaced with one or more bases to accommodate longer targets with the same number of probes. Longer probes can be used to target directly, without amplification or separation from the rest of the DNA in the sample. If the results obtained indicate mutagenesis (or pathogen), additional pools of probes can be used to detect mutations or pathogen subtypes. This process is very cost effective in prophylactic diagnosis where part of the patient is infected or mutated.

In the process described in the examples, various sensing methods can be used, for example, large molecules or particles that can be detected by radiolabels, fluorescent labels, Hyogo, antibodies (chemical luminescence), light dispersion or interferometric processes. Can be used.

Example 13; Sequencing Targets with Octamers and Nanomers

Sequencing by hybrid from hybrid reaction data of octamer and nanomeric oligonucleotides shows that it provides a significant degree of accuracy. In this experiment, known sequences can be used to predict overlap with octamer and nanomeric oligonucleotides.

In addition to fully paired oligonucleotides, targets are examined for mismatch olinonucleotides with internal or end mismatches in the double helix formed by the oligonucleotides. In such an analysis, the lowest experimental temperature can be used to maximize hybrid reaction formation. Washes are performed at the same or lower temperatures to maximize identification using the larger dissociation rates of mismatched oligonucleotide / target hybrid reactions and matched oligonucleotide / target hybrid reactions. While the absolute yield of hybrids depends on the sequence, such conditions can apply to all sequences.

Destabilizing at least mismatches is a simple end mismatch, where sequencing tests by hybrid reactions are oligonucleotides / targets that are completely matched from end-mismatched oligonucleotides / target duplexes. The ability to identify double helices.

102 identifications out of 105 hybridized oligonucleotides in dot blot format allow for highly accurate sequence generation of two or more. Such a system can also assess the impact of sequences on hybrid reaction formation and hybrid reaction instability.

100 base pairs, such as 100 bp target sequences, of the known portion of the human-interferon gene prepared by PCR are obtained from data obtained by hybridizing 105 oligonucleotide probes having a known sequence to the target nucleic acid. The oligonucleotide probes used were 72 octamers and 21 nanomeric oligonucleotides, which sequence is completely complementary to the target. The 93 probe sets provide a frame that continuously overlaps the target sequence with one or two bases substituted.

To assess the impact of mismatches, hybrid responses were examined for 12 additional probes containing at least one terminal mismatch when hybridized to 100 bp test target sequences. By testing whether the hybrid reaction of 12 probes with the target is end-mismatched against 4 selected reference nucleic acids, the 12 oligonucleotides formed are completely matched with the 4 reference DNAs and the double hybrid. Therefore, hybrid reactions of mismatched oligonucleotides with target double helix pairs, end-mismatched oligonucleotides with target double helix pairs, and fully matched oligonucleotides with target double helix pairs were tested. Each oligonucleotide used was evaluated. The effect of absolute DNA target concentrations in hybrid reactions with test octamer and nanomeric oligonucleotides is that different oligonucleotide probes detect hybrid responses to one non-target site in co-amplified plasmid DNA to target DNA concentrations. Determine by definition.

The results of this experiment show that all oligonucleotides that contain complementary sequences completely matched to the target or reference DNA hybridize more strongly than oligonucleotides with mismatches. To reach this conclusion, the H _p and D values for each probe were examined. H _p represents the amount of hybrid double helix formed between the first target and the oligonucleotide probe. Assigning a value of 0 to 10 to the hybrid reactions obtained for 105 probes, 68.5% of the 105 probes were found to have _Hp values of 2 or more.

Identification (D) includes (1) a dot comprising a fully mesoformed double helix formed between a test oligonucleotide and a target or reference nucleic acid, and 2) a mismatch formed between different sites and the same oligonucleotide within the target or reference nucleic acid. This value can be obtained by indicating the signal intensity ratio between dots including a double helix. Changes in the D value can either: 1) change the hybrid effect to see a signal above background; 2) due to mismatch types found between the test oligonucleotide and the target. The D value obtained in this experiment is 102 to 102 of the 105 oligonucleotide probes tested. Computing the D value of 102 oligonucleotides generally, the average D is 10.6.

There are 20 oligonucleotide / target double helices that exhibit end-mismatches. In five of these cases, D was 10 or more. In this case, large D values are mostly due to hybrid reaction instability due to causes other than stable (G / T and G / A) terminal mismatches. Another possibility is an error in the oligonucleotide or target sequence.

In the case of probes with low H _p values, errors in the target are excluded because such errors affect the hybrid reaction of each of the other eight overlapping oligonucleotides. There is no real instability due to sequence mismatches to other overlapping oligonucleotides, indicating that the target sequence is correct. After retesting the hybrid reaction of the seven newly synthesized oligonucleotides, the errors in the oligonucleotides are excluded. Only 1 of 7 olinonucleotides had a better D value. Low hybrid formation values are due to the lack of hybrid instability or the ability to form a hybrid double helix. The inability to form a hybrid double helix is due to 1) the self complementarity of the selected probe or 2) the target / target self hybrid reaction. Similarly, target / target association works well when the target is self-complementary or forms a palindrom therein. In assessing this possibility, probe analysis showed that the probes in question were not hybridized with themselves. In addition, examination of the target / target hybrid response distribution reveals that one of the oligonucleotide probes in question is ineffectively hybridized with two different DNA containing the same target. If two different DNAs are unlikely to have self-complementary portions to the same target sequence, then the target / target hybrid reaction does not contribute to the formation of a low hybrid reaction. Thus, this conclusion indicates that the instability of the hybrid and the ability to not form a hybrid is responsible for the low hybrid formation observed in certain oligonucleotides. In addition, the results show that the low hybrid formation is due to specific sequences of specific oligonucleotides. As a result, octamer and nanomeric oligonucleotides can be used to obtain reliable results in the construction of sequences.

These results show that using the methods described herein, maximally unique overlaps of constituent oligonucleotides can produce long sequences of arbitrary specific target nucleic acids. Such sequencing methods are dependent on the individual components of the oligomer and indicate that they are independent of their frequency and location.

Sequences created using the algorithm described below have high fidelity. The algorithm ignores the false positive signal generated at the hybrid response dot, indicating that the sequence from the 105 hybrid response values, including four somewhat less reliable values, is accurate. This fidelity, which is sequenced by hybrid reactions, is characterized by the “all or none” kinetics of the short oligonucleotide hybrid reactions and the double helix that exists between the fully mesmerized double helix and the mismatched double helix. Because of the stability difference. The double helix stability ratio of the acquired double helix and the end-mismatch double helix increases as the length of the double helix decreases. In addition, reducing the length of the double helix reduces the binding energy, which lowers the hybrid reaction effect. However, according to the results provided, the octamer hybrid reaction balances factors affecting the stability and identification of the double helix, providing a highly accurate sequencing method by the hybrid reaction. In the results presented in another example, oligonucleotides with 6, 7, 8 nucleotides can be used effectively to achieve reliable sequences at targets of 0.5 kb (hexamer), 2 kb (septamer) and 6 kb (octamer) Make Long fragment sequences overlap to form a complete genomic sequence.

Example 14; Analysis of the data obtained

Image files are analyzed using a phase analysis program such as the DOTS program (Drmanac et al., 1993) and evaluated using statistical functions such as the SCORES program (Drmanac et al. 1994). From the signal distribution, the optimal initiation to convert the signal to +/- is determined. The sequences of labeled probes corresponding to the labeled positions with known fixed probes can be combined to determine F + P nucleotide sequences from fragments. From the overlapping F + Ps determined by the computer, the complete nucleic acid sequence or subfraction sequence of the native molecule, such as a human chromosome, can be coalesced.

One option is to convert the hybrid reaction signal (number) to +/- during the sequence merging process. In this case, coalescing starts with a very large number of F + Ps where the F + P sequence is AAAAAATTTTTT. Compare the four possible overlapping probes AAAAATTTTTTA, AAAAATTTTTT, AAAAATTTTTTC, AAAAATTTTTTG and three additional probes with different start (TAAAAATTTTTT, CAAAAATTTTTT, GAAAAATTTTTT) and confirm three results; (i) only one of the four overlapping probes with the starting probe has a relatively positive record compared to the other six probes, and in the case of AAAAAATTTTTT, one nucleotide is extended to the right; (ii) no probes with significant positive records except start probes, and stop coalescence, eg, the AAAAAATTTTT sequence becomes the end of the DNA molecule being sequenced; (iii) one or more significant positive probe abnormalities are found among the overlapping or three other probes; The coalescence is stopped because of errors or branching (Drmanac et al., 1989).

Computer-based analogy processes use computer programs that use existing algorithms (Pevzner, 1989; Drmanac et al., 1991; Labat and Drmanac, 1993).

In addition to F + P, if F (space 1) P, F (space 2) P, F (space 3) P, and F (space 4) are determined, an algorithm is used to map all the data to find possible errors. Resolve situations that are corrected or have branching problems (Drmanac et al., 1989; Bains et al., 1988;).

Example 15: Sequencing method by two-stage occlusion

The following examples show sequencing methods contemplated by the inventors. First, the entire chip is hybridized with a complex DNA mixture of about 100 million bp (one human chromosome). Guidelines for performing hybridization include Drmanac (1990); Khrapko (1991); Found in a paper such as Broude (1994). These documents suggest wash steps, buffers and hybridization temperature ranges suitable for use in the early stages of Format 3 SBH.

The inventors have determined that the hybridization should be performed for up to several hours at low temperatures (-2-5 ° C.), high concentration salts, due to the very low concentration of target DNA that can be provided. For this purpose SSC buffers are used instead of sodium phosphate buffer (Drmanac, 1990) which precipitates at 10 ° C. The wash does not need to be excessive due to the second step and can be completely eliminated when hybridization is used for (moisture) highly complex DNA sample sequencing. The same buffer is used during the hybridization and wash steps to continue the second hybridization step with the labeled probe.

One labeled probe (6-mer) is added after appropriate washing using a simple robotic device on each array (8 × 8 mm). A 96-tip or 96-pin device is used to accomplish this in 42 processes. Determination conditions in the range described in the known scientific literature can be used.

The present inventors use the following conditions especially. First, the labeled probe is added and incubated at low temperature (0-5 ° C.) for only a few minutes (since the added oligonucleotide is high concentration), then the temperature is increased to 3-10 ° C. depending on F + P length and washing buffer is added do. The wash buffer used at this moment is compatible with the ligation reaction (ie 100 mM salt concentration). After ligase addition the temperature is raised back to 15-37 ° C. to allow rapid ligation (less than 30 minutes) and discrimination of complete and inconsistent hybrids.

The use of cationic detergents in format 3 SBH is also contemplated as published by Pontius & Berg (1991). They use two simple cationic detergents, dodecyl- and cetyl-trimethyl ammonium bromide (DTAB and CTAB), for DNA regeneration.

DTAB and CTAB are modified compounds of (DTAB) quaternary amine tetramethylammonium bromide (TMAB) with methyl groups substituted with 12-carbon alkyl groups or (CTAB) with 16-carbon alkyl groups. TMAB is a bromine salt of tetramethylammonium ions and is a reagent used in nucleic acid regeneration experiments to reduce the melting temperature due to G-C content. DTAB and CTAB are similar in structure to sodium dodecyl sulfate (SDS) when the negatively charged sulfate of SDS is replaced by a positively charged quaternary amine. SDS is commonly used to reduce nonspecific binding and inhibit nucleases in hybridization buffers but does not significantly affect the rate of regeneration.

Enzymes can be added with labeled probes or after appropriate washing steps to reduce background when using the ligation process. Although not used in all SBH methods, ligase technology is well established in the field of molecular biology. For example, Hood et al. Present a ligase-mediated gene detection technique (Landegren, 1988) that can be readily used in format 3 SBH. Wu and Wallace also use bacteriophage T4 DNA ligase to link two adjacent short oligonucleotides. Their oligo ligation reaction is carried out in 50 mM Tris HCl, pH 7.6, 10 mM MgCl ₂ , 1 mM ATP, 1 mM DTT and 5% PEG. The ligation reaction involves heating to 100 ° C. for 5-10 minutes and cooling to 0 ° C. prior to the addition of T4 DNA ligase (1 unit; Bethesda Laboratories). Most ligation reactions are carried out at 30 ° C. and terminated by heating to 100 ° C. for 5 minutes.

A final wash suitable for the detection of hybridized contiguous or ligation reacted oligonucleotides of length (F + P) is then performed. This washing step is carried out in water at 40-60 ° C. for several minutes to rinse off all labeled probes and other compounds that are not ligated to maximize background. Detection is simplified because of covalently labeled labeled oligonucleotides (it does not take time and no low temperature limit is required).

Depending on the label used, chip imaging with various devices is possible. In the case of radioactive labels, an imager and in-store screen technology can be used as scanners (Molecular Dynamics, Sunnyvale, CA). Place the chips in a cassette and cover with an in-screen. After 1-4 hours of exposure, the screen is scanned and the image file is saved on your computer's hard disk. For the detection of fluorescent labels, CCD cameras and epi-fluorescence or confocal microscopes are used. In the case of a chip generated directly on the pixel of the CCD camera, the detection method published by Eggers (1994) is performed.

Charge pair device (CCD) detectors serve as active solid carriers that image and quantitatively detect the distribution of labeled target molecules in probe based assays. The device takes advantage of the unique characteristics of microelectronics that allow for very similar analysis, UV-sensitive detection, high throughput, integrated data recognition and computation. Egger (1994) discloses a CCD for use in probe based analysis, such as the Format 3 SBH of the present invention, which allows for quantitative evaluation in seconds due to sensitivity and direct coupling.

Integrated CCD detection methods enable the detection of molecular binding dictionaries on a chip. The detector quickly generates a two-dimensional pattern that allows samples to be uniquely analyzed. In the operation of the CCD elementary molecular detector, different biological probes may be immobilized directly on the pixels of the CCD or attached to a disposable cover slip located on the CCD surface. Sample molecules may be labeled with radioactive isotopes, fluorescent or chemiluminescent tags.

Exposure of the sample to the CCD based probe array results in photon or radioisotope decay products at the pixel point where the sample is bound to two complementary probes in the case of format 3. Alternatively, electron-hole pairs are generated in silicon when radiated energy from charged particles or labeled samples enters the CCD gate. The electrons are then collected under the adjacent CCD gate and read sequentially on the display module. The number of photons generated at each pixel is directly proportional to the number of molecular binding events at this point. As a result, molecular binding can be determined quantitatively (Eggers, 1994).

By placing the imaging array adjacent to the sample, the collection efficiency is more than 10 times improved over the lens based techniques found in conventional CCD cameras. That is, a sample (emitter) is contacted with a detector (imaging array) to remove conventional imaging optics such as lenses and mirrors.

Highly energetic particles are detected when radioactive isotopes are attached to target molecules as reporter groups. Several reporter groups that emit particles of various energies, such as ³² P, ³³ P, ³⁵ S, ¹⁴ C and ¹²⁵ L, have been successfully used in microfabricated detectors. High energy particles, such as ³² P, offer the best molecular detection sensitivity, while low energy particles, such as ³⁵ S, provide better resolution. Radioisotope reporters can be selected as needed. Once a particular radioisotope label is selected, detection performance can be predicted by calculating the signal-to-noise ratio (SNR) as published by Egger (1994).

Luminescent detection methods use fluorescence or chemiluminescent reporter groups attached to a target molecule. Fluorescent labels are inherently bound or attached through interaction. Best suited for CCD devices in which fluorescent dyes, such as ethidium bromide, have a strong absorption band in near UV (300-350 nm) and a main emission band in visible light (500-650 nm), because quantum efficiency is This is because it is many times lower at the activation wavelength than the fluorescence signal wavelength.

In terms of luminescence detection, polysilicon CCD gates have the capacity to filter the incident light contribution in the UV range, but are very sensitive to the visible light emission generated by the fluorescent reporter group. This large discriminant for UV activation allows a large SNR (over 100) to be achieved by the CCD as published by Egger (1994).

In the case of probe immobilization on the detector, a hybrid matrix can be produced on cheap SiO ₂ wafers, which are placed on the CCD surface and hybridized and dried. This format is economically efficient as DNA hybridization is performed on cheap disposable SiO ₂ wafers to allow reuse of more expensive CCD detectors. Alternatively, the probe can be immobilized directly on the CCD to create a dedicated probe matrix.

A uniform epoxide layer is connected to the film surface using an epoxy-silane reagent and standard SiO ₂ modification chemistry to immobilize the probe on the SiO ₂ coating. The amine modified oligonucleotide probe is then connected to the SiO ₂ surface by means of a secondary amine composition having an epoxide ring. The resulting linkage provides 17 rotatable separation bonds between the three base oligonucleotides and the SiO ₂ surface. In order to completely remove the protons of the amine to minimize secondary structure formation during coupling, the reaction is preferably carried out at 0.1 M KOH and incubated at 37 ° C. for 6 hours.

In Format 3 SBH, the signal is scored every 100 million (bp). There is no need to hybridize all the arrays (4000 5 x 5 mm) at a time and fewer arrays can be used continuously.

Periodic hybridization is one way of increasing the hybridization signal. Most of the probes immobilized in one cycle hybridize with DNA fragments having tail sequences that are not complementary to the labeled probes. By increasing the temperature, the hybrid melts. In the next cycle, some (˜0.1%) hybridize with the appropriate DNA fragment and additional labeled probes are linked. In this case, a distinct melting of the DNA hybrid that is inconsistent with the two probe sets occurs.

In cycle hybridization, all components prior to initiation of cycling are added at 37 ° C. for T4 and at higher temperatures for thermostable ligase. The temperature is then reduced to 15-37 ° C. and the chip is incubated for up to 10 minutes, after which the temperature is increased above 37 ° C. for several minutes and then reduced. The cycle can be repeated up to 10 times. In one variant, longer optimal coupling reactions can be carried out (1-3 hours) if an optimal higher temperature (10-50 ° C.) is used without cycling.

The procedure described herein requires relatively few oligonucleotides, thus enabling complex chip fabrication using standard synthesis and accurate spotting of oligonucleotides. For example, if all 7-mer oligos were synthesized (16384 probes) a 260 million 14-mer list could be determined.

Another variation is to use one or more differently labeled probes per base array. This is done for two purposes; Determining multiplexing or lists of longer oligo sequences, such as 3 × 6 or 3 × 7, to reduce the number of separately hybridized arrays. If two labels are used in this case, the specificity of three consecutive oligonucleotides is almost absolute since the positive site must have sufficient signal for both labels.

Another variant is to use a chip containing BxNy (y is 1 to 4). Such chips allow sequence readings in different frames. This may also be achieved using a suitable set of labeled probes or both F and P probes may have non-specific end positions (ie some terminal degenerate elements). A universal base may also be used to link the probe of defined sequence to the solid carrier as part of the linker. This makes the probe more used for hybridization, making the structure more stable. If the probe has five bases, three universal bases can be used as the linker (FIG. 4).

Example 16: Sequence Determination from Hybridization Data

Sequence assembly may interfere if a given overlap (N-1) mer overlaps more than once. One of two different N-mers in the last nucleotide can then be used for sequence extension. This branch point limits the apparent assembly of the sequence.

Sequence reassembly of known oligonucleotides that hybridize to a target nucleic acid for the complete sequence of the target nucleic acid cannot be performed in any case. This is because some information may be lost if the target nucleic acid is not fragmented to an appropriate size relative to the size of the oligonucleotide used for the hybridization. The amount of information lost is proportional to the length of the target being sequenced. However, if a sufficiently short target is used, the sequence can be determined explicitly.

The incidence of duplicate sequences that interfere with sequence assemblies distributed along a particular length of DNA can be calculated. This requires the introduction of parameter definitions that must be sequence organized: sequence subfragment (SF). The sequence subfragment determines which portion of the target nucleic acid sequence starts and ends with a (N-1) mer repeated more than once in the target sequence. Thus, subfragments are sequences generated between two branching points during sequence assembly in the method of the invention. The sum of all subfragments is longer than the actual nucleic acid due to the overlapping short ends. In general, the subfragments share the (N-1) mer at the end and the starting point and thus cannot be assembled in a linear order without additional information. Depending on the number of repeated (N-1) mers, different numbers of subfragments are obtained for each nucleic acid target. This figure depends on the length of N-1 and the target.

Probability calculations allow us to evaluate the correlation of two factors. Length N-1 using the nested sequence or the average distance A _o of the ranking of the two N- Murray fragment when performed on N-1 of the base length, Lf formula _{N sf = 1 + A o ×} K × P (K, L of _f )

If K is 2 or more, P (K, L _f ) represents the probability that N-mers occur K times on the fragment Lf base length. Also described in Example 18 is a computer program capable of forming subfragments from N-mers for a given sequence.

The longer fragment length at a given probe length increases the number of subfragments. The subfragments obtained cannot be uniquely sequenced. Although not complete, this information is very useful for recognition and comparative sequencing of functional sequences. This kind of information is called partial sequence. Another way of obtaining partial sequences is to use only a subset of oligonucleotide probes of a given length.

There is a good agreement between the theory of random DNA sequences and the predicted sequences from computer simulations. 200 base targets, for example when N-1 = 7 (using 8-mers or 16 5 '(A, T, C, G) B ₈ (A, T, C, G) 3' 10-mers) Nucleic acids have an average of three subfragments. However, because of the variance around the mean value, the target nucleic acid library has an insert of 500 bp so that less than 1 has at least 3 subfragments at 2000 targets. Thus, libraries with short enough target nucleic acid inserts can be used in ideal cases of long nucleic acid sequencing of random sequences. For such inserts, each target can be rebuilt by this method. The entire sequence of large nucleic acids is obtained by overlapping each defined insert sequence.

In the case of very short fragments, such as 8-mer probes, information contained in overlapping fragments present in random PCR fragmentation processes such as cloning or random PCR is used to reduce the need for 50 bases. Short physical nucleic acid fragment pools can also be used. Using 11-mers or 8-mers such as 5 '(A, T, C, G) N8 (A, T, C, G) 3' for 1 megabase sequencing instead of 20,000 50bp fragments, only 2100 A sample is enough. This figure consists of 700 random 7 kb clones (base library), 1 500 pools of 500 bp 20 clones (subfragment sequencing library) and 150 clones from the jumping library. The developed algorithm (Example 18) generates a sequence using the hybridization data of the described sample.

Example 17: Algorithm

This example describes an algorithm for the generation of long sequences written in the four letter alphabet from the minimum number of separated randomly defined constituent K-set words (K is the length of the oligonucleotide probe) of the starting nucleic acid sequence. This algorithm is mainly used for sequencing by hybridization (SBH) process. This algorithm is based on the possibility of using physical nucleic acid sequence pools for small fragment (SF), information fragment (IF) and information fragment definition.

Small fragments may be caused by branching points in the assembly process due to repetition of the K-1 oligomer sequence in the target nucleic acid. Small fragments are sequence fragments found between two repeat units of length K-1 occurring in the sequence. Multiple occurrences of the K-1 word are a source of interference in the overlapping sequencing of K-words during sequencing. Interference results in sequences remaining in the form of small fragments. Thus, the apparent segment between branches where the sequence is not inherently determined is called the sequence small fragment.

An information fragment is defined as a sequence fragment determined by the most recent end of the overlapping physical sequence fragment.

A certain number of physical fragments can be gathered without losing the possibility of information fragmentation. The total length of the randomly gathered fragments depends on the length of the K-set used in the sequence process.

The algorithm consists of two main units. The first part is used to generate small fragments from the K-collections contained in the sequence. Small fragments may occur within coding regions of physical nucleic acid sequences of a particular size or within defined information fragments within long nucleic acid sequences. Two kinds of fragments are components of the base library. This algorithm does not describe the determination of the contents of the information fragment K-sets in the base library. That is, it does not describe the information fragment manufacturing steps to be used in the sequence generation process.

The second part of the algorithm determines the linear order of the small fragments obtained for the purpose of regenerating the complete sequence of the nucleic acid fragments of the base library. For this purpose a second ordered library made from fragments of randomly collected starting sequences is used. This algorithm does not include a base fragment sequence combining step that regenerates the entire megabase plus sequence. This can be done using fragment linkage of the underlying library, which is essential for generating information fragments. Alternatively, the detection may be performed after generating the sequence of the base library fragment by an algorithm by detecting the overlap based on the common end-sequence.

This algorithm does not require knowledge of the number of k-set occurrences given in the nucleic acid sequences of the basic and ordered libraries, nor does it require information that the k-set words are at the end of the fragment. This algorithm works with hybrid content of k-sets of varying lengths. The concept of this algorithm makes it possible to operate with k-sets that include wrong positive and negative k-sets. Only in special cases the content of the wrong k-set affects the accuracy of the sequence generated. This algorithm can be used for optimization of parameters in simulations and for sequence generation in real SBH experiments, such as for generation of genetic DNA sequences. Selection of oligonucleotide probes (k-sets) or optimal length and number of fragments for the limited probes is particularly important for practical and convenient cross sections in parameter optimization.

This part of the algorithm plays a central role in the generation of sequences from the k-set contents. This is based on the unique ordering of the k-sets by means of maximum overlap. The main obstacle to sequence development is the special repeating sequence and the wrong positive or negative k-set. The purpose of this part of the algorithm is to obtain the minimum number of maximum long fragments with the correct sequence. This part of the algorithm consists of one basic step and several control steps. Some information is only available after every address fragment has occurred, so a two-step process is necessary.

The challenge of sequence generation is to obtain a repeated sequence from the word content that, by definition, does not have information about the number of occurrences of a particular k-set. The concept of the whole algorithm depends on whether this problem is solved. In principle there are two opposing methods: 1) the repeated sequence is initially obtained during pSF generation, and 2) the repeated sequence is obtained later in the small fragment final sequencing process. In the first case the pSF contains excess sequence and in the second case there is a lack of sequence. The first method requires the removal of the excess sequence generated and the second method requires the use of some small fragments several times during sequence final assembly.

The difference between the two methods lies in the precision of the inherent nesting rules of the k-set. A less precise rule is that k-set X overlaps maximally with k-set Y only if the rightmost k-1 end of k-set X is at the leftmost end of k-set Y. This rule allows for repetitive sequencing and formation of excess sequences.

The stricter rule used in the second method is the addition procedure: when the rightmost k-1 end of k-set X is at the far left of k-set Y and the leftmost k-1 end of k-set Y K-set X overlaps maximally with k-set Y only if is not present at the rightmost end of the other k-set. Algorithms based on stricter rules are simpler.

The stretching process of a given small fragment is stopped when the last k-set right k-1 end involved does not exist at the left end of all k-sets or is present in more than one k-set. If only one k-set exists, the second part of the rule is tested. If there are k-sets different from those previously included in addition, the assembly of a given small fragment terminates only in the first leftmost position. If no additional k-set exists, the condition for unique k-1 overlap is met, so the small fragment extends to the right by one element.

In addition to the basic rules, complementary rules are used for the use of k-sets of different lengths. The maximum overlap is the length of k-1 of the shorter k-sets of the overlapping pairs. The generation of pSF is performed starting from the first k-set from the file where the k-set is randomly presented regardless of the sequence of the nucleic acid sequences. Thus, the first k-set of the file need not be at the beginning of the sequence or at the beginning of a particular small fragment. The small fragment generation process is performed by ordering the k-sets by unique overlap defined by the rules described. The k-set used is deleted from the file. When there is no longer a k-set nested with the last included, the short fragment construction ends and another pSF construction begins. Most small fragment generations do not start at the beginning, so the formed pSF is added to the k-set pile and is considered a longer k-set. Another possibility is to form small fragments that run in both directions from the starting k-set. This process ends when further overlapping, i.e., the extension of the small fragments, becomes impossible.

pSF can be divided into three groups: 1) small fragments of maximum length and correct sequence in case of correct k-sets; 2) short fragments formed from the use of maximum overlapping rules for incomplete sets or sets with wrong amounts of k-sets; 3) pSF of incorrect sequence. The incompleteness of the set in 2) is caused by the wrong negative result of the occlusion experiment or the use of an incorrect k-set. They are formed because of wrong positive and negative k-sets and a) misconnected small fragments; b) small fragments with wrong ends; c) can be the wrong amount of k-sets represented by the wrong smallest fragment.

In the case of wrong amounts of k-sets, there is the possibility of a k-set containing one wrong base in the middle or one or more wrong bases and a k-set having the wrong base at the end. Short, misconnected small fragments are caused by the latter k-set. The former two kinds of k-sets represent wrong pSFs of the same length as the k-set lengths.

In the case of a false negative k-set, the maximum overlap is not possible, resulting in pSF. In the case of a wrong amount of k-sets with wrong base on the leftmost or rightmost end, pSF is generated since no apparent overlap is possible. PSF is generated if there are wrong positive and negative k-sets with consensus k-1 sequences in the file and one of the pSFs contains an incorrect k-set at the relevant end.

The binding of the pSF linked to the erroneous small fragment modification process of the sequence is performed in the small fragment sequencing process after small fragment generation. The first step of obtaining the final small fragment by means of explicit linkage of the pSF and cutting off the mislinked pSF is described below.

There are two ways to form the wrong fragments. Mistakes occur when an erroneous k-set occurs at repeated sequence assembly points of first length k-1. Second, it occurs when the repeated sequence is shorter than k-1. Each of these situations can occur in two variants. One of the sequences repeated in the first modification represents the end of the fragment. In the second modification the repeated sequence occurs at any position in the fragment. In the first case, the absence of some k-sets (false negatives) in the file is necessary to make the wrong connection. The second case requires the presence of wrong positive and negative k-sets in the pile. In the case of repetition of the k-1 sequence, only one k-set lack is sufficient when one end is internally repeated. Two shortages are needed in the case of strict internal iterations. The reason is that the ends of the sequence can be regarded as an infinite linear array of wrong negative k-sets. Repeated sequences of length k-2 that require two or three erroneous k-sets are considered "when less than k-1." These are detected in real experiments and less frequently in other cases.

Recognition of mislinked small fragments is more strictly limited when repeated sequences do not appear at the end of the fragment. In this situation two further fragments can be detected, one of which contains the leftmost end k-2 sequence, which is also present in the misconnected small fragment, and the other contains the rightmost end k-2 sequence. When the repeated sequence is on the end of the fragment, there is one small fragment that causes a mistake in forming the small fragment on the rightmost or leftmost end, including the k-2 sequence.

Removal of incorrectly linked small fragments by cleavage is performed according to the following rules: If the leftmost or rightmost sequence of small fragments of length k-2 is present in the other small fragments, the small fragments are cut into two small fragments and each Comprises a k-2 sequence. This rule does not apply to repeated ends when there is one or more false negative k-1 sets at points of the repeated k-1 sequence. Mismatched subfractions of this type can be recognized using information from nested fragments or from information fragments from basic and ordered libraries. In addition, poorly linked small fragments are maintained when two or more false negative k-sets occur at two points that contain the same k-1 sequence. This is a very rare situation because it requires at least four specific wrong k-sets. Additional rules can be introduced to cut small fragments on a sequence of length k if a given sequence can be obtained by combining a sequence shorter than k-2 from the end of one small fragment with another beginning.

Strict application of the described rules results in a loss of some integrity to ensure the accuracy of the output. Some small fragments will fit into the pattern of misconnected small fragments and will be cut even if they are not mislinked. There are several situations. For example, the fragment as well as the at least two identical k-1 sequences may comprise any k-2 sequence from k-1 or at least one incorrect negative k-set comprising at least two repeated k-2 sequences. It contains the k-2 sequence in the center.

The purpose of this algorithm is to reduce the number of pSFs to the smallest minimum number of shorter sequences. The generation of longer small fragments or complete sequences is possible in two situations. The first situation relates to the specific order of repeated k-1 words. Some or all of the maximum extended pSFs (first group) may be uniquely ordered. For example, format S-R1-a-R2-b, where S and E are the beginning and end of the fragment, a, b, c are different sequences belonging to each subfragment, and R1 and R2 are two k-1 sequences repeated in series Five small fragments occur at -R1-c-R2-E (S-R1, R1-a-R2, R2-b-R1, R1-C-R2, RE). They are ordered in two ways: in the original sequence or in S-R1-c-R2-b-R1-a-R2-E contrasting fragments having the same number and type of repeated sequences, but differently ordered, That is, there is no other sequence that includes all of the small fragments in S-R1-a-R1-b-R2-c-R2-E. This kind can only be recognized after the pSF generation process. These indicate a two step need in the pSF generation process. When the file contains an incorrect negative or positive k-set, the situation of occurrence of a false short fragment on the position of the unrepeated k-1 sequence is more important.

The method for two pSF groups consists of two parts. First, erroneous amounts of k-sets that appear as non-existent minimal fragments are eliminated. All k-set small fragments of length k not overlapping on either end longer than k-a at one end and longer than k-b at the other end can be removed to form the maximum connection. In experiments the values of a and b of 2 and 3 were considered suitable for removing a sufficient number of incorrect amounts of k-sets.

The integration of small fragments which can be uniquely connected is carried out in the second step. The linking rules are as follows: Two small fragments can be explicitly linked only if there is no overlapping sequence at the beginning or end of the two small fragments at the beginning or end of the other small fragment.

The exception is when one small piece of the considered pair has the same start and end. In this case the connection is permitted even if there is another small piece with the same end present in the pile. The problem is the correct definition of overlapping sequences. For only a pair of small fragments the linkage is not allowed if there is an additional small fragment with an overlapping sequence of length less than k-2 or longer than k-2 or longer, but longer than k-4. Also the end after omitting one or a small number of the last bases and the normal end of the pSF are considered overlapping sequences.

After this step some small fragments with wrong ends and wrong amounts of k-sets (as the smallest minor fragments) can survive. In addition, an error concatenation can occur in very rare cases where a certain number of erroneous specific k-sets exist simultaneously. This case is detected and resolved in the small fragment sequencing process and in an additional control step for handling uncut "misconnected" small fragments.

The short small fragments obtained are of two types. In common cases these small fragments may be explicitly linked due to the distribution of repeated k-1 sequences. This is done after the pSF generation process and is a good example of the need for two steps in the pSF generation process. Short pSF is obtained on the site of an unrepeated k-1 sequence when using a file containing an incorrect positive or negative k-set. In the case of an incorrect amount of k-set, the k-set may contain one or more wrong bases (or one wrong base in the middle) and so on. The occurrence of short, misconnected small fragments is caused by the latter k-set. The former k-set represents the wrong pSF with the same length as the k-set length.

The purpose of pSF integration is to reduce the number of pSFs into longer, smaller fragments with the least number of correct sequences. All k-set small fragments that are longer than k-a at one end and longer than k-b at the other end and not overlapping at either end are removed to allow for maximum connection. In this way most wrong amounts of k-sets are discarded. The linking rule is as follows: Two small fragments may be linked only if there is no overlapping sequence at the beginning or end of the two small fragments at the beginning or end of the other small fragment. The exception is small fragments with the same start and end. In this case the connection is only permitted if there is another small piece with the same end in the pile. The problem is the precise definition of overlapping sequences. The presence of at least two false negative k-sets and the wrong positive and negative k-set combinations on the repeat points of the k-1 or k-2 sequences destroy or "block" some overlapping sequences and result in obvious but incorrect pSF linkages. Can provide. To prevent this, integrity must be sacrificed for accuracy: linkage is not allowed in the presence of end-sequences shorter than k-2 and additional overlapping sequences longer than k-2. The overlapping sequence is defined from the pSF end or omits one or several last bases.

In very rare situations where there is a certain number of incorrect positive and negative k-sets, some small fragments with wrong ends may survive, and some incorrect positive k-sets (with the smallest minor fragments) may be left and the wrong connections This can happen. This case is detected and resolved in an additional control process that handles small fragment sequencing and unconnected small fragments that are not cut.

The small fragment sequencing process is similar to the generation process. If the small fragments are longer k-sets, they are ordered by explicit connection through the overlapping ends. Information for explicit linkage is divided into small fragments generated in fragments in the base library into groups representing fragment segments. This method is similar to the biochemical solution of the problem based on hybridization with longer oligonucleotides with related linkage sequences. The linking sequence is generated as a small fragment using a k-set of appropriate segments of the base library fragment. Associated segments are defined by fragments of the ordered library that overlap each fragment of the base library. The shortest segment is the information fragment of the ordered library. Longer is the number of neighboring pieces of information or the total overlap of fragments of the ordering and base libraries. To reduce the number of separated samples, fragments of the ordered library are randomly collected and unique k-set contents are determined.

A large number of fragments in the sequencing library can be used to generate very short segments, reducing the likelihood of multiple occurrences of the k-1 sequence, which is the reason for small fragment generation. In addition, longer segments consisting of various regions of fragments of the base library do not include portions of the repeated k-1 sequence. In every segment, a linking sequence (linking small fragment) is generated for a particular small fragment from a given fragment. The ordering process consists of three steps: (1) generating the k-set contents of each segment; (2) small fragments in each segment; (3) Small piece connection of segment. The first segment is defined as the difference and overlap between the k-set contents of the fragments of the base library and the k-set contents of the pool of the ordered library. The second (shorter) segment is defined as the difference and overlap of the k-set contents of the first segment.

There is a problem of accumulating wrong positive and negative k-sets in difference and overlap. False negative k-sets from the starting sequence accumulate at the overlap and false positive k-sets occur randomly in the two sequences, but not at the overlap regions. On the other hand, most of the wrong amounts of k-sets from the starting sequence are not taken at overlap. This is an example of using the information from the overlapping fragments to reduce the experimental error of the fragments. Incorrect k-sets accumulate as differences for another reason. False negative k-sets from the original sequence are expanded for false positive k-sets that are not included in the overlap by error. If the starting sequence contains 10% false negative data, then the first and second overlaps contain 19% and 28% false negative k-sets, respectively. On the other hand, mathematical expectations for 77 incorrect amounts of k-sets can be predicted if the base fragment and the pool are 500bp and 10,000bp in length, respectively. However, there are ways to recover most of the "lost" k-sets and to remove the wrong amount of k-sets.

First, we need to determine the base content of the k-set for a given segment as the overlap of the contents of the k-set of a given pair. The overlap then includes all k-sets of the starting k-set content, where the k-1 sequence at one end of the two k-sets of the base set includes the k- + sequence at the other end. This is done before the difference occurs so that the wrong amount of k-set expansion is applied to the difference in this process. All leased k-sets are removed from the nested file as an incorrect amount of k-sets.

The overlap, which is a common k-set, is defined as X (ordered library pool) for each pair (base fragment). If the number of k-sets is large, they are expanded incorrectly according to the rules described. The first difference set is obtained by subtracting the overlap set obtained from a given base fragment. The wrong negative k-set is attached to the difference set from the nested set and removed from the nested set as the wrong positive k-set. When the base fragment is longer than the collected fragments, this difference can represent two separate segments that slightly reduced usability in an additional step. The first segment is the generated overlap and difference of the pair (base fragment) X (ordering library) containing a significant number of k-sets. The k-set of the second segment is obtained by comparing the k-sets of all possible pairs of the first segment. Two differences are defined from each pair, with a large number of k-sets creating overlap. Most of the information available from the overlapping fragments is rarely obtained from the third round, which is recovered at this stage to form overlaps and differences.

(2) Small fragment generation of segments is performed as described for fragments of the base library.

(3) The small fragment linking method includes sequentially determining a pair of small fragments that are correctly connected among the small fragments coming from the basic library fragment having some overlapping ends. In the case of four related small fragments, there are four different small fragment pairs that can be connected because two have the same beginning and two have the same end. Generally two are correct and two are wrong. To find the correct one, the presence of each pair of linking sequences is tested in small fragments resulting from all the first and second segments for a given base fragment. The length and position of the linking sequences are chosen to avoid interference with sequences that occur by chance. They are at least k + 2 in length and comprise at least one element 2 as well as overlapping sequences in a given fragment of small fragments. If only two connection sequences are found and the other two do not exist, the connection is allowed. The two linked small fragments replace the previous small fragment in the file and the process is repeated periodically.

Repeated sequences are generated at this stage. This means that some small fragments are included in the linked small fragments more than once. These are identified by finding the linking sequence connecting two different small fragments with one small fragment.

Recognition of mislinked small fragments resulting from pSF construction and incorporation of pSF into longer fragments is a test of whether the sequence of small fragments from the base fragment is present in the sequence of small fragments generated in the segment for this fragment. Based. The sequence at the incorrectly linked position is not found, indicating that it is a poorly linked small fragment.

In addition to the three steps described above, additional control steps applicable to specific sequences in small fragment sequencing are necessary for more complete sequence generation without mistakes.

Determination of which segment a small fragment belongs to is performed by comparing the contents of the k-set in the segment and the small fragment. Accurate distinction of small fragments is not possible due to errors in the k-set contents due to the first error of the pool and the statistical error of the frequency of k-set occurrences. Therefore, the probability P (sf, s) coming from a given small fragment is measured for each small fragment. This probability is a function of the k-set length, the small fragment length, the length of the ordered library fragment, the size of the pool, and the ratio of bad k-sets in the file: P (sf, s) = (Ck-F) / Lsf

Where Lsf is the length of the small fragments, Ck is the number of common k-sets for a given small fragment / segment pair, and F is a parameter containing the relationship between k-set length, base library fragment, pool size, and error rate .

Small fragments contributing to a particular segment are treated as excessive short pSFs and undergo an explicit linking process. The definition of the apparent connection is slightly different in this case, since the small fragments with overlapping ends are based on the possibility that they belong to the segment considered. In addition, the accuracy of the apparent connection is controlled by connecting to other segments of these small fragments. All small fragments obtained after linking in different segments are combined and the short small fragments contained in the longer small fragments are removed and the remainder is subjected to conventional linking procedures. If the sequence is not completely reoccurring, the separation and small fragment ligation process is repeated and an explicit ligation is carried out using less stringent probability criteria belonging to the particular segment.

No information is used when using strict criteria that define explicit overlap. Several small fragments are obtained for a given fragment instead of the complete sequence. Less stringent criteria are used to generate accurate and complete sequences. In some situations (eg, with erroneous linkages), a complete but incorrect sequence may occur or a "monster" small fragment with no linkages may occur. Thus, for each fragment of the base library, one gets some possible solutions and the most likely correct solution. Also, in rare cases, errors in the small fragment generation process or a certain percentage of the probability of belonging do not produce clear answers. In this case an obvious solution is obtained by keeping the incomplete sequence or comparing the data of other nested fragments of the base library.

The algorithm described was tested against randomly generated 50 kb sequences containing 40% GC mimicking the GC content of the human genome. In the center of the sequence, various All and repeat sequences of about 4 kb in total were inserted. For ex vivo SBH experiments the following process is carried out to obtain appropriate data.

The positions of 60 5kb overlapping "clones" are randomly defined to mimic basic library preparation:

The locations of 1000 500 bp “clones” are randomly determined to mimic the ordered library preparation. These fragments are extracted from the sequence. Twenty fragments of random pools are formed and the k-sets of the pools are determined and stored on the hard disk. These data are used in the small fragment sequencing step: for clones of equal density, 4 million clones in the base library and 3 million clones in the sequence library are used for the entire human genome. A total of 7 million clones are several times less than a few kb long clones for all genomic DNA cloning and sequencing by gel-based methods.

From the data at the beginning and end of the 5 kb fragment, 117 "information fragments" are determined to be in sequence. This is followed by the determination of the overlap k-sets in which a single "information" fragment exists. Only k-set subsets matching the intended list are used. The list includes 65% 8-mer, 30% 9-mer, 5% 10-12-mer. Small fragment generation and sequencing processes were performed based on these data.

Algorithm tests are performed on the data from the two experiments. The sequence of 50 information fragments was regenerated with a 100% correct data set (20,000 bp or more) and 26 information fragments (10,000 bp) were regenerated with 10% incorrect k-sets (5% positive, 5% negative).

In the first experiment, all the small fragments are accurate and the sequence in one of the 50 information fragments does not completely regenerate and remains in the form of five small fragments. Positional analysis of nested fragments in the ordering library shows that they lack information about the unique ordering of the five subfractions. The small fragments can be joined in two ways, 1-2-3-4-5 and 1-4-3-2-5, based on the overlapping ends. The only difference is the positional exchange of subsections 2 and 4. Since subfractions 2, 3, and 4 are relatively short (total 100bp), none of the ordered library fragments will begin or end in subfragment 3.

In order to simulate the actual sequencing process, some incorrect ("crossover") data is included as input in many experiments. The only situation that produces unreliable data under the conditions proposed in oligomeric hybridization experiments is the end mismatch for exact match hybridization. Therefore, only k-sets that differ in a single element on one end in the simulation were considered to be false positives. The "wrong" set is formed as follows. A subset of 5% false positive k-sets is added to the first k-sets of information fragments. A false positive k-set is formed by randomly copying the k-set to replicate and altering the nucleotides on the start or end. Subtraction of the 5% randomly selected k-sets is then performed. In this way the most complex case arises where the correct k-set is replaced with a k-set with the wrong base on the end.

k-set occurrences result in up to 10% incorrect data. This value varies from case to case because of the randomness of the k-set selection to be replicated, changed and deleted. Nevertheless, this ratio exceeds 3-4 times the amount of unreliable data in actual hybridization experiments. The introduced error of 10% doubles the number of small fragments in the basic library fragment (basic library information fragment) and segments. About 10% of the final small fragments have the wrong base at the end as expected in the k-set containing the wrong positive. Neither small fragment mislinking nor small fragments with incorrect sequences are observed. Complete sequences do not regenerate in four of the 26 information fragments tested in the sequencing process. In all four cases, sequences are obtained in the form of several longer and shorter fragments contained in the same segment. The results show that the algorithmic principle works for a large proportion of incorrect data.

The success of sequencing from the k-set content can be described in terms of completeness and accuracy. Two special situations can be defined in the development process: 1) a part of the information falls into the generated sequence but the vague location and type is known; 2) The situation in which the regenerated sequence does not match the sequence in which the k-set contents occurred, but no mistakes can be detected. Assuming the algorithm has been developed to theoretical limits, as in the use of the correct k-set, the first situation can occur. Incompleteness results in a problem of determining the exact length (number of complete series repetitions) of a certain number of small fragments and forging sequences that cannot be explicitly ordered.

Incorrect sequences can be generated using incorrect k-sets. The cause of the error is not a flaw in the algorithm but the fact that the content of the k-set clearly represents a sequence that differs from the original. There are three classes of errors depending on the type of invalid k-sets present in the file. Bad negative k-sets (without bad positives) produce "delete". False positives followed by false negatives result in "insert" or "delete". Deletion occurs when the two beginnings of a small fragment are false negatives. Since all positions in the sequence are defined by the k-set, the occurrence of deletion requires k consecutive false negatives (10% false negatives and a situation where k = 8 occurs after 108 elements). This situation rarely occurs in mammalian genome sequencing operations using a random library containing ten genomic equivalents.

Elongation of sequence ends caused by erroneous positive k-sets is a special "insertion" case since the sequence ends can be regarded as an infinite linear array of erroneous negative k-sets. Consider a group of false positive k-sets that produce small fragments longer than one k-set. This situation can be detected if small fragments occur in overlapping fragments, such as random fragments in an ordered library. Insertion instead of insertion or deletion can occur as a result of a combination of false positive and negative k-sets. In the first case, the number of consecutive negative negatives is less than k. Both cases require several overlapping false positive k-sets. Insertion and deletion are theoretical possibilities. Because the number and specificity of the wrong k-sets are too large.

In other situations where the minimum number of theoretical conditions are not met, a false positive or negative in the k-set content results in a sequence of low integrity.

SBH, sample nucleic acid is sequenced by exposing the sample to a labeled carrier with a carrier-binding probe of known sequence in solution. Probe ligase is introduced into the mixture of probes and samples such that the two probes are chemically bound by the action of ligase when the carrier has a hybridized and labeled probe hybridized along the sample. After washing, chemically linked carrier bonds and labeled probes are detected by the presence of labeled probes. By identifying the type of carrier binding probe and the type of labeled probe at a particular location in the arrangement, a portion of the sample sequence can be determined by the presence of the label at a point in the formatal arrangement with three substrate samples. There is no case where the maximum nested sequence of all ligated probe pairs is not working. None of the samples to be sequenced are nucleic acid fragments or 10 base pairs (“bp”) oligonucleotides. The sample has 4 to 1,000 bases.

The probe is a fragment having less than 10 bases, in particular 4-9 bases. In this manner, the carrier binding probe arrangement includes all oligonucleotides of a given length or includes specific test oligonucleotides. The number of central oligonucleotides is 4 ^N when all oligonucleotides of a given length are used. Where N is the length of the probe.

Example 18: Sequencing Chip Reuse

Conventional oligonucleotide chips cannot be reused immediately when ligation is used in the sequencing process. This problem is overcome by the inventor in various ways.

Ribonucleotides are used as the second probe, probe P, which can be subsequently removed by RNAse treatment. RNAse treatment may utilize RNAse A as an endoribonuclease that pulls short-chain RNA 3 to pyrimidine residues to break phosphate bonds to adjacent nucleotides. The final product is an oligonucleotide with pyrimidine 3 phosphate and terminal pyrimidine 3 phosphate. RNAse A acts in the absence of coenzymes and divalent cations.

To utilize the RNAse, the chip is positively loaded into the appropriate RNAse containing buffer as published by Sambrook (1989). It is recommended to use 30-50 ul of RNAse-containing buffer for every 8 x 8 mm or 9 x 9 mm array at 37 ° C. for 10 to 60 minutes. Afterwards wash with hybridization buffer.

Although not widely available, uracil bases may be used as published by Craig (1989). Destruction of ligation-reacted probe combinations for reusable chip generation is accomplished using E. coli repair enzyme uracil-DNA glycosylase, which removes uracil from DNA.

It is also possible to generate cleavable bonds between probes and cleave the bonds after detection. For example, this can be achieved by chemical ligation reactions published by Shabarova (1991) and Dolinnaya (1998).

Shabarova (1991) discloses the condensation reaction of oligodioxyribonucleotides using cyanogen bromide as a condensing agent. In a one-step chemical ligation reaction, the oligonucleotide is heated to 97 ° C. and slowly cooled to 0 ° C., followed by the addition of 1ul 10 mM BrCN to acetonitrile.

Dolinnaya (1988) shows how to incorporate phosphoamidiate and pyrophosphate nucleotide bonds into DNA double helices. They use a chemical ligation reaction to denature the glycophosphate backbone of DNA using water-soluble carbodiimide (CDI) as coupling agent. Selective cleavage of phosphoamide bonds involves contacting 95 ° C. 15% CH ₃ COOH for 5 minutes. Selective cleavage of pyrophosphate bonds involves contacting the pyridine-water (9: 1) mixture with distilled (CF ₃ CO) ₂ O.

Example 19: Diagnosis-Mutant Scoring or Whole Gene Resequencing

In the simplest case, the goal is to find out whether the selected known mutation occurs in the DNA segment. Less than 12 probes are sufficient for this purpose, with five probes positive for one allele, five positive for the other allele, and two negative for both. Since the number of probes to be scored per sample is small, a large number of samples can be analyzed simultaneously. For example, using 12 probes in a three-hybrid cycle, genes from 96 different gene positions or from 64 patients could be found on membranes containing 12 × 24 subarrays with 64 dots representing the same DNA segment from 64 patients. × 9 can be analyzed. In this example the samples are prepared in 64 96-well plates. Each plate is for one patient and each well represents one of the DNA segments to be analyzed. Samples of 64 plates are spotted in 4 replicates as 4 quarts of the same membrane.

One set of 12 probes for each of the 96 segments can be selected by a single channel pipetting or single pin delivery (or by individually controlled pipette or pin arrangement) and the selected probes can be arranged in 12 96-well plates . The probes are reveled and the probes from the four plates are mixed with the hybridization buffer and added to the subarray by a 96 channel pipetting device. After one hybridization cycle, the previously applied probe may be stripped off by incubating the membrane at 37-55 ° C. in an undistilled hybridization or wash buffer.

A probe that is positive for one allele is positive and a probe that is positive for another allele can be used to determine whether either of the two alleles is likely to be negative. In this excessive scoring scheme, some level of error (10%) in the hybridization of each probe can be tolerated.

One or two probes may be used for most allele scoring, demonstrating presence or absence in a sample of one of the two alleles. For example, using 4,000 8-oligomers, the probability of finding at least one probe positive for one of the two alleles for a randomly selected site is 91%. Incomplete probe sets can be optimized to reflect G + C content and the like in the analyzed samples.

Genes can be amplified in the appropriate number of segments for overall gene sequencing. One set of probes (one probe per 2-4 bases) is selected and hybridized for each segment. These probes can identify mutation sites in the analyzed segment. Segments in which one or more mutation sites are detected (ie, a small array comprising these segments) are hybridized with additional probes to find the correct sequence at the mutation site. If the DNA sample is tested by the 6-mer and surrounded by the positively hybridized probes TGCAA and TATTCC and the mutation is located at the point covered by the three negative probes CAAAAC, AAACTA and ACTATT, the mutated nucleotide occurs at the normal sequence at this position. A or C. These can be changed by single base mutations or by removal of one or more nucleotides or by insertion between bases AA, AC, CT.

One method is to select a probe that extends the positive hybridized probe TGCAAA to the right for one nucleotide and the probe TATTCC to the left by one nucleotide. Two nucleotides are determined using these eight probes (GCAAAA, GCAAAT, GCAAAC, GCAAAG, ATATTC, TTATTC, CTATTC, GTATTC).

The most plausible hypothesis for mutation can be determined. For example, A is found to be mutated to G. There are two ways to satisfy this result. The only change in which A has been replaced by G is the insertion of some base between the newly determined G and the next C. If the results of linking probes are negative, this option is reviewed by one or more linking probes (AAGCTA) containing the mutated position and by eight additional probes (CAAAGA, CAAAGT, CAAAGC, CAAAGG, ACTATT, TCTATT, CCTATT, GCTATT). Can be. There are many other ways to select mutation resolution probes.

In the case of embryos, score comparisons to test samples and homozygous criteria can be performed to identify heterozygotes. Several consecutive probes will have approximately two times less signal if the segments covered by these probes are mutated on one of the two chromosomes.

Example 20 Identification of Genes (Mutations) Responsible for Genetic Disorders and Other Properties

DNA fragments as long as 5-20 kb can be sequenced without subcloning using longer sets of probes (8-mer or 9-mer) on a fixed sample array. In addition, the sequencing rate is 10 million bp / day / hybridizer. This capability allows for the substantial re-sequencing of human genes or genomes from scientific or medically interesting individuals. Approximately 100 million bp are tested to resequence 50% of the human genes. This can be done in a relatively short period of time at a reasonable cost.

This infinite resequencing ability can be used to identify genes or mutations that encode disorders or other features in a variety of ways. Basically, genomic DNA of a patient with a specific disorder or mRNA from a specific tissue (convertible to cDNA) is used as a starting material. Separate genes or genomic fragments of appropriate length from two DNA sources can be prepared by cloning procedures or in vitro amplification procedures (PCR). If cloning is used, the minimum set of clones to be analyzed is selected from the library prior to sequencing. This can be done effectively by hybridization of a few probes, especially if a few clones longer than 5 kb can be sorted. Cloning increases about twice the amount of hybridization data but does not require tens of thousands of PCR primers.

In one modification procedure, a gene or genomic fragment can be prepared by restriction digest using an enzyme such as Hga I to cleave DNA in the following manner: GACGC (N5 ′) / CTGCG (N10 ′) Protruding end of 5 bases Is different for different fragments. One enzyme produces an appropriate fragment for a certain number of genes. All genes of interest are appropriately excised by cleaving the cDNA or genomic DNA with different enzymes in separate reactions. In one method, the cleaved DNA is separated by size. DNA fragments prepared in this manner (treated with exonuclease III, which removes nucleotides from the 3 'end to increase the specificity and length of the end) are dispensed into tubes or multiwell plates. A pair of adapters can be selected for the gene fragments that need to be amplified from a set of DNA adapters having variable lengths and consensus portions of appropriate length. These adapters are ligation reactions and PCR is performed by general purpose primers. One million pairs are generated from 1000 adapters and one million different fragments can be amplified under the same conditions using universal primer pairs complementary to the common ends of the adapters.

If multiple DNA differences are found in many patients and sequence changes are insignificant or can alter the function of the protein, the mutated gene is the cause of the disorder. By analyzing several individuals with particular characteristics, functional allele changes in a particular gene can be associated with that particular characteristic.

This method eliminates the need for expensive genetic maps for blood tones and has particular value in the absence of such genetic data or materials.

Example 21: Single Nucleotide Polymorphic Scoring in Gene Maps

The technique disclosed in this application is suitable for efficient identification of genomic fragments with single nucleotide polymorphisms (SNUPs). A sufficient number of DNA segments with SNUPs can be identified in 10 by applying the sequencing process described above to genomic fragments of known sequences that can be amplified by ex vivo amplification or cloning. Polymorphic fragments are also used as SNUP markers. Such markers may be mapped in the foregoing manner or through a screening procedure in the manner described below.

By amplifying the markers and arranging them in subarray form, SNUPs can be scored in each individual from the family or population concerned. The subarray includes the same markers amplified from the analyzed individuals. As in the diagnosis of known mutations, for each marker, up to six probe sets positive for one allele and up to six probe sets positive for the other allele are selected and scored. The chromosomal location of the responsible gene can be determined from the group of markers with the disorder. Thousands of markers can be scored for thousands of people because of high throughput and low cost. These data enable localization of genes at resolutions below one million bp and localization of genes involved in multiple gene diseases. Localized genes can be identified by scoring mutations by sequencing specific regions from normal individuals and patients.

PCR is preferred for amplification of markers from genomic DNA. Each marker requires a specific primer pair. Existing markers may be converted or new markers prepared by genomic DNA cleavage by Hga type I restriction enzyme or by ligation reaction using a pair of adapters may be defined.

SNUP markers can be amplified as pools reducing the number of individual amplification reactions. In this case more probes are scored for one sample. When four markers are collected and spotted on 12 replicates, 48 probes (12 per marker) can be scored in 4 cycles.

Example 22 Detection and Verification of Kinds of DNA Fragments

DNA fragments generated by restriction digestion, cloning or ex vivo amplification (PCR) are identified in the experiment. Identification is performed by identifying DNA bands of specific size on gel electrophoresis. Alternatively, specific oligonucleotides are prepared and used to validate the DNA sample in question by hybridization. The procedure developed here allows for more effective identification of large numbers of samples without preparing specific oligonucleotides for each fragment. Positive and negative probe sets can be selected from the universal set for each fragment based on known sequences. A probe selected to be positive may form one or several overlapping groups and the negative probe spreads over the entire insert.

This technique can be used to identify the STS during mapping on the YAC clone. Each STS can be tested on 100 YAC clones or YAC clone pools. DNA from 100 reactions is spotted in one subarray. Different STSs represent consecutive subarrays. In several hybridization cycles, one symbol is generated for each DNA sample and used to confirm the presence of a specific STS in a given YAC clone at the required significance level.

In order to reduce the number of independent PCR reactions or the number of samples for spotting, several STSs may be amplified simultaneously in one reaction or PCR samples may be mixed. In this case more probes have to be scored for one dot. The pooling of the STS is independent of the YAC pooling and can be used for a single YAC or YAC pool. This scheme is particularly beneficial when several labels labeled with different colors are hybridized together.

The amount of DNA can be assessed using the hybridization intensity of several separate probes or one or more probe pools in addition to confirming the presence of DNA fragments in one sample. The DNA amount of all spotted samples is measured simultaneously by comparing the intensity of the DNA with the strength of the known comparative sample. Since only a few probes are needed for DNA fragment identification and there are N possible probes that can be used for N base length DNA, the identification of DNA segments does not require many probe sets. An average of 30 exact match probes from a thousand 8-mers can be selected for a 1000 bp fragment.

Example 23 Identification of Infectious Disease Organisms and Variants thereof

DNA-based tests for detecting viruses, bacteria, bacteria and other parasites are more reliable and less expensive than other methods. The main advantage of DNA testing is that it can identify specific strains and mutations and allow for more effective treatment.

Twelve antibody resistance genes in bacterial infections can be tested by amplifying these genes. The amplified product from 128 patients was spotted in two subarrays and 24 subarrays for 12 genes were repeated four times on an 8 × 12 cm membrane. Twelve probes for each gene can be selected for positive and negative scoring. Hybridization is performed in three cycles. Much fewer probe sets are common for testing. For example, an average of 30 probes from a thousand 8-mer set is positive in a 1000 bp fragment and 10 positive probes are sufficient for high reliability identification. As in Example 9 several genes can be amplified or spotted together and the amount of DNA given can be measured. The amount of gene amplified can be used to assess the level of transmission.

Another example relates to the entire genome or one gene sequencing of the HIV virus. Because of the rapid change, this virus presents a great deal of difficulty in the selection of an optimal treatment. DNA fragments in viruses isolated from up to 64 patients can be amplified and resequenced by the described procedure. The optimal treatment can be selected based on the sequences obtained. Mutations can be identified by quantitatively comparing hybridization scores with scores of other samples (particularly comparative samples containing only basic viral species) in the presence of two kinds of viral mixtures having a base sequence (similar to a heterologous conjugate). A score twice as low can be obtained for three to four probes covering the mutated site in one of the two viruses present in the sample.

Example 24 Forensic and Parental Rights Identification

Sequence polymorphism makes each genomic DNA unique. This allows blood or other body fluids or tissue from the crime scene to be analyzed and compared with samples taken from the suspect. A sufficient number of polymorphic sites are scored to create unique symbols of the sample. SBHs generate this symbol by easily scoring a single nucleotide polymorphism.

One set of DNA fragments (10-100) can be amplified from the sample and the suspect. The suspect from one fragment and the DNA from the sample are spotted into one or several subarrays and each subarray is replicated four times. Twelve probes in three cycles determine the presence of allele A or B in each sample including the suspect for each DNA site. Pattern matching of the sample and the suspect makes it possible to detect the offender.

The same procedure can be applied to parental identification of a child. DNA is prepared and polymorphic sites are amplified from children and adults. Hybridization determines the pattern of the A or B allele. The pattern obtained determines kinship compared to positive and negative comparison samples. In this case, much of the allele needs to match the parent. A large number of scored sites can prevent the masking effects or statistical errors of new mutations.

Example 25 Assessment of Genetic Diversity of Populations or Species and Biological Diversity of Ecological Sites

At many sites (eg, several genes or whole mitochondrial DNA), measuring allelic frequency can lead to various conclusions about the effects of the environment on genotype, species evolution or history, or the likelihood of disease or extinction. This assessment can test for specific known alleles or fully resequence sites to define new mutations that may indicate the presence of microscopic changes or mutagens.

In addition, biological diversity in the microbial world can be studied by evolutionarily conserved DNA resequencing, such as genes of ribosomal RNA or genes of highly conserved proteins. DNA is taken from the environment and specific genes are amplified using primers corresponding to conservative sequences. DNA fragments are cloned in plasmid vectors or diluted to one molecule level per well in multiwell plates and amplified ex vivo. Clones thus prepared are resequenced in the manner described. Two pieces of information are obtained. First, catalogs of species different from the density of each species are defined. Another piece of information can be used to measure the effects of ecological factors or pollution on ecosystems. This reveals whether any species are extinct or the proportions of species change due to contamination. This method is applicable to DNA sequencing from fossils.

Example 26 Detection or Quantification of Nucleic Acid Species

DNA or RNA species can be detected and quantified using probe pairs comprising unlabeled probes immobilized on a substrate and labeled probes in solution. This species can be detected and quantified by exposure to unlabeled probes in the presence of labeled probes and ligase. In particular, the formation of extended probes by the ligation of labeled and unlabeled probes on the sample nucleic acid backbone indicates the presence of the species to be detected. Thus, the presence of a label at a particular point in the matrix array after removal of the unligated labeled probe indicates the presence of the sample species and the amount of label indicates the expression level of the species.

Alternatively, one or more unlabeled probes can be arranged on the substrate with one or more labeled probes to be introduced into the solution as a pair of first elements. According to one method, multiplexing of labels on an array can be performed using dyes fluorescent at distinguishable wavelengths. In this way, the cDNA mixture applied to the array with labeled and unlabeled probe pairs specific for the species to be identified is examined for assessing the presence or expression level of the cDNA species. In one embodiment, the method can sequence a portion of the cDNA by selecting labeled and unlabeled probe pairs that include overlapping sequences along the cDNA sequence to be detected. The presence and amount of specific pathogen genomes can be detected by including in the composition selected pairs of probes that appear in combination in the target pathogenic genomic organism. Thus, no single pair of probes need to be specific for the pathogen genome but the combination of pairs should be. Similarly, when cDNA detection or sequencing may occur, certain probes are not specific for cDNA or other species. Nevertheless, the presence and amount of a particular species is measured such that a combination of selected probes located in different configurations indicates the presence of the particular species.

Progenitors with DNA greater than 10 kb can be detected using a carrier binding detection chip without using PCR or other target amplification procedures. According to another method, genomes of infectious agents, including bacteria and viruses, are assessed by amplifying single target nucleotide sequences via PCR and hybridization of labeled probes specific for the target sequence. Since this evaluation is specific to a single target sequence only, it is necessary to amplify the gene by methods such as PCR to provide enough targets for detectable signaling.

This example provides an improved method for detecting the nucleotide sequence properties of an infectious agent via a format 3-type reaction, wherein a solid-state detection chip comprising various immobilized oligonucleotide probe arrays specific for the infectious agent is prepared. A single dot comprising an unlabeled probe mixture complementary to the target nucleic acid enhances sensitivity to diffusion or single probe labeling by concentrating the species specific label in one location. Such multiple probes may be overlapping sequences of target nucleotide sequences but may be sequences that are not contiguous and not overlapping. Such probes have a length of 5 to 12 nucleotides.

The nucleic acid sample exposed to the target sequence and probe array present in the sample is hybridized using several immobilized probes. Labeled probe pools selected to specifically bind to target sequences adjacent to the immobilized probes are applied to the unlabeled oligonucleotide probe mixture array with the sample. The ligase is then applied to the chip to connect adjacent probes on the sample. After that, the detection chip may be washed to remove the hybridized and unlinked probe and the sample nucleic acid, and the presence of the sample nucleic acid may be detected depending on the presence of the label. This method makes it possible to reliably detect samples with 1000-fold reductions in sample concentration.

As another aspect of the invention, the signal of a labeled probe can be amplified by providing a common tail to a free probe specifically binding to a chromosome, enzyme or radiolabel or by multiple labeled additional probes. In this way, the second round signal amplification is performed. Labeled or unlabeled probes are used for the second signal amplification. Long DNA samples with multiple labels in the second amplification increase the amplification signal by 10 to 100 times resulting in a total of 100,000 signal amplifications. An approximately 100,000-fold signal can result in probe-DNA binding without the need for PCR or other amplification procedures.

According to another aspect of the invention an array of, for example, 4096 6-mer probes can be produced. This arrangement is universal in that it can be used for the detection or complete sequencing of all nucleic acid species. Each spot in the array may comprise a single probe species or a N (1-3) B (4-6) N (1-3) type mixture synthesized in a single reaction. N is four nucleotides, B is one nucleotide and 1-3 in the relevant figures represent 1-3 bases. This mixture collects signals from different portions of the nucleic acid species molecules of the same length to provide stronger signals for nucleic acid species present at low concentrations. This universal probe set can be divided into several subsets spotted as unitary units separated by barriers that prevent the diffusion of hybridization buffers with samples and labeled probes.

One or more oligonucleotide sequences can be selected that include both labeled and immobilized unlabeled probes in solution for detection of known nucleic acid species. Labeled probes are selected or synthesized from presynthesized such as 7-mer sets. Labeled probes are added to a fixed probe unit array such that a pair of immobilized and labeled probes hybridize adjacent to the target sequence such that the probes are covalently bound upon ligase administration.

If the unit array comprises one or more immobilized probes positive for a given nucleic acid species (as separate spots or within the same spot) all labeled probes are mixed and added to the same unit array. Mixtures of labeled probes are more important when the nucleic acid species mixture is tested. An example of a complex mixture of nucleic acid species is mRNA in a cell or tissue.

According to one embodiment of the invention the unit arrangement of immobilized probes allows all possible immobilized probes to be used with a mixture of small amounts of labeled probes. More complex mixtures of labeled probes can be used in the case of multiplex labeling schemes. Preferred multiplexing methods may use different fluorescent dyes or molecular tags that can be separated by mass spectroscopy.

Alternatively, relatively short fixed probes can be selected that frequently hybridize to many nucleic acid sequences. Such short probes are used in combination with labeled probe mixtures in which at least one labeled probe can be prepared to correspond to each immobilized probe. Preferred mixtures are none of the labeled probes corresponding to one or more immobilized probes.

Example 27 Reaction of Segments of HIV Virus with All Possible 10-mers

In format III SBH arrangement occurs on all possible bonded 5-mer (1024 possible pentamers) nylon membranes. Bound pentameric oligonucleotides are synthesized with 5 'tails of 5'-TTTTTT-NNN-3' (N = A, C, G, T and equal molar amounts of 4 bases added at this stage). This oligonucleotide is spotted on the nylon membrane, fixed and dried by treating the dried spot with UV light. A density of up to 18 oligonucleotides per square nanometer is obtained. After UV treatment the nylon membrane is treated with detergent-containing buffer at 60-80 ° C. Oligonucleotide spots are placed in 10 × 10 spot subarrays and each array has 64 pentamer spots and 36 control spots. The 16 subarrays provide 1024 pentamers, including all possible pentamers.

The subarrays in the array are separated from each other by physical barriers such as hydrophobic strips and each subarray can be hybridized to the sample without contamination due to adjacent subarrays. In one embodiment, the hydrophobic strip is made of a silicone solution (eg, household silicone adhesive and sealing paste) in a suitable solvent. This silicone grease solution is applied between small arrays to form a line that acts as a hydrophobic strip separating the cells after solvent evaporation.

In this format III free (unbound) or solution pentamers are synthesized with 3 'tails of 5'-NN-3' (N = A, C, G, T). Pentamers bound with free pentamers combine to produce all possible 10-mers that sequence DNA sequences of less than 20 kb. 20kb of double stranded DNA is denatured to 40kb of single stranded DNA. 40kb of ss DNA hybridizes up to 4% of all possible 10-mers. This low 10-mer binding frequency and target sequence allows the collection of free or solution pentamers for each subarray treatment without loss of sequence information. In one embodiment 16 probes are collected for each subarray and all possible pentamers appear in 64 pools of free pentamers. Thus all possible 10-mers are probed for DNA samples using 1024 subarrays (16 subarrays for each free pentamer pool).

In this embodiment the target DNA represents two 600 bp segments of HIV virus. The 600 bp segment is represented by 60 overlapping 30-mer pools (30-mer overlap by 20 nucleotides of adjacent 30-mers). The 30-mer pool mimics the treated target DNA, shearing and digesting the target DNA, resulting in very few fragments of random pools.

Free pentamers are labeled with radioactive isotopes, biotin, and fluorescent dyes, as described earlier in Format III. Labeled free pentamers are hybridized and linked with pentamers bound to the target DNA. In a preferred embodiment 300-1000 units of ligase are added to the reaction. Hybridization conditions are similar to the previous embodiment. After the ligation reaction and removal of the target DNA and excess free probe, the sequence is analyzed to determine the position of the labeled probe.

The DNA sequence of the target and the knowledge of the free and bound pentamers reveal that the bound pentamers in each subarray are linked to the labeled free pentamers. The signal from 20 of the predicted dots is lost and 20 new signals are obtained from the predicted sequence for the target DNA change. The overlapping sequence of bound pentamers of ten new dots identifies free labeled pentamers that are bound to the new dots.

Test HIV DNA sequences are probed using all possible 10-mers using free, labeled pentamer sequences and pools. This Format III method was used to identify the "wild-type" sequence of the segment tested and to identify the sequence "mutant inducer" incorporated into the segment.

Example 28 Repeat DNA Sequence Sequencing

In one embodiment the repeat DNA sequence in the target DNA is sequenced using “spacer oligonucleotides” in a modified Format III method. Spacer oligonucleotides of variable length repeating DNA sequences (repeated sequences identified by the first SBH implementation) are hybridized to the target DNA along with the first oligonucleotide and the second oligonucleotide connecting the other side of the spacer. Two adjacent oligonucleotides are linked to the spacer when a spacer matching the length of the repeating DNA segment is hybridized to the target. When the first oligonucleotide is immobilized on the substrate and the second possible oligonucleotide is labeled, a bound linkage product comprising a labeled second oligonucleotide is formed when the spacer of the appropriate length is hybridized to the target DNA.

Example 29 Branch Point Sequencing by Format III SBH

Branching points in the target DNA are sequenced using a third oligonucleotide and a modified Format III method. Several branches can be identified when the sequence is collected after the first SBH run. This is solved by hybridizing oligonucleotides overlapping one of the known sequences to the branching point, and then hybridizing additional oligonucleotides to the target that correspond to one of the sequences labeled and derived from the branching point. Labeled oligonucleotides can be linked to one another when appropriate oligonucleotides are hybridized to the target DNA. In one embodiment a second oligonucleotide is selected that is offset by one to several nucleotides from the branch point and a second oligonucleotide is selected that is read from the first and branch point sequences and selected by one to several nucleotides, A third oligonucleotide is selected that corresponds to all possible branches of overlap. These oligonucleotides hybridize to the target DNA and have a third oligonucleotide with the appropriate branching sequence to form a linkage product with the first and second oligonucleotides.

Example 30: Multiplexing Probes for Target Nucleic Acid Analysis

In this embodiment, the probe set is labeled with various labels so that each probe in the set can be distinguished from other probes in the set. Thus, a probe set can be contacted with a target nucleic acid in a single hybridization without loss of probe information. In one embodiment the different labels are different radioisotopes, different fluorescent labels or EML. These probe sets can be used for formats I, II and III SBH.

Differently labeled probe sets in Format I SBH are hybridized to a target nucleic acid immobilized on a substrate under conditions that distinguish between perfect match and one base pair mismatch. Specific probes that bind to the target nucleic acid are identified by various labels and a perfect match is determined from this binding information.

In format II SBH target nucleic acids are labeled with different probes and hybridized to the probe array. Specific target nucleic acids that bind to the probe are identified by different labels and a perfect match is determined from the binding information.

In Format III SBH, differently labeled probes and a fixed set of probes are hybridized to the target nucleic acid under conditions that distinguish a perfect match from a mismatch of one base pair. Labeled probes adjacent to the immobilized probe on the target are bound to the immobilized probe and the product is detected and distinguished by different labels.

In one embodiment, the different label is EML and can be detected by electron capture mass spectrometry (EC-MS). EMLs are prepared from various skeletal molecules and aromatic backbones are particularly preferred (Xu, J. Chromatog. 764: 95-102 (1977). EMLs are attached to the probes in a reversible and stable manner and after the probes are hybridized to the target nucleic acid EML is removed from the probe and identified by standard EC-MS (EC-MS can be performed by gas chromatography-mass spectrometer).

Example 31 Detection of Low Frequency Target Nucleic Acids

Format III SBH has sufficient discrimination to identify the sequences present in the sample in the 1-99 portions of the analogous sequences that differ in a single nucleic acid. Thus, Format III can be used to identify nucleic acids present in very low concentrations of nucleic acid samples (eg blood samples).

In one embodiment, the two sequences are for cystic fibrosis and the sequences are different from each other by three nucleotide removals. The probes for the two sequences are as follows: a probe that distinguishes deletions from wild species immobilized on a substrate, labeled probes common to both. Using these targets and probes, deletion mutagenesis can be detected using format III SBH when present in one-hundredth of wild species.

Example 32: Target Nucleic Acid Assay and Polaroid Apparatus

The nucleic acid analysis device may be constructed using auxiliary materials that prevent the two nucleic acid sequences from being mixed until mixing with the two nucleic acid sequences is required. The arrangement of the device can be supported by various substrates, including nylon membranes, nitrocellulose membranes or other materials disclosed above. In a preferred embodiment one of the substrates is a sector separated member by a suitable support material having wells which may comprise hydrophobic strips or gels or sponges. In this embodiment, the probe is placed on a sector of the membrane or in a well, gel or sponge and a solution (where target nucleic acid may be present) is added to the membrane or well to dissolve the probe. The solution with the dissolved probe is then contacted with the second nucleic acid sequence. The nucleic acid may be an oligonucleotide probe or target nucleic acid and the probe or target nucleic acid may be labeled. Nucleic acids can be labeled with labels used in the art, such as radioisotopes, fluorescent labels or electrophoretic labels.

Materials that prevent nucleic acid mixing can be placed between the two arrays in such a way that the two nucleic acid arrays are mixed when the material is removed. Such materials may be in the form of sheets, membranes or other barriers and may consist of substances which prevent mixing of nucleic acids.

Such devices can be used in Format I SBH as follows: The first array of devices is a target nucleic acid immobilized on a substrate and the second array has a nucleic acid probe that can be labeled and removed to react to the target nucleic acid of the first array. . The two arrays are separated into sheets of material that prevent the probe from contacting the target nucleic acid and when the sheet is removed the probe responds to the target. The target array can be “read” to determine if the probe formed after the appropriate incubation and washing steps is fully consistent with the target. Such reading can be done automatically or manually (eg visually checking the autogram). The following procedure will be similar to the procedure described above, except that the target is labeled in the Format III SBH and the probe is fixed.

This device can be used in Format III SBH as follows: Two nucleic acid probe arrays are formed, nucleic acid probes are labeled and one of the arrays is attached to a substrate. The two arrays are separated into sheets to prevent probe mixing. The format II reaction is initiated by adding the target nucleic acid and removing the sheet to allow the probes to mix with each other and the target. Probes bound to adjacent sites on the target are bound to each other by covalent bonds to base accumulation interactions or backbones and the results are read to determine probes bound to the target at adjacent sites. When one set of probes is immobilized on a substrate, the immobilized array is read to determine which of the probes from the other array is associated with the immobilized probe. In this way, the reading is either automated (ELISA reader) or manually (visually checking the autoradiogram).

Example 33; 3-D array of probes

In a suitable embodiment, the oligonucleotide probe is immobilized in a three dimensional array. The three-dimensional array consists of a plurality of layers, each layer separated from the other layers and analyzed separately. Three-dimensional arrays take many forms, for example, the array is placed on a substrate having multiple depressions and probes located at different depths in the depression (each level is placed on a substrate having a similar depth in the depression) or arrangement Is placed at the bottom of the depression or at a peak that separates the depression or on a substrate having a depth of depression different from the located probe where the peak and depression are combined; Or placed on a substrate composed of multiple layers layered to create a three-dimensional array.

Materials for synthesizing three-dimensional arrays are known in the art and include those mentioned as suitable for support for probe arrays. In addition, other suitable materials capable of supporting oligonucleotide probes, suitably flexible materials, are used as the substrate.

Example 33; Signaling process to collect cDNA clones

Multiple distinct nucleic acid sequences were obtained from the cDNA library using standard PCR, SBH sequence signal analysis and Sanger sequencing techniques. The library insert was amplified by Ekfk in PCR using primers specific for the vector sequence linked to the insert. These samples were spotted on nylon membranes, provided signals with the appropriate number of oligonucleotide probes, and the strength of the positive binding probes measured with the given sequence signal. Clones are classified into similar or identical sequence signal groups, and single clones are selected from each gel sequence group. The 5 'sequence of the amplified insert is inferred using reverse M13 sequencing primers using a typical Sanger sequence protocol. The PCR product is purified and subjected to fluorescent dye termination cycle sequencing reactions. Single pass gel sequencing uses a 377 Applied Biosystems (ABI) sequencer. Most clones selected and sequenced by this method have different sequences, with very few having the same sequence.

Example 35; Mass production of chips

In a suitable embodiment, the apparatus for making a large amount of probe arrangement comprises an ink-jet deposition apparatus such as a rotating drum or plate with a microdrop dosing head coupled thereto; It consists of a suitable robotic system such as Anrad Gantry. Specific examples of the apparatus are described in FIGS. 1-3.

The device consists of a cylinder 1 to which a suitable substrate is fixed. The substrate will be any of the materials described above suitable for making probe arrays. In a suitable embodiment, the substrate is a flexible material and the arrangement is made directly on the substrate. In another embodiment, the flexible substrate is fixed to the cylinder and the individual chips are fixed to the substrate. Arrays are made from each individual chip.

In a suitable embodiment, a physical barrier is provided to the substrate or chip, which barrier is the well arrangement. The physical barrier is provided by the device to the substrate or chip, or the physical barrier is provided to the substrate or chip prior to fixing to the cylinder. Single spot oligonucleotide probes are located in each well, where probes located in individual wells have the same sequence or probes that are spotted in individual wells have different sequences. In a more suitable embodiment, probes spotted in each individual well in an array are different from probes spotted in other wells of the array. Guseoodin sequencing chips are assembled from these arrays into multiple wells.

After the substrate and the chips are fixed to the cylinder 1, the cylinder is rotated using a motor (not shown). The rotational speed of the cylinder is accurately determined by any method known in the art, for example using a fixed optical sense and a light source that rotates the cylinder. The dispensing device 3 moves along the arm 2 to move the probe or other reagent through the dispensing tip 8 to the correct position on the substrate or chip, as described above using methods known in the art. To use the correct rotational speed. The dispensing device receives a probe or reagent from the reservoir 6 via the supply line 7. Reservoir 6 has all the probes and other reagents needed to make an array.

3 illustrates a dispensing device. The dispensing device has one or multiple dispensing tips 14 & 8 dmf. Each dispensing tip has a sample well 13 in the body 12 that receives a probe or other reagent through the sample line 10. Pressure line 11 applies psi pressure to chamber 9 to force the probe or reagent through the dispensing tips 14, 8. Sample lines 10, wells 13, and dispensing tips 14, 8 must be filled with a probe or reagent between each change. Appropriate wash buffer is supplied to sample well 13 via sample line 10 or optionally designated wash line (not shown) or filled in some or all of chamber 9 in wash buffer. The wash buffer is then withdrawn from the sample wells and chambers as needed, either via delivery line (not shown) or via sample line 10 and dispensing tips 14, 8.

When the dispensing means provides the probe to the appropriate site in each array or chip, the substrate (with or without chips) is removed from the cylinder and the new substrate is fixed to the cylinder.

Example 36; Analysis of Target Nucleic Acids Having Probe Complexed to Separate Particles

In one embodiment, the target nucleic acid is provided with a signal complexed (covalent or non-covalent) to a plurality of separate particles. These separate particles are distinguished from each other based on their physical properties (or complex physical properties), which particles have different probes. In a suitable embodiment, the probe is an oligonucleotide having a known sequence and length. Thus, probes are identified by the physical properties of the discrete particles. Suitable probes suitable for this embodiment also include probes having a shorter information sense than all previously described probes and full length probes.

The physical properties of the discrete particles are used to distinguish the particles into sets with any properties known in the art. For example, the particles can be distinguished by their size, fluorescence, absorbance, charge in the electromagnetic field, weight, dye, radionuclides or particles labeled by EMLs. Other suitable labels include ligands that act as binding elements specific to labeled antibodies, chemiluminescent materials, antibodies that can act as specific binding pairs of enzymes and labeled ligands, and the like. Use a variety of labels used for ready-to-use immunoassays. Other labels include antigens, groups with specific reactivity, electrochemically detectable moieties, and the like. Another label includes a previously described label. Such labels and properties are not quantitatively measured by methods known in the art, for example, using previously described methods, and the particles may be characterized by signal strength, signal type (signal shape and strength for labels, such as , Different dye intensities to the particles or different types of dyes). In a suitable embodiment, several physical properties are combined and different properties are combined to distinguish particles (eg, if 10 sizes and colors are combined, 100 particles can be distinguished).

Particle-probes can be used to develop standard complex methods, for example, all possible 10-mers can be synthesized using about 2000 reaction vessels. A first set of 1024 reactions are conducted to synthesize all possible 5-mers from 1024 differently labeled particles. The resulting probe-particle mixtures are mixed together and divided into another 1024 reaction vessel set. A second set of reactions is carried out using these samples to synthesize all possible 5-mer extensions on the probe to the particle pool. Physical properties identify the first five nucleotides of each probe, and reaction vessels identify the second five nucleotides of all probes. Thus, all possible 10-mers can be synthesized using 2048 reaction vessels. By easily modifying this approach, it is possible to make all possible n-mers for a larger range of probes.

In suitable embodiments, the particles are separated into sets according to the fluorescence intensity of the particles. The particles in each set are prepared with varying densities of fluorescent labels so that the particles have different fluorescence intensities. The fluorescence intensity of floresin relates to concentrations ranging from 1: 300 to 1: 300,000 (Lockhart et al., 1986), preferably between 1: 3000 and 1: 300,000, with a linear relationship (fluorescence intensity is about 1 Linear over the range -300). In the detection of the linear range, 256 sets of particles are labeled with floresin (e.g., 30-259). With 256 set particles, it is possible to attach all possible 4-mers to different set particles. When these particles are added together, we have all possible 4-mer pools, each extending by A, G, C, and T in each pool to produce all possible 5-mers. Similarly, a probable 6-mer has all the 4-mers likely, and each 4-mer pool is made to extend into one of 16 probable two base variants of A, G, C, and T. For example, 64 pools for 7-mer, 256 pools for 8-mer ..)

A 5-mer probe (four pools) is used to provide signal to the target nucleic acid. The target nucleic acid is labeled with another fluorescent dye or another different label (described above). The labeled targets are mixed into four pools, and the complementary probes in each pool are hybridized to the target nucleic acid. Such hybrid diphabe can be detected using a method known in the art, the positively hybridized probe can be confirmed by sensing the fluorescence intensity of the particles. In the most suitable embodiment, the probe-particle and target nucleic acid mixture is supplied to one particle at a time through a flow cytometer or other separation device, and the particle label and target are measured to determine the probe complementary to the target nucleic acid.

In another embodiment, the probe set is labeled with another fluorescent dye or another label (described above), individual free probes are mixed with each 5-mer pool (four pools), and then the mixture is mixed with the target nucleic acid. Hybrid. When the free probe is bound to a site in the target nucleic acid adjacent to the site to which the 5-mer probe is bound (the free probe site must be adjacent to the end of the ligation 5-mer probe), the substance is added to the free probe covalently bind to the mer probe (see the description of the appropriate material). The particles are then inspected using known methods to measure particles covalently bound to the free probe, ie, particles with free probe labels, wherein the 5-mer probe is identified by the fluorescence intensity of the particle. In the most suitable embodiment, the probe-particle, free probe, target nucleic acid mixture is supplied to one particle at a time through a flow cytometer and the particle label and free probe label are measured to determine the probe complementary to the target nucleic acid.

In a suitable embodiment, one device retains all or most of the adjustment means for target nucleic acid analysis with probe-particle complexes. The device has one or more reagent chambers in which the buffer and labeled target nucleic acid are thoroughly mixed (target nucleic acid can be added by hand or automatically). The mixture is divided from reagent chambers into a number of reaction chambers, each having a probe-particle complex pool. The probe-particle and the target nucleic acid react under conditions that allow the complementary probe to bind to the target nucleic acid. Excess target nucleic acid, such as unbound, is removed from the reaction chamber (using washing, etc.), the probe bound to the target nucleic acid is identified using a target nucleic acid label with particles, and the probe is identified by the physical properties of the particle. . In a suitable embodiment, after removing the excess target, the particles are moved in a single transmission through a channel leading from the reaction chamber to the sensing device. When a single particle is used as a sensing device, they measure the physical properties of the target nucleic acid and the particle. In another suitable embodiment, before or after removing an excess of targets, the particles may have one of their physical properties, such as size (eg, extrusion chromatography), charge (eg, ion exchange chromatography), density-weight, or the like. Sort into sets based on complex properties. The particles thus separated are examined by a sensing device.

In another embodiment, the main reaction chamber is supplied with a buffer, target nucleic acid, particle-probe complex, chemical or enzymatic ligation material, and the like. These components are mixed thoroughly and divided into a plurality of reaction chambers in the reagent chamber. Each reaction chamber has a labeled free probe. Alternatively, the probe-particle complex pool is placed in the free reaction probe and containment reaction chamber instead of adding them to the reagent chamber. In addition, free probes can be added to the reagent chamber and probe particle exit can be added to the reaction chamber. The probe-particle, target nucleic acid, and free probe react under conditions such that the free probe and the particle-probe can bind to adjacent sites in the target nucleic acid so that the free probe is ligated to the probe particle. Excess free probes (e.g., ligated) and target nucleic acids are removed from the reaction chamber (using washing), the ligated probes are identified using free probe labels and particles, and probes conjugated to the particles are used to Check using properties. In suitable embodiments, after removal of excess probes and targets, particles are transferred once through the channel leading from the reaction chamber to the sensing device. When a single particle is used as a sensing device, they measure the physical properties of the target nucleic acid and the particle. In another suitable embodiment, before or after removing an excess of targets, the particles may have one of their physical properties, such as size (eg, extrusion chromatography), charge (eg, ion exchange chromatography), density-weight, or the like. Sort into sets based on complex properties. The particles thus separated are examined by a sensing device.

In a suitable embodiment, the device has a second reaction chamber and the probe-particle pool is located in the second reaction chamber. The buffer and target are mixed in the reagent chamber and they are fed to a first reaction chamber containing a labeled free probe. The probe and the target are mixed and optionally the probe can be hybridized to the target. Such labeled probes and targets are transferred to a second reaction chamber containing a probe-particle pool. The free probe and probe-particle are hybridized to the target and the appropriate probe is ligated to the second reaction chamber. Ligation material may be added to the reagent chamber or to each reaction chamber, suitably adding the ligation reagent to the second reaction chamber. The probe-particle hybrid reaction product in the second reaction chamber is analyzed as above.

In one embodiment, the target nucleic acid is not amplified prior to analysis (in a vector such as a PCR or lambda library). Suitably longer free probes and particle probes are used in this embodiment because the sequence complexity of the sample is increased (to distinguish positives over background).

The probe-particle embodiments described in this example can be used in various described applications, for example, but not limited to the described diagnostic and sequencing fields. In addition, such probe particle embodiments may be modified using variables or modifications previously described.

Example 37; Interaction of complementary polynucleotides in the presence of a substance that modifies the binding between polynucleotides

In this embodiment, the substance can be added to control the distinction of completely mesothelial from mismatches in the binding of complementary polynucleotides. In suitable embodiments, the complementary polynucleotides are polynucleotides and polynucleotide probes. The addition of a substance can control the differentiation of the mismatched one from the complete one, in which the substance is a trialkyl ammonium salt (eg, TMAC, Ricelli et al., Nucl. Acids Res. 21: 3785-3788 (1993). ), Sodium chloride, phosphate, boronate, formamide, glycol, dimethyl sulfoxide, dimethylformamide, urea, organic solvents such as guanidium, amino acid derivatives such as betaine (Henke et al., Nucl.Acids Res. 19: 3957-3958 (1997); Rees et al., Biochemistry 32: 137-144 (1939), polyamines such as spermidine and spermine (Thomas et al., Nucl.Acids Res. 25: 2396- 2402 (1997)), other positively charged molecules that neutralize the negative charges of the phosphate structure, surfactants such as sodium dodecyl sulfate, sodium lauyl sulfate, small / large groove binders, positively charged polypeptides, acridine, ethidium Inserts such as bromide and anthracyc. In a suitable embodiment, the substance mixture can be added to the hybrid reaction to control the distinction between perfectly mismatched and mismatched ones. Some such materials reduce the melting entropy between two complementary strands, thus affecting the distinction.

In suitable embodiments, the addition of these materials can better distinguish the mesmerized from the mismatched ones. For example, formamide, which is commonly used as a denaturant, has been shown to preferentially destabilize the mesmetheted and fully mesomethed in format III reactions. As described above, set up the format III reaction and add varying amounts of formamide (0%, 10%, 20%, 30%, 40%, 50%). At 0%, a fully embellished signal is detected and the background (mismatch) is quite high. In the case of 10% formamide, there is a good meth signal, and the background / mismethod signal is reduced. At 20% formamide, the perfectly mesmerized signal was reduced (detected) and the background / mismatched signal was lost. At 30% -50% formamide there was no perfectly medled signal and no background / mismatched signal.

In another embodiment, material was added to reduce or increase the T _m of complementary oligonucleotide pairs. In a more suitable embodiment, the material mixture was used to increase or decrease the T _m of the complementary oligonucleotides. Substances modify T _{m in a number} of ways, including two examples: (1) a substance that breaks the hydrogen bond between two complementary polynucleotide bases (Goodman, Proc. Nat'l Acad. Sci. 94: 10493 10495 (1997); Moran et al., Proc. Nat'l Acad. Sci. 94: 10506-10511 (1997); Nguyen et al., Nucl.Acids Res. 25: 3059-3065 (1997); (2) substances that neutralize or block the negative charge of phosphate in the sugar-phosphate framework of polynucleotides (Thomas et al., Nucl. Acids Res. 25: 2396-2402 (1997)), but are not limited thereto . Enhance or attenuate (1) or (2) to control the T _m of the complementary polynucleotide pair.

In the most suitable embodiment, the substance can be added to reduce the binding energy of the GC base pair, increase the binding energy of the AT base pair, or both. In suitable embodiments, a substance is added such that the binding energy of the AT base pair is approximately equivalent to the binding energy of the GC base pair. Thus, the binding energy between two complementary polynucleotides depends entirely on length. The binding energy of such complementary polynucleotides can be increased by the addition of a substance that can neutralize or block the negative charge of the phosphate groups in the polynucleotide framework.

The invention is not limited to the examples for describing a single aspect of the invention, but the scope of the invention includes compositions and methods that are functionally equivalent thereto. In addition, those of ordinary skill in the art will envision various modifications and variables in the practice of the present invention in light of the examples that are appropriate to the art. As a result, the scope of the invention is defined by the appended claims.

All documents mentioned in this specification should be incorporated by reference in their entirety.

Claims (64)

For a plurality of oligonucleotide probe arrays, -temperament; A material forming a physical barrier; The material is then placed in a substrate forming a grid having a plurality of wells; And Wherein a plurality of oligonucleotides are arranged to form an array in a plurality of wells, wherein each well comprises one probe spot fixed to a substrate. 2. The oligonucleotide probe array of claim 1, wherein each individual spot comprises a probe having a sequence that is different from other probes in the other spot in the array. The probe array of claim 1, wherein the probes in each individual spot have the same sequence. The probe arrangement of claim 1, wherein a distance from the center of one spot to the center of another adjacent spot is at least 325 μm. A sequence chip comprising a plurality of arrangements according to claim 1. A sequence chip comprising a plurality of arrangements according to claim 2. Sequence chip comprising a plurality of the arrangement according to claim 3. 2. The oligonucleotide probe arrangement of claim 1, wherein the oligonucleotide probe consists of an information portion and a reactor, wherein the reactor is used to attach the probe to a substrate. 10. The oligonucleotide probe array of claim 8, wherein the oligonucleotide probe further comprises at least one random position. 10. The oligonucleotide probe array of claim 9, wherein the oligonucleotide probe further comprises a spacer. A plurality of oligonucleotide probe arrays, comprising a plurality of levels of substrates, wherein the plurality of probes are anchored to different levels of substrates. 12. The oligonucleotide probe array of claim 11, wherein each level can be analyzed separately. 12. The oligonucleotide probe array of claim 11, wherein different levels are analyzed simultaneously. 12. The oligonucleotide probe arrangement of claim 11, wherein the oligonucleotide probe consists of an information portion and a reactor, wherein the reactor is used to attach the probe to a substrate. 15. The oligonucleotide probe array of claim 14, wherein the oligonucleotide probe further comprises at least one random position. The oligonucleotide probe array of claim 15, wherein the oligonucleotide probe further comprises a spacer. In a method for analyzing a target nucleic acid, The target nucleic acid is brought into contact with a plurality of oligonucleotide probes, where the probe is complexed with several different numbers of separate particles, which are distinguished according to their physical properties, and the different probes are complexed with each of these separate particles; -Detecting such probes complementary to the target nucleic acid; And -Analyzing the target nucleic acid from each set of complementary probes. 18. The method of claim 17, further comprising fractionating the discrete particles, wherein the discrete particles are separated based on physical properties. 19. The method of claim 18, wherein the complementary probe set has at least two probes that overlap. 19. The method of claim 18, wherein the target nucleic acid sequence is collected in an analysis step. 19. The method of claim 18, wherein the complementary probe is identified based on the physical properties of the discrete particles. 19. The method of claim 18, wherein the physical property is associated with a molecule selected from dyes, radionucleotides, EMLs, fluorescent molecules. 19. The method of claim 18, wherein the physical property is selected from size, charge, absorbance, weight. The method of claim 23 wherein the physical property is dependent on the strength of the physical property. The method of claim 23, wherein the physical property is associated with a number of different molecules. 19. The method of claim 18, wherein the information portion of the probe is shorter than the full length probe. 19. The method of claim 18, wherein the target nucleic acid comprises a label and the complementary probe is detected by a label on the target nucleic acid. 18. The method of claim 17, wherein the sensing step is performed on the individual particles by passing the individual particles through the detector. 29. The method of claim 28, wherein the complementary probe set has at least two probes that overlap. The method of claim 28, wherein the target nucleic acid sequence is collected in an analysis step. 29. The method of claim 28, wherein the complementary probe is identified based on the physical properties of the discrete particles. 29. The method of claim 28, wherein the physical property is associated with a molecule selected from dyes, radionucleotides, EMLs, fluorescent molecules. 29. The method of claim 28, wherein the physical property is selected from size, charge, absorbance, weight. 34. The method of claim 33, wherein the physical property is in accordance with the strength of the physical property. 34. The method of claim 33, wherein the physical property is associated with a plurality of different molecules. 29. The method of claim 28, wherein the information portion of the probe is shorter than the full length probe. The method of claim 28, wherein the target nucleic acid comprises a label and the complementary probe is detected by a label on the target nucleic acid. 29. The method of claim 28, further comprising the steps of contacting a plurality of free oligonucleotides with a target nucleic acid and complementary free probes bound to a site in the target nucleic acid are linked to a site at the target nucleic acid adjacent to the position to which the free probes are bound. Covalently binding to a complementary probe complexed to a separate particle, wherein the sensing step is performed by identifying a free probe covalently bound to the probe of the separate particle. The method of claim 38, wherein the complementary, covalently coupled probe set comprises at least two covalently linked probes that overlap. The method of claim 38, wherein the target nucleic acid is collected at the assay step. 39. The method of claim 38, wherein the probe complexed to the separate particles is identified by the physical properties of the separate particles. 39. The method of claim 38, further comprising fractionating the discrete particles, wherein the discrete particles are separated based on physical properties. 43. The method of claim 42, wherein the discrete particles are fractionated using a flow cytometer. 39. The method of claim 38, wherein the physical property is associated with a molecule selected from dyes, radionucleotides, EMLs, fluorescent molecules. 39. The method of claim 38, wherein the physical property is selected from size, charge, absorbance, weight. 46. The method of claim 45, wherein the physical property is in accordance with the strength of the physical property. 46. The method of claim 45, wherein the physical property is associated with a number of different molecules. 39. The method of claim 38, wherein the information portion of the free probe is shorter than the full length probe. 39. The method of claim 38, wherein the information portion of the probe complexed to the discrete particles is shorter than the full length probe. The method of claim 38, wherein the information portion of the probe complexed to the separate molecule and the information portion of the free probe are shorter than the full length probe. In a method for analyzing a target nucleic acid, Contacting the target nucleic acid with the probe under conditions that are fully mesophilic, distinct from those that are mismatched, wherein a substance is added to better distinguish between the mismatched and completely mesentized; -Detecting whether the probe is complementary to the target nucleic acid. The material of claim 51, wherein the substance is a salt, an organic solvent, urea, guanidine, an amino acid derivative, a polyamine, another molecule having a positive charge to neutralize the negative charge of phosphate, a surfactant, a small / large groove binding material, a positively charged polypeptide, Intercalating agent material and the like. 53. The method of claim 52, wherein the salt is selected from trialkyl ammonium salts, sodium chloride, phosphate, borate. 53. The method of claim 52, wherein the organic solvent is selected from formamide, glycol, dimethylsulfoxide, dimethylformamide. 53. The method of claim 52, wherein the amino acid derivative is betaine. 53. The method of claim 52, wherein the polyamine is selected from spermidine, spermine. 53. The method of claim 52, wherein the surfactant is selected from sodium dodecyl sulfate, sodium lauryl sulfate. 53. The method of claim 52, wherein the intercalating agent is selected from acridine, ethidium bromide, anthracene. 53. The method of claim 51, wherein a plurality of materials are added. 60. The method of claim 59, wherein the substance is selected from salts, organic solvents, urea, guanidine, amino acid derivatives, polyamines, other molecules with positive charges to neutralize the negative charges of phosphates, surfactants, small / large groove binding materials, positively charged polypeptides, Intercalating agent material and the like. In a method for analyzing a target nucleic acid, Providing a plurality of immobilized oligonucleotide probe arrays, Providing a plurality of labeled oligonucleotide probes, Contacting the target nucleic acid with the immobilized probe and the labeled probe under conditions such that the probe bound to the target nucleic acid is completely distinguished from the probe that is completely embedded in the target nucleic acid with one base mismatch to the target nucleic acid, wherein Substances are added to better distinguish between mismatched and completely embroidered; A probe covalently linked to a labeled probe at the site in the target nucleic acid, wherein the labeled probe is hybridized to a site adjacent to the site to which the anchored probe at the target nucleic acid is bound; And -Identifying the covalently bound immobilized and labeled probes. 62. The method of claim 61, wherein the substance is a salt, organic solvent, urea, guanidine, amino acid derivative, polyamine, other molecule having a positive charge to neutralize the negative charge of the phosphate, surfactant, small / large groove binding material, positively charged polypeptide, Intercalating agent material and the like. 62. The method of claim 61, wherein the substance is formamide. 62. The method of claim 61, wherein a plurality of materials are added.