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WO1999039004A1 - Resequençage automatique - Google Patents

Resequençage automatique Download PDF

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Publication number
WO1999039004A1
WO1999039004A1 PCT/US1998/005438 US9805438W WO9939004A1 WO 1999039004 A1 WO1999039004 A1 WO 1999039004A1 US 9805438 W US9805438 W US 9805438W WO 9939004 A1 WO9939004 A1 WO 9939004A1
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WIPO (PCT)
Prior art keywords
sequence
nucleic acid
probes
target nucleic
probe
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PCT/US1998/005438
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English (en)
Inventor
Mark Chee
Joseph G. Hacia
Francis S. Collins
Keith Edgemon
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Affymetrix, Inc.
The Government Of The United States Of America, Secretary, Department Of Health And Human Services
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Application filed by Affymetrix, Inc., The Government Of The United States Of America, Secretary, Department Of Health And Human Services filed Critical Affymetrix, Inc.
Publication of WO1999039004A1 publication Critical patent/WO1999039004A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C12Q1/6874—Methods for sequencing involving nucleic acid arrays, e.g. sequencing by hybridisation

Definitions

  • the invention resides in the technical fields of molecular genetics, genomics and comparative sequence analysis.
  • Arrays of probes provide a more efficient means of analyzing variant sequences once a prototypical or reference sequence has been determined. Analysis of the hybridization pattern of probes to a target nucleic acid reveals the position, and optionally the nature, of differences between the target and reference sequence.
  • WO 95/11995 describes arrays comprising four probe sets. Comparison of the intensities of four corresponding probes from the four sets to a target sequence reveals the identity of a corresponding nucleotide in the target sequences aligned with an interrogation position of the probes. The corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity.
  • hybridization intensities for multiple targets from different sources can be classified into groups or clusters suggested by the data, not defined a priori , such that isolates in a give cluster tend to be similar and isolates in different clusters tend to be dissimilar (see WO 97/29212, incorporated by reference in its entirety for all purposes) .
  • Array-based resequencing has been used, for example, in the identification of large numbers of human polymorphisms in mitochondrial DNA and ESTs, the identification of drug- 3 induced mutations in HIV, and analysis of mutations in p53 correlated with human cancer.
  • FIG. 1 Outline of sequence analysis algorithm using first and second level base calling strategies.
  • Figs. 2 A-F Chimpanzee and human chip image comparisons. Magnified digitized false colored red images showing human and chimpanzee BRCA1 target hybridization patterns to high density oligonucleotide arrays evaluating antisense strands (array size is 1.2 cm x 1.2 cm with 50 micron probe feature sizes) . Contrast and brightness parameters were changed in each panel to increase image clarity. Probes designed to detect single nucleotide insertions are not shown for clarity. Nucleotide identities, determined through dideoxysequencing analysis, are given under the respective column, underlined if differing from human, and colored red or blue if correctly or incorrectly identified by level one hybridization analysis, respectively.
  • Figs. 3 A-G Primate chip image comparisons. Digitized false colored red images showing hybridization 4 pattern of BRCAl fluorescent targets to high density oligonucleotide arrays evaluating antisense target strands. Magnification of the region (50 micron feature size) corresponding to nucleotide positions 3374-3388 of human BRCAl cDNA is given for each species; specific insertion probes are not shown for clarity. The arrangement of sequencing probes is given in Fig. IB. Nucleotide identities, determined through dideoxysequencing analysis, are given under each column and colored or underlined as described in Fig. 2. Hybridization patterns of (A) human ⁇ Hsa, Homo sapiens) , (B) chimpanzee
  • Figs. 4 A-D Representative chip images of alternative second order tiling schemes for orangutan target sites. Magnified digitized false colored red images showing hybridization pattern of BJ?CA1 fluorescent orangutan targets to high density oligonucleotide arrays evaluating sense and antisense target strands. Nucleotide identities, determined through dideoxysequencing analysis, depicting coding strand sequence are given under the respective column and underlined if differing from the human consensus sequence. For the 2731 C->T and 3667 A->G base substitutions relative to human sequence, hybridization to nucleotides 2724-2728 and 3660-3674 using human cDNA numbering are given respectively.
  • Hybridization patterns of orangutan (A) sense target with standard 2731 C tiling, (B) , sense target with alternative 2731 T tiling, (C) , antisense target with standard 3667 A tiling, (D) , antisense target with alternative 3667 G tiling. 5
  • Antisense strand hybridization data is given relative to coding strand sequence.
  • a nucleic acid is a deoxyribonucleotide or ribonucleotide polymer in either single-or double-stranded form, including known analogs of natural nucleotides unless otherwise indicated.
  • An oligonucleotide is a single-stranded nucleic acid ranging in length from 2 to about 500 bases, and is typically, about 8-40, and more typically, 10-25 bases.
  • a probe is an oligonucleotide capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • An oligonucleotide probe may include natural (i.e. A, G, C, or T) or modified bases (e.g., 7-deazaguanosine, inosine) .
  • the bases in oligonucleotide probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • oligonucleotide probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Stringent conditions are conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence- dependent and are different in different circumstances. Longer sequences hybridize specifically at higher 6 temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • the Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50% of the probes are occupied at equilibrium) .
  • stringent conditions include a salt concentration of at least about 0.01 to 1.0 M
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide .
  • destabilizing agents such as formamide .
  • 5X SSPE 750 mM NaCl , 50 mM Na phosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30°C are suitable for allele-specific probe hybridizations.
  • a perfectly matched probe has a segment perfectly complementary to a particular target sequence.
  • Complementary base pairing means sequence-specific base pairing which includes e . g. , Watson-Crick base pairing or other forms of base pairing such as Hoogsteen base pairing.
  • Probes typically have a segment of complementarity of 6-20 nucleotides, and preferably, 10-25 nucleotides. Leading or trailing sequences flanking the segment of complementarity can also be present.
  • the term "mismatch probe” refer to probes whose sequence is deliberately selected not to be perfectly complementary to a particular target sequence. Although the mismatch (s) may be located anywhere in the mismatch probe, terminal mismatches are less desirable as a terminal mismatch is less likely to prevent hybridization of the target sequence. Thus, probes are often designed to have the mismatch located at or near the 7 center of the probe such that the mismatch is most likely to destabilize the duplex with the target sequence under the test hybridization conditions.
  • Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population.
  • a polymorphic marker or site is the locus at which divergence occurs.
  • Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population.
  • a polymorphic locus may be as small as one base pair.
  • An array including a pooled probe means that a cell in the array is occupied by pooled mixture of probes .
  • a cell might be occupied by probes ACCCTCCA and ACCCCCCA, in which case, the underline position is described as a pooled position.
  • the identity of each probe in the mixture is known, the individual probes in the pool are not separately addressable.
  • the hybridization signal from a cell is the aggregate of that of the different probes occupying the cell.
  • species variant refers to a gene sequence that is evolutionarily and functionally related between species.
  • the human CD4 gene is the cognate gene to the mouse CD4 gene, since the sequences and structures of these two genes indicate that they are highly homologous and both genes encode a protein which functions in signaling T-cell activation through MHC class II- restricted antigen recognition.
  • Percentage sequence identity is determined between optimally aligned sequences from computerized implementations of algorithms such as GAP, BESTFIT, FASTA, and TFASTA in the 8
  • the invention provides iterative methods of analyzing a target sequence, which represents a variant of a reference sequence.
  • the methods employ an array of probes which includes a probe set comprising probes complementary to the reference sequence.
  • a target nucleic acid is hybridized to the array of probes.
  • the relative hybridization intensities of the probes to the target nucleic acid are then determined.
  • the relative hybridization intensities are used to estimate a sequence of the target nucleic acid.
  • a further array of probes is then provided comprising a probe set comprising probes complementary to the estimated sequence of the target nucleic acid.
  • the target nucleic acid is then hybridized to the further array of probes and the relative hybridization of the probes to the target nucleic acid is determined.
  • the sequence of the target nucleic acid is then reestimate from the relative hybridization intensities of the probes.
  • the cycles of hybridization and estimating the sequence of the target nucleic acid can be reiterated, if desired, until the reestimate sequence of the target nucleic acid is the true sequence of the target nucleic acid.
  • the methods are particularly useful for analyzing a target nucleic acid that represents a species variant of a known reference sequence.
  • the reference sequence can be from a human and the target sequence from a primate .
  • the target nucleic acid shows 50-99% sequence identity with the reference sequence.
  • the methods are also particularly useful in situations where a target sequence 9 differs from a reference sequence by more than one mutation within a probe length.
  • the methods can readily accommodate a reference sequence of at least 1 or 10 kb long or even a complete or substantially complete human chromosome or genome.
  • a probe set for use in the methods typically includes overlapping probes that are perfectly complementary to and span the reference sequence, and the further array comprises probes that are perfectly complementary to and span the estimate sequence.
  • the array of probes comprises four probe sets.
  • a first probe set comprises a plurality of probes, each probe comprising a segment of at least six nucleotides exactly complementary to a subsequence of the reference sequence, the segment including at least one interrogation position complementary to a corresponding nucleotide in the reference sequence.
  • third and fourth probe sets each comprise a corresponding probe for each probe in the first probe set, the probes in the second, third and fourth probe sets being identical to a sequence comprising the corresponding probe from the first probe set or a subsequence of at least six nucleotides thereof that includes the at least one interrogation position, except that the at least one interrogation position is occupied by a different nucleotide in each of the four corresponding probes from the four probe sets.
  • the target sequence can be estimated by comparing the relative specific binding of four corresponding probes from the first, second, third and fourth probe sets. A nucleotide in the target nucleic acid is then assigned as the complement of the interrogation position of the probe having the greatest 10 specific binding. Other nucleotides in the target sequence are assigned by similar comparisons.
  • the invention also provides methods of analyzing a target nucleic acid comprising the following steps.
  • An array of probes is designed to be complementary to an estimated sequence of the target nucleic acid.
  • the array of probes is hybridized to the target nucleic acid.
  • the target sequence is reestimated from hybridization pattern of the array to the target nucleic acid.
  • the steps are the repeated at least once.
  • the invention provides improved methods for analyzing variants of a reference sequence using arrays of probes.
  • the methods are particularly useful for target sequences showing substantial variation from a reference sequence, as may be the case where target sequence and reference sequence are from different species.
  • the methods involve designing a primary array of probes based on a known reference sequence. Effectively, the reference sequence serves as a first estimate of sequence of the target nucleic acid.
  • the primary array of probes is hybridized to a target nucleic acid, and the sequence of the target is estimated as well as possible from its hybridization pattern to the primary array.
  • a secondary array of probes is then designed based on the estimated sequence of the target nucleic acid.
  • the target nucleic acid is then hybridized with the secondary array of probes, and the sequence is reestimated from the resulting hybridization pattern. Further cycles of array design and estimation of target sequence can be performed in an iterative 11 fashion, if desired, until the estimated sequence is constant between successive cycles.
  • Reference sequences for polymorphic site identification are often obtained from computer databases such as Genbank, the Stanford Genome Center, The Institute for Genome Research and the Whitehead Institute. The latter databases are available at http://www-genome.wi.mit.edu; http://shgc.stanford.edu and http://ww.tigr.org. Reference sequences are typically from well-characterized organisms, such as human, mouse, C. elegans , Arabidopsis, Drosophila, yeast, E. coli or Bacillus subtilis . A reference sequence can vary in length from 5 bases to at least 1,000,000 bases. References sequences are often of the order of 100-10,000 bases . The reference sequence can be from expressed or nonexpressed regions of the genome.
  • RNA samples are used, highly expressed reference sequences are sometimes preferred to avoid the need for RNA amplification.
  • the function of a reference sequence may or may not be known.
  • Reference sequences can also be from episomes such as mitochondrial DNA. Of course, multiple reference sequences can be analyzed independently.
  • Targets can represent allelic, species, induced or other variants of reference sequences. Considerable diversity is possible between reference and target sequence. Target sequences usually show between 50-99%, 80-98%, 90-95% sequence identity.
  • a human reference sequence can be used as the starting point for analysis of primates, such as 12 gorillas, orangutans, other mammals, reptiles, birds, plants, fungi or bacteria.
  • the nucleic acid samples hybridized to arrays can be genomic, RNA or cDNA. Nucleic acid samples are usually subject to amplification before application to an array. An individual genomic DNA segment from the same genomic location as a designated reference sequence can be amplified by using primers flanking the reference sequence. Multiple genomic segments corresponding to multiple reference sequences can be prepared by multiplex amplification including primer pairs flanking each reference sequence in the amplification mix. Alternatively, the entire genome can be amplified using random primers (typically hexamers) (see Barrett et al . , Nucleic Acids Research 23, 3488-3492 (1995)) or by fragmentation and reassembly (see, e.g., Stemmer et al . , Gene 164, 49-53
  • Nucleic acids can also be amplified by cloning into vectors and propagating the vectors in a suitable organism.
  • YACs, BACs and HACs are useful for cloning large segments of genomic DNA.
  • Genomic DNA can be obtained from virtually any tissue source (other than pure red blood cells) .
  • tissue samples include whole blood, semen, saliva, tears, urine, fecal material, sweat, buccal, skin and hair.
  • RNA samples are also often subject to amplification. In this case amplification is typically preceded by reverse transcription. Amplification of all expressed mRNA can be performed as described by commonly owned WO 96/14839 and WO 97/01603. In some methods, in which arrays are designed to tile highly expressed sequences, amplification of RNA is unnecessary. The choice of tissue from which the sample is obtained affects the relative and absolute levels of different 13
  • RNA transcripts in the sample For example, cytochromes P450 are expressed at high levels in the liver.
  • Nucleic acids in a target sample are usually labelled in the course of amplification by inclusion of one or more labelled nucleotides in the amplification mix. Labels can also be attached to amplification products after amplification e.g., by end- labelling.
  • the amplification product can be RNA or DNA depending on the enzyme and substrates used in the amplification reaction.
  • LCR ligase chain reaction
  • NASBA nucleic acid based sequence amplification
  • ssRNA single stranded RNA
  • dsDNA double stranded DNA
  • An array of probes contain at least a first set of probes that are complementary to a reference sequence (or regions of interest therein) .
  • the probes tile the reference sequence. Tiling means that the probe set contains overlapping probes which are complementary to and span a region of interest in the reference sequence.
  • a probe set might contain a ladder of probes, each of which differs from its predecessor in the omission of a 5 ' base and the acquisition of an additional 3' base.
  • the probes in a probe set may or may not be the same length.
  • the number of probes can vary widely from about 5, 10, 20, 50, 100, 1000, to 10,000 or 100,000.
  • the arrays do not contain every possible probe sequence of a given length.
  • the first probe set comprises a plurality of probes exhibiting perfect complementarily with a reference sequence, as described above.
  • Each probe in the first probe set has an interrogation position that corresponds to a nucleotide in the reference sequence. That is, the interrogation position is aligned with the corresponding nucleotide in the reference sequence, when the probe and reference sequence are aligned to maximize complementarily between the two.
  • For each probe in the first set there are three corresponding probes from three additional probe sets. Thus, there are four probes corresponding to each nucleotide in the reference sequence .
  • the probes from the three additional probe sets are identical to the corresponding probe from the first probe set except at the interrogation position, which occurs in the same position in each of the four corresponding probes from the four probe sets, and is occupied by a different nucleotide in the four probe sets.
  • a substrate bearing the four probe sets is hybridized to a labelled target sequence, which shows substantial sequence similarity with the reference sequence, but which may differ due to e.g., species variations.
  • the amount of label bound to probes is measured. Analysis of the pattern of label revealed the nature and position of differences between the target and reference sequence. For example, comparison of the intensities of four corresponding probes reveals the identity of a corresponding nucleotide in the target sequences aligned with the interrogation position of the probes. The corresponding nucleotide is the complement of the nucleotide occupying the interrogation position of the probe showing the highest intensity. The comparison can be performed between successive columns of four corresponding probes to determine the identity of successive nucleotides in the target sequence .
  • one of the four probes clearly has a significantly higher signal than the other three, and the identity of the base in the target sequence aligned with the interrogation position of the probes can be called with substantial certainty.
  • two or more probes may show similar but not identical signals. In these instances, one can simply score the position as ambiguous. Alternatively, can still call a base from the probe that has the higher signal but must recognize a significant possibility of error. In general, if the ratio of signals of two probes is less than 1.2, a base call has a significant possibility of error.
  • Ambiguous positions are most frequently due to closely spaced multiple points of variation between target and reference sequence (i.e., within a probe length). Ambiguities 16 can also arise due to low hybridization intensity because of base composition effects.
  • a secondary array of probes is constructed based on the same principles as the first array, except that the first probe set is tiled based on the newly estimated sequence rather than the original reference sequence.
  • the estimated sequence includes the best estimate of base present at positions of ambiguity as noted above. If there is equal probability of two or more bases occupying a particular position in the estimated sequence, one can arbitrarily decide to include one of the bases, provide alternate tilings corresponding to the different possible bases, or include multiple pooled bases at the position.
  • the secondary array typically has second, third and fourth probe sets designed according to the same principles as in the primary array.
  • the secondary array is hybridized to the same target nucleic acid as was the primary array.
  • Bases in the target sequence are called using the same principles as described above by comparison of probe intensities to give rise to a reestimated target sequence.
  • the process can be repeated through further iterations, if desired. Further iteration is desirable if the estimated sequence contains a substantial number of positions, which have been estimated with a low degree of confidence (e.g., from a comparison of probe intensities differing by a factor of less than 1.2) . After sufficient iterations, the estimated sequence from one cycle should converge with that from the subsequent cycle. In some instances, positions of ambiguities may remain through many cycles. These positions may. be due to effects such as heterozygosity, and should be checked by other means (e.g., conventional dideoxy sequencing 17 or de novo sequencing by hybridization to a complete array of probes a given length) .
  • a low degree of confidence e.g., from a comparison of probe intensities differing by a factor of less than 1.2
  • arrays tile both strands of a reference sequence. Both strands are tiled separately using the same principles described above, and the hybridization patterns of the two tilings are analyzed separately. Typically, the hybridization patterns of the two strands indicates the same results (i.e., location and/or nature of variation between target sequence and reference sequence) . Occasionally, there may be an apparent inconsistency between the hybridization patterns of the two strands due to, for example, base-composition effects on hybridization intensities. Combination of results from the two strands increases the probability of correct base calling and can decrease the number of iterations required to determine the correct base sequence of a target .
  • duplicate arrays are synthesized to allow analysis of hybridization between target sequence and probes under conditions of high and low stringency.
  • high stringency is generally most useful
  • Statistical combination of base calls from conditions of high and low stringency can increase the overall probability of correct base calling.
  • Arrays of probe immobilized on supports can be synthesized by various methods.
  • a preferred methods is VLSIPSTM (see Fodor et al . , US 5,143,854; EP 476,014, Fodor et al., 1993, Nature 364, 555-556; McGall et al . , USS ⁇ 08/445,332), which entails the use of light to direct the synthesis of oligonucleotide probes in high-density, miniaturized arrays (sometimes known as chips) .
  • Algorithms for design of masks to reduce the number of synthesis cycles are described by Hubbel et al . , US 5,571,639 and US 5,593,839.
  • Arrays can also be synthesized in a combinatorial fashion by delivering monomers to cells of a support by mechanically constrained flowpaths. See Winkler et al . , EP 624,059. Arrays can also be synthesized by spotting monomers reagents on to a support using an ink jet printer. See id. ; Pease et al. , EP 728,520.
  • hybridization intensity for the respective samples is determined for each probe in the array.
  • hybridization intensity can be determined by, for example, a scanning confocal microscope in photon counting mode. Appropriate scanning devices are described by e.g., Trulson et al . , US 5,578,832; Stern et al . , US 5,631,734.
  • a minimal overlapping set of physical clones is first obtained. For example, random bacterial artificial chromosome clones are generated, and ordered by hybridization or conventional methods. If necessary, regions mapping to related positions in the genome are determined. E.g., pools of clones are hybridized to an array of mapped markers. Pools of clones are then generated for hybridization (e.g., 300 pools if the resequencing capacity is 1 Mb/chip and 300 chip designs are used to analyze 1/lOth a mammalian genome) .
  • sequences differences between differences species allows correlation between form and function. For example, the sequence of chimpanzee and human differ by -1% overall. Further, the present methods allow comparison of a range of primate sequences, to see which sequences have evolved the most rapidly and which are highly conserved. It will be apparent from the above that the invention includes a general concept which can be expressed concisely as follows. The invention entails the use of iterative cycles of designing an array of probes to be complementary to an estimated sequence of a target nucleic acid, and using the hybridization pattern of the array to the target nucleic acid sequence to determine a more accurate reestimated target sequence.
  • base 21 calling was made with at least 99.91% accuracy covering a minimum of 97% of the sequence.
  • PCR from genomic DNA and transcription PCR reactions were performed on genomic samples using the EXPANDTM Long Range PCR Kit (Boehringer Mannheim) with intronic primers 11PIF 5 ' -CCTTGTTATTTTTTGTATATTTTCAG-3 ' and 11PIR 5 ' -CAAAAACCTGGTTCCAATAC-3 ' , directly overlapping the underlined 5 ' -AG acceptor and 3 ' -GT donor splice sites.
  • PCR reactions using the templates generated from the 11PIF and 11PIR primer set, were performed with primers 11PIFT3 5'- ATTAACCCTCACTAAAGGGACCTTGTTATTTTTTGTATATTTTCAG- .3 ' and 11PIRT7 5 ' -TAATACGACTCACTATAGGGACAAAAACCTGGTTCCAA TAC-3' containing T3 and T7 RNA polymerase promoter sequences respectively.
  • Test sample transcription product was diluted to a final concentration of 100 nM in a 25 ⁇ l solution of 30 mM MgCl 2 .
  • the reaction was incubated at 94 degrees C for 60 minutes to hydrolyze target into fragments ranging from
  • hybridization buffer 3 M TMAC (tetramethylammonium chloride), IX TE pH 7.4, 0.005% Triton X-100, 1 nM 5 ' -fluorescein-labelled control oligonucleotide 5'CGGTACCATCTTGAC-3' ) .
  • the control oligonucleotide hybridizes to specific surface probes aiding in image alignment.
  • Target was hybridized with the appropriate sense or antisense strand reading array in a 250 ⁇ l volume for 4 hours at 35 degrees C.
  • the array surface was washed with 5 ml of wash buffer (6X SSPE, 0.005% Triton X-100) and stained with phycoerythrin-streptavidin conjugate (Molecular Probes) (2 ⁇ /ml in wash buffer containing 2 mg/ml acetylated BSA (GIBCO BRL)) for 20 minutes at room temperature.
  • wash buffer 6X SSPE, 0.005% Triton X-100
  • phycoerythrin-streptavidin conjugate Molecular Probes
  • Each array was washed with 5 ml of wash buffer and imaged using a 488 nm argon laser equipped with a scanning confocal microscope (GeneChip Scanner, Affymetrix) .
  • Fluorescent hybridization signals were detected by a photomultiplier tube using a 560 nm longpass emission filter.
  • oligonucleotide array The synthesis and design of the oligonucleotide array has been described previously 9 . Briefly, DNA phosphoramidites bearing 5 ' -photolabile protecting groups are coupled to a derivatized glass surface using modified DNA synthesis protocols. Spatially addressable oligonucleotide synthesis is obtained through photolithographic techniques with selective oligonucleotide photodeprotection for each 23 coupling cycle. Thirty identical high density array chips containing over 48,000 oligonucleotides were simultaneously produced in a single 8 hour synthesis.
  • GeneChip Software created digitized fluorescence images by converting photomultiplier tube output signal into proportional spatially addressed pixel values. The probe intensity is calculated from the mean of the non-outlier photon counts for each feature (i.e. per probe). Background corrected fluorescent hybridization signal to each probe was extracted from test images using AVI Software (Affymetrix) and imported into ViewSeq Software (Affymetrix) which quantitates ratios of fluorescent target hybridization signal to each set of 8 oligonucleotide probes (4 per strand) interrogating each nucleotide. Data from 4 sets of experiments reading both target strands were averaged to produce a composite file.
  • Template PCR products were purified using the Wizard DNA Purification Kit (Promega) .
  • Conventional fluorescent dye terminator 3 pass dideoxysequencing analysis was performed using the AB1377 System.
  • Human BRCAl exon 11 primers were used for first pass sequencing of all templates, except the canine ortholog of known sequence 18 . Sequence gaps were filled by a primer walking strategy.
  • This sequencing and template generation strategy is not sensitive towards detecting all possible heterozygous single nucleotide polymorphisms; however, it is quite sensitive to detection of heterozygous sequences causing chain length differences in dideoxysequencing products. Nevertheless, in cases of 24 heterozygous base substitutions the identity of one allele is reported.
  • a nested amplicon within the flying lemur template was generated using Amplitaq GOLD (Perkin Elmer) and the manufacturers protocols to clarify a suspected heterozygous sequence.
  • PCR product was subcloned using Zero Blunt Cloning Kit (Invitrogen) and inserts from individual colonies were sequenced.
  • a heterozygous in-frame 3 base pair deletion was found in flying lemur target which aligns with bases 2192-2194 of human BRCAl cDNA sequence and results in the removal of a single serine from a tract of three serine residues.
  • High density arrays have been used to screen the 3.43-kb exon 11 of the human hereditary breast and ovarian cancer BRCAl gene 13 for all possible heterozygous polymorphisms and mutations 9 .
  • level one analysis quantitates the ratios of fluorescent target hybridization signal for eight probes (four per sense and antisense strands, respectively) querying each nucleotide position.
  • the identity of the brightest signal is assigned to the target nucleotide, using human sequence numbering. If the brightest probe signal in each set is less than or equal to a factor of 1.2 of the next brightest, an IUPAC ambiguity designation is assigned.
  • 1,363 nucleotide positions had single nucleotide mismatch specificity ratios (the ratio between the two highest probe signals within each averaged composite set of four) greater than 9.0, 1,346 positions had ratios between 5.0 and 9.0, 708 positions had ratios between 2.0 and 5.0, 5 positions had ratios between 1.2 and 2.0, and 4 positions had ratios less than 1.2 (giving an ambiguous level one base call) .
  • Figs. 3 A-D Human, chimpanzee, gorilla, and orangutan targets with identical sequence tracts showed similar hybridization patterns (Figs. 3 A-D) .
  • a single nucleotide substitution between rhesus and human targets is correctly identified by level one analysis; however, the 3' -adjacent nucleotide is incorrectly assigned (Fig. 3E) .
  • Level one hybridization data identifies two red howler monkey nucleotide substitutions, but cannot accurately read adjacent sequences (Fig. 3F) .
  • Galago target contained 3 closely spaced nucleotide substitutions causing diminished hybridization signals and lower fidelity nucleotide assignments (Fig. 3G) .
  • level one sequence information was determined from the least (dog) and most (chimpanzee) highly conserved targets by referring to dideoxysequencing data.
  • level one dog sequence Upon inspecting level one dog sequence, it was evident that base calling was poor quality in regions of predicted multiple substitutions. Furthermore, it was apparent that the most accurate level one base calls occurred in sequence tracts predicted identical to human reference sequence. Therefore, such tracts ranging from 4 to 8 nucleotides in length were systematically evaluated for base-calling fidelity.
  • predicted single nucleotide substitutions flanked by these tracts were included in this evaluation since the array has the capacity to correctly identify them (Fig. 2B) .
  • a second order tiling scheme with probes designed to match anticipated base substitutions in the level one data based upon single nucleotide mismatch probe hybridization signals, clarifies most or all ambiguities.
  • All chimpanzee, gorilla, and orangutan miscalls made adjacent to base substitutions can be clarified using second order tiling schemes since the sequence accuracy was at least 99.88% when the tiling pattern matches the target sequence.
  • red howler monkey, galago, and dog orthologs provided less level two quality hybridization data (Table 1) . This was primarily caused by an increased number of closely spaced nucleotide substitutions along with insertions and deletions. Of the 26 level-two red 29 howler target miscalls, 23 were found nearby an almost exact 21-bp target duplication while 3 were due to a 3 base pair deletion.
  • Completion of the Human Genome Project allows use of DNA chips for rapid genome-wide determination of non-human primate sequences 14 . This approach is particularly powerful when scanning for conserved sequence tracts, important for phylogenetic footprinting of promoter regions 2 .

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  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne des techniques itératives permettant d'analyser un acide nucléique cible qui représente un variant d'un acide nucléique de référence. On construit une matrice de sondes complémentaire d'une séquence estimée d'un acide nucléique cible. On hybride ladite matrice avec ledit acide nucléique cible. On procède à une nouvelle estimation de la séquence cible à partir du motif d'hybridation de la matrice avec l'acide nucléique cible. On construit une autre matrice de sondes complémentaire de la séquence nouvellement estimée, puis on utilise cette matrice pour obtenir une nouvelle estimation de la séquence de l'acide nucléique cible. En effectuant des cycles répétés de construction des sondes et d'estimation de la séquence cible, on obtient une séquence estimée de la cible qui se rapproche de la véritable séquence.
PCT/US1998/005438 1998-02-02 1998-03-19 Resequençage automatique WO1999039004A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US7385398P 1998-02-02 1998-02-02
US7334598P 1998-02-02 1998-02-02
US60/073,345 1998-02-02
US60/073,853 1998-02-02

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WO1999039004A1 true WO1999039004A1 (fr) 1999-08-05

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DE19957320A1 (de) * 1999-11-29 2001-05-31 Febit Ferrarius Biotech Gmbh Dynamische Sequenzierung durch Hybridisierung
WO2001040509A3 (fr) * 1999-11-29 2001-12-06 Febit Ferrarius Biotech Gmbh Determination dynamique d'analytes
WO2001057278A3 (fr) * 2000-02-04 2003-01-09 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hela humaines ou d'autres cellules epitheliales humaines du col de l'uterus
EP0972078A4 (fr) * 1997-03-20 2003-05-28 Affymetrix Inc Resequen age iteratif
US6632641B1 (en) 1999-10-08 2003-10-14 Metrigen, Inc. Method and apparatus for performing large numbers of reactions using array assembly with releasable primers
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US7179638B2 (en) 1999-07-30 2007-02-20 Large Scale Biology Corporation Microarrays and their manufacture by slicing
US7211390B2 (en) 1999-09-16 2007-05-01 454 Life Sciences Corporation Method of sequencing a nucleic acid
US7244559B2 (en) 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid

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US5698391A (en) * 1991-08-23 1997-12-16 Isis Pharmaceuticals, Inc. Methods for synthetic unrandomization of oligomer fragments

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US5698391A (en) * 1991-08-23 1997-12-16 Isis Pharmaceuticals, Inc. Methods for synthetic unrandomization of oligomer fragments
US5683881A (en) * 1995-10-20 1997-11-04 Biota Corp. Method of identifying sequence in a nucleic acid target using interactive sequencing by hybridization

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0972078A4 (fr) * 1997-03-20 2003-05-28 Affymetrix Inc Resequen age iteratif
US7144699B2 (en) 1997-03-20 2006-12-05 Affymetrix, Inc. Iterative resequencing
US7179638B2 (en) 1999-07-30 2007-02-20 Large Scale Biology Corporation Microarrays and their manufacture by slicing
US6887701B2 (en) 1999-07-30 2005-05-03 Large Scale Proteomics Corporation Microarrays and their manufacture
US6846635B1 (en) 1999-07-30 2005-01-25 Large Scale Proteomics Corp. Microarrays and their manufacture
US7335762B2 (en) 1999-09-16 2008-02-26 454 Life Sciences Corporation Apparatus and method for sequencing a nucleic acid
US7264929B2 (en) 1999-09-16 2007-09-04 454 Life Sciences Corporation Method of sequencing a nucleic acid
US7244559B2 (en) 1999-09-16 2007-07-17 454 Life Sciences Corporation Method of sequencing a nucleic acid
US7211390B2 (en) 1999-09-16 2007-05-01 454 Life Sciences Corporation Method of sequencing a nucleic acid
US6632641B1 (en) 1999-10-08 2003-10-14 Metrigen, Inc. Method and apparatus for performing large numbers of reactions using array assembly with releasable primers
EP1650314A1 (fr) * 1999-11-29 2006-04-26 febit AG Détermination dynamique d'analytes en utilisant une puce localisée sur une surface interne
WO2001040510A3 (fr) * 1999-11-29 2001-12-06 Febit Ferrarius Biotech Gmbh Sequençage dynamique par hybridation
DE19957320A1 (de) * 1999-11-29 2001-05-31 Febit Ferrarius Biotech Gmbh Dynamische Sequenzierung durch Hybridisierung
WO2001040509A3 (fr) * 1999-11-29 2001-12-06 Febit Ferrarius Biotech Gmbh Determination dynamique d'analytes
GB2382814B (en) * 2000-02-04 2004-12-15 Aeomica Inc Human genome-derived single exon nucleic acid probes useful for analysis of gene expression in human hela cells or other human cervical epithelial cells
WO2001057278A3 (fr) * 2000-02-04 2003-01-09 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hela humaines ou d'autres cellules epitheliales humaines du col de l'uterus
WO2001057270A3 (fr) * 2000-02-04 2003-02-13 Aeomica Inc Sondes d'acide nucleique a un seul exon derivees du genome humain utiles pour analyser l'expression genique dans des cellules hbl 100

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