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WO1992016657A1 - Procede d'identification d'un nucleotide present dans un acide nucleique en une position definie - Google Patents

Procede d'identification d'un nucleotide present dans un acide nucleique en une position definie Download PDF

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Publication number
WO1992016657A1
WO1992016657A1 PCT/US1992/001691 US9201691W WO9216657A1 WO 1992016657 A1 WO1992016657 A1 WO 1992016657A1 US 9201691 W US9201691 W US 9201691W WO 9216657 A1 WO9216657 A1 WO 9216657A1
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Prior art keywords
nucleotide
nucleic acid
probe
interest
dna
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PCT/US1992/001691
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English (en)
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Kenneth J. Livak
Jan Antoni Rafalski
Nancy Faye Stacy Shepherd
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E.I. Du Pont De Nemours And Company
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Publication of WO1992016657A1 publication Critical patent/WO1992016657A1/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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6858—Allele-specific amplification
    • 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
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers

Definitions

  • This invention relates to a rapid, convenient process to identify a nucleotide present at a specific position in a nucleic acid chain (DNA or RNA) of a biological sample.
  • nucleic acid content of any organism is the essence of that organism, and differences in the nucleic acid are known to be of primary importance in distinguishing one from another.
  • the science of genetics is based on the identification and characterization of differences in nucleic acid sequence. These differences, or polymorphisms, are often termed "mutations" and may be due to nucleotide substitution, insertion or deletion.
  • many techniques have been developed to compare homologous segments of DNA or RNA to determine if the segments are identical or if they differ at one or more nucleotides.
  • Identification of genetic polymorphisms is useful for genetic diagnoses in medicine, identification of individuals in forensic science, identification of pathogenic organisms, construction of genetic polymorphism maps for locating genes important in disease and in agriculture and for breeding of plants and animals.
  • the most definitive method for comparing DNA segments is to determine the complete nucleotide sequence of each segment. Examples of how sequencing has been used to study mutations in human genes are included in the publications of Engelke, et. al., Proc. Natl. Acad. Sci. U.S.A. 85:544-548 (1988) and Wong, et al.. Nature 330:384-386 (1987).
  • nucleotide sequence information at any one point in a nucleotide chain is one of several hundred such pieces of information. That is, if one is only interested in determining the nucleotide at a specific point in the polynucleotide chain (e.g. a position known from previous genetic analysis to be involved in a disease phenotype) , that information must be retrieved from a larger volume of data.
  • the most commonly used screen for DNA polymorphisms consists of digesting DNA with restriction endonucleases and analyzing the resulting fragments by means of Southern blots, a method known as "Restriction Fragment Length Polymorphism” or RFLP mapping, as described by Botstein, et al.. Am. J. Hum. Genet. 32:314-331 (1980); White, et al., Sci. Am. 258:40-48 (1988) . Mutations that affect the recognition sequence of the endonuclease will preclude enzymatic cleavage at that site, thereby altering the cleavage pattern of that DNA. DNAs are compared by looking for differences in restriction fragment lengths.
  • RFLP detection in a genomic DNA sample is very labor intensive for it requires preliminary steps of genomic DNA isolation, restriction, gel electrophoresis and Southern transfer steps, before hybridization to a probe that is generally radioactively labeled for sensitive detection of homologous sequences.
  • a major problem associated with RFLP detection is the necessity of the polymorphism to affect cleavage with a restriction endonuclease, therefore many mutations cannot be detected with this method (Jeffreys, Cell 18:1-18, 1979) .
  • RFLP and several other methods in the prior art e.g. Wallace et al., Nucl. Acids Res. 9:879-894, 1981 or Saiki, et al., U.S. Pat. No.
  • a technique involving amplification and mismatch detection AMD, described by Montandon et al., Nucl. Acids Res. 17:3347-3358, 1989, utilizes amplification of the DNA region of interest from two samples, followed by denaturation and reannealing to form homo- and heteroduplexes between DNA molecules of the two samples.
  • nucleotide position that is different between the two amplified DNAs can be identified with the use of hydroxylamine and osmium tetroxide to modify mispaired cytosines and thymines, respectively, followed by piperidine-catalysed cleavage of the modified heteroduplexes, and subsequent gel electrophoresis to identify cleavage products.
  • this technique and the analogous technique of EP0329311 to Campbell and Cotton are useful for both detecting and identifying all point mutations within a nucleic acid segment, they share some of the same, serious disadvantages of the chemical degradation method for DNA sequencing by Maxam and Gilbert (Proc. Natl. Acad. Sci. 74:560, 1977). These techniques require dangerous chemicals that modify and cleave nucleic acids, they involve several different chemical reactions, and require a time-consuming gel assay.
  • oligonucleotide probes designed to overlap the position of interest at the 3'prime terminus are reviewed below.
  • the technique described in Landegren, et al.. Science 241:1077-1080 (1388) uses an enzymatic detection of polymorphisms.
  • oligonucleotide probes are constructed in pairs such that their junction corresponds to the specific nucleotide site which is of interest. These oligonucleotides are then hybridized to the DNA being analyzed. Base pair mismatch between either oligonucleotide and the target DNA at the junction location prevents the efficient joining of the two oligonucleotide probes by DNA ligase.
  • 4,851,331 also depends upon an enzymatic reaction that requires one end of the oligonucleotide probe to form a perfect, complementary matched basepair with the target nucleotide sequence.
  • an oligonucleotide probe is designed such that the 3-prime end of the complementary probe includes the specific nucleotide position of interest. After annealing this oligonucleotide probe to the template DNA, a polymerase that replicates nucleic acid strands in a template directed fashion is used to incorporate modified nucleotides into a newly synthesized strand.
  • the polymerase cannot begin the replication process.
  • the amount of incorporation is a measure of the amount of the specific template DNA in the biological sample.
  • ASPCR Allele-specific Polymerase Chain Reaction
  • reaction products were run on an agarose gel and detected by ethidium bromide staining.
  • fluorescently-labeled oligonucleotide primers may also be used for detection in ASPCR (e.g. Chehab and Kan, Proc. Natl. Acad. Sci. 86:9178-9182, 1989).
  • the patent of Caskey and Gibbs (EP0333465A2) for a process involving Competitive Oligonucleotide Priming (COP) is essentially the same. In COP, two differentially labeled oligonucleotide primers that differ at the 3' nucleotide and overlap the position of interest are present in the same, rather than in separate reactions, and thus compete for template molecules in the hybridization reaction.
  • a general problem shared by the various techniques mentioned in the preceding paragraph is that the difference in duplex stability of a perfectly vs nonperfectly matched oligonucleotide to its target DNA is dependent upon the length and sequence of the oligonucleotide. Therefore, regardless of the assay method, a different set of emperically determined experimental reaction conditions may be required in order to assay different genomic loci for a polymorphism. Secondly, since their assay is a +/- assay (a reaction product should be formed or absent) , it is necessary to perform several assays on a single target DNA so that an inference can be made concerning the nature of the nucleotide at a given position.
  • specific mutations can be detected by first hybridizing a labeled DNA probe to the target nucleic acid in order to orm a hybrid in which the 3' end of the probe is positioned adjacent to the specific base being analyzed. Then, a DNA polymerase is used to add a nucleotide analog, such as a thionucleotide, to the probe strand, but only if the analog is complementary to the specific base being analyzed. Finally, the probe- target hybrid is treated with exonuclease III. If the nucleotide analog has been incorporated, the labeled probe is protected from nuclease digestion.
  • a nucleotide analog such as a thionucleotide
  • nucleotide analog may be given as the sole substrate for incorporation, or it may be added as one of a maximum of three nucleotide substrates. It is critical to note that all four bases cannot be given as substrate, since this would allow chain elongation to occur until eventually the modified nucleotide analog would be complementary and thus incorporated. This limits the utility of the assay to certain DNA sequences that can have the appropriate positioning of primers. For it is well known that if the correct base is not supplied in a reaction that a "wobble" base pair between G and T will occur to a significant degree (Boosalis et al., J. of Biol.
  • the reaction of the present invention can include all four nucleotide substrates, thus leading to a lower incorporation of non-complementary nucleotide.
  • This invention uses chain-terminating nucleotides as substrates in the reaction, therefore preventing incorporation of several of the same nucleotide in the primer extension product if there are several of the same nucleotides present in a row on the template.
  • the analysis of Sokolov required four separate reactions whereas the present invention would need only one reaction to gain the same amount of information.
  • (4) As mentioned above, if the correct, complementary nucleotide substrate is not present in the reaction, then significant misincorporation can occur in the Sokolov reaction. Misincorporation is substantially prevented in the present invention.
  • each ASPCR reaction is performed using one biotin-labeled primer and one fluorescently- labeled primer.
  • the biotinylated, double-stranded amplification products are then separated from unincorporated fluorescent primer using streptavidin coated magnetic beads.
  • the color of the amplified DNA would then be determined fluorometricly through a fiber optic bundle, or alternatively, by separation and detection on a sequencing gel as is currently performed for DNA sequencing using fluorescently labeled primers. The differences between this method and that of the present invention are significant.
  • reaction products due to amplification from the "wrong" primer are also biotinylated and will be retained in the capture process. Furthermore, these products due to misincorporation will electrophorese to the same position in the sequencing gel, thus interfering with the analysis. Finally, the size of the amplified products will in general be larger than an oligonucleotide, thus requiring a longer time for gel electrophoresis and detection than that of the present invention.
  • a technique that identifies a nucleotide at a given position in a nucleic acid sample should be fast, simple, reliable, and avoid radioactive or nucleic acid cleaving compounds.
  • the currently available detection techniques discussed above are deficient in one or more of these areas. All of these problems are overcome by the present invention.
  • the process of the present invention exploits some of the same principles and advantages as described for sequencing of nucleic acids using fluorescent dideoxynucleotide substrates (Prober et al., EPO 252638; Mitchell and Merril WO 89/12063; Innis et al., WO 9003442) . It is, however, different from that process in that one of the components of the process namely the dNTP substrates allowing multiple lengths of primer elongation product is missing in this invention.
  • the method of the present invention generally requires a knowledge of the nucleic acid sequence in the region of interest, but it does not require the mutation to be at a restriction enzyme cleavage site. The method is capable of giving unambiguous results.
  • the present invention provides a process for identifying the nucleotide present at a specific position in a nucleic acid sequence. It is based upon the selective attachment of one of four chain- terminating nucleotides, that are detectably labeled and distinguishable, onto a probe in a complementary, template dependent fashion.
  • the probe is designed to selectively hybridize to a target nucleotide sequence and oriented such that a one nucleotide extension of the probe, usually in the 3-prime direction, will base pair to the nucleotide position of interest.
  • the oligonucleotide probe, the nucleic acid containing the target nucleotide sequence, or both, may contain a site for specific immobilization to facilitate separation from unincorporated nucleotides and primers, such that the labeled nucleotides incorporated into the reaction product can be measured without use of a gel system such as agarose or acrylamide.
  • the present invention provides a method for the identification of the nucleotide present at a single, defined position in the nucleic acid which comprises the following steps:
  • nucleic acid analyte (a) contacting a nucleic acid analyte with a probe oligonucleotide of sufficient length and appropriate sequence under conditions sufficient for the probe to bind preferentially to a target nucleotide sequence and form a hybrid having a double-stranded portion including the 3-prime end of the probe.
  • the nucleotide position of interest is the first base of the nucleic acid analyte which extends in a 3* to 5' direction beyond the 3" end of the probe nucleotide sequence and is immediately adjacent to the hybrid formed ( Figure 1) .
  • nucleotide of interest (d) identifying the nucleotide of interest as the nucleotide complementary to the incorporated chain- terminating nucleotide.
  • the present invention also provides a kit.
  • the kit includes reagents in packaged form.
  • the package may include extension primers, a probe polynucleotide, chain-terminating nucleotides and extension enzymes.
  • the package may include attachment moieties and solid supports. Any of the reagents may include attachment moieties.
  • a package may include any combination of reagents as necessary for a particular purpose.
  • a package may include an insert such as a standard or direction for handling specific reagents. DESCRIPTION OF FIGURES
  • Figure 1 comprising Figures la-lh, illustrate in various schematic forms, the location of various components of the process of this invention.
  • Figure la illustrates an analyte strand (An) which contains the nucleotide position of interest (N) , the identity of which is to be determined by the assay.
  • a target nucleotide sequence (TNS) immediately 3' of, but not including the nucleotide position of interest is illustrated.
  • a double strand nucleic acid region forms when a probe binds to analyte strand An by complementary base pairing to the target nucleotide sequence TNS.
  • Figure lb illustrates the incorporation of a chain terminating nucleotide (N*) complementary to the nucleotide of interest (N) after contacting the double stranded region in Figure la with a polymerase capable of primer extension. (The * in this and subsequent figures is used to illustrate a detectable label attached to the nucleotide) .
  • Figure lc illustrates the same features as Figure la, but with a specific example showing the nucleotide of interest as a thymidine (T) .
  • T thymidine
  • Figure Id illustrates the same features as Figure lb, but using the same specific example as Figure lc, to show the result of enzymatic incorporation of a detectably labeled adenosine at the 3' terminus of the probe as the nucleotide complementary to the nucleotide of interest (thymidine) .
  • Figure le illustrates the incorporation of a detectably labeled guanosine at the 3 1 terminus of the probe and complementary to the nucleotide of interest (cytidine) .
  • Figure If illustrates the use of another analyte strand for the assay (the complementary strand of the analyte strand shown in Figure lc) .
  • the target nucleotide sequence is chosen to be immediately 3 ⁇ of the nucleotide of interest (in this example shown as an adenosine)
  • the probe is complementary to the target nucleotide sequence.
  • Figure Ig illustrates the incorporation of a detectably labeled thymidine at the 3 1 terminus of the probe and complementary to the nucleotide of interest (adenosine) .
  • Figure Ih illustrates the incorporation of a detectably labeled cytidine at the 3' terminus of the probe and complementary to the nucleotide of interest (guanosine) .
  • Figure 2 illustrates an example of steps that can be used in the practice of this invention when it is desired to use a nucleic acid strand immobilized on a solid support as the analyte strand.
  • Figure 3 illustrates an example of steps that can be used in the practice of this invention when it is desired to use a nucleic acid strand in solution as the analyte strand.
  • Figure 4 illustrates identification of the nucletide of interest (N) when it is located at the 5' terminus of the analyte strand.
  • Figure 5 illustrates identification of a difference between two analyte strands, when that difference is part of a nucleotide insertion or deletion.
  • Figure 6 illustrates how the number of assays must increase if the number of distinguishably labeled chain terminating nucleotides in the reaction are decreased, if the identity of all possible nucleotides at the position of interest is to be determined.
  • Figure 7 illustrates the output signals obtained when the detectably labeled substrates used in the examples are detected with the Genesis 2000 DNA analysis system.
  • Figure 7a illustrates the output signal [ratio of the green line to red line peak height, +/- one standard deviation] of data obtained as in Figure 7b and 7c for each of the four detectably labeled nucleotides used in the examples detected either through a gel or through a capillary.
  • Figure 7b is representative data showing the position and relative peak heights of the two photomultiplier tube signals (red and green lines) when SF-ddGTP-505 or SF-ddTTP-526 are electrophoresed through a urea-polyacrylamide slab gel mounted on the Genesis 2000.
  • Figure 7c illustrates the output signal obtained when SF-ddGTP-505 or SF-ddCTP-519 are each passed four times (therefore four peaks) through an empty capillary mounted for detection on the Genesis 2000 unit.
  • Figure 8 illustrates the double stranded portion of the mouse RNA polymerase II gene that was amplified using PCR primer 1 and PCR primer 2, as well as the position and sequence of the various oligonucleotide probes used in Examples 1-5.
  • Figure 9 illustrates the sequence of the Wildtype and the Mutant allele of the RNA polymerase II gene between nucleotides 5395 and 5454, with the difference between the two alleles indicated by boldface type at position 5430.
  • Figure 10 illustrates the data obtained in Example 1: Incorporation of either labeled SF-ddATP-512, SF- ddGTP-505, or both in approximately equal amounts, when probe A is used on nucleic acid samples known to be either Wildtype, Mutant, or Heterozygous at nucleotide position 5430 of the RNA polymerase II gene.
  • Figure 11 illustrates the data obtained in Example 2: Incorporation of either labeled SF-ddTTP-526 or SF-ddCTP-519 when probe B is used on nucleic acid samples known to be either Wildtype or Mutant at nucleotide position 5430 of the RNA polymerase II gene.
  • Figure 12 illustrates the data obtained in Example 3: The level of misincorporation that may occur if the correct, complementary nucleotide is not included in the reaction.
  • Figure 13 illustrates the data obtained in Example 4: Correct incorporation of SF-ddTTP-526 in the Wildtype allele and SF-ddCTP-519 in the Mutant allele in the presence of all four, distinguishably labeled ddNTPs in the reaction at lower nucleotide concentrations than in previous examples.
  • Figure 14 illustrates the double stranded nucleic acid region of the Wildtype Al gene of Zea mays that is amplified using PCR primers A and B, with primer C indicating the sequence and position of the oligonucleotide probe used in Examples 5-6.
  • Figure 15 illustrates the data of Example 5: Correct incorporation of SF-ddGTP-505 when a mutant allele, a-dt f of the maize Al gene (carrying a G-C base pair rather than C-G base pair) is assayed.
  • Figure 16 illustrates the data of Example 6: Use of a modified T7 polymerase (Sequenase) , to give the correct incorporation of approximately equal amounts of SF-ddCTP and SF-ddGTP in a sample heterozygous for the maize allele.
  • Tequenase modified T7 polymerase
  • the present invention discloses a process for identifying the nucleotide present at defined nucleotide position in a nucleic acid sequence.
  • This process has utility as a rapid, convenient means to genotype a biological sample with respect to specific, nucleic acid sequence information (e.g. nucleotide positions correlated with phenotypic differences among individuals between species or in tissues) .
  • nucleic acid sequence information e.g. nucleotide positions correlated with phenotypic differences among individuals between species or in tissues
  • Single base pair mutations such as transitions, transversions, insertion, deletion as well as more complex rearrangements can be assayed using the method of the present invention if the appropriate oligonucleotide probe is designed ( Figure 5) .
  • the presence of a target nucleic acid in a biological sample may be detected generally as the presence or absence of an incorporated nucleotide.
  • nucleic acid sequences DNA or RNA
  • Any source of nucleic acid, in purified or nonpurified form can be utilized as the starting nucleic acid or acids, if it contains, or is suspected of containing, the target nucleic acid sequence.
  • the target nucleic acid can be only a fraction of a larger molecule or can be present initially as a discrete molecule. Additionally, the target nucleic acid may constitute the entire nucleic acid or may be a fraction of a complex mixture of nucleic acids.
  • the method of this invention requires formation of a hybrid between an oligonucleotide primer (referred to herein as the oligonucleotide probe) and the target nucleic acid sequence.
  • Probes of relatively short length e.g. 10-100 nucleotides are preferred in that they can be chemically synthesized.
  • the probe can consist of DNA, RNA, a contiguous DNA-RNA polynucleotide, or a nucleic acid chain containing one or more modified nucleotides. Under the appropriate conditions the probe should selectively form a hybrid with the target nucleotide sequence.
  • the probe of this invention terminates one nucleotide prior to the position of interest such that the first nucleotide to be added to the 3' terminus of the oligonucleotide probe in a template-dependent, primer extension reaction will be a nucleotide complementary to the nucleotide position of interest ( Figure 1) .
  • the specific target nucleic acid of a biological sample must be present in sufficient quantity such that hybrid molecules formed with the probe oligonucleotide are detectable by the label incorporated onto the probe.
  • Some samples may contain a sufficient number of target nucleic acid strands, but other samples may not.
  • molecular cloning of a region surrounding the nucleotide position of interest would suffice as a means to increase the number of target molecules, but it is tedious and time-consuming.
  • Methods to clone nucleic acid fragments see Sambrook, J. et al., 1989, Molecular Clonin ⁇ : A Laboratory Manual.
  • U.S. Pat. No. 4,683,202 may also be utilized. It is necessary to either remove or inactivate the unincorporated nucleotides and primers of the amplification reaction. Non-labeled, unincorporated nucleotides will allow primer extension to occur beyond the nucleotide of interest. Similarly, amplification primers that are free in solution can be extended and provide incorporation at positions other than the position of interest. Various methods obvious to those skilled in the art of molecular biology are available for removing unincorporated nucleotides and primers. However, since we desire a method that is rapid and automatable, the preferred form of separation is one utilizing attachment of the amplified nucleic acid product to a solid support with subsequent washing steps. An avidin-biotin system is preferred.
  • the template may be RNA or DNA, and may be double or single stranded. If double stranded, it is necessary to denature the strands to allow hybridization between the template strand and the oligonucleotide probe. Methods for this denaturation and subsequent hybridization step are well known to those skilled in the art of sequencing. However, since it is well known that formation of the hybrid between the oligonucleotide probe and the nucleic acid strand containing the target nucleic acid sequence can be inhibited by the complementary, non-template strand, the preferred method is to physically separate the template and the non- template strand after a denaturation step.
  • the template strand can either be the strand present on the solid support ( Figure 2) , or a strand that is free in solution ( Figure 3) .
  • the probe that has formed a duplex (hybrid) within the template is then subjected to enzymatic primer extension with enzyme such as primer-dependent DNA
  • 4,683,202 and include definition of a primer, size of primers, preparation of oligonucleotide primers, methods for separating strands of double stranded .nucleic acid, preferable ratio of primer to template, conditions for mixing and annealing primer to template strand, and conditions for extending the primer in a 5' to 3' direction.
  • the enzyme used in the primer extension reaction should not exhibit exonuclease activity upon the components of the reaction.
  • the enzyme should be free of 3' to 5' exonuclease activity or the probe should be of such composition as to resist such a degradation activity. Examples of this patent were performed under the former condition.
  • Double stranded nucleic acid targets can be used to generate both the template and primer strands, thereby eliminating the primer-template annealing step.
  • molecules can be produced that have a recessed 3' strand and an overhanging 5' strand and thus are substrates for nucleotide addition by a DNA polymerase.
  • cleavage of DNA with many restriction enzymes generates 5' overhangs that are substrates for DNA polymerases.
  • there are 3' exonucleases that remove 3" nucleotides from double-stranded DNA producing molecules with 3 1 recessed strands and 5* overhanging strands.
  • the hybridizing and extending steps can be performed in solution or in solid phase reactions .
  • the detection can also be in solution, after attachment to a solid phase, or after passing through a gel such as acrylamide or agarose.
  • the first two methods are preferred for they avoid the time- consuming gel assay. Without a gel assay, it is necessary to separate the unincorporated labeled chain- terminators after the elongation step. Note that in the present invention, it is not necessary to wash away the excess oligonucleotide probe that did not hybridize, since the unextended probe does not contain a label.
  • Form (1) is the preferred method for it offers improved specificity and signal concentration in that a binding group can be captured specifically by a solid phase material.
  • a pendant biotin or biotin-dUTP incorporated into the probe can be specifically captured by avidin-coated materials such as avidin-agarose, avidin-coated magnetic beads, or avidin- coated microtiter wells.
  • avidin-coated materials such as avidin-agarose, avidin-coated magnetic beads, or avidin- coated microtiter wells.
  • Another example might be the use of an oligonucleotide probe with a 5'-extension that is nonhomologous to the target sequence.
  • This portion of the probe can then be used to capture the elongated probe (or hybrid) to a solid support that contains the complementary sequence.
  • elongated probe or hybrid can be captured specifically and in high concentration on the solid phase, with the major other material captured (unhybridized probe) not causing non ⁇ specific signal.
  • Form (3) is also superior to many conventional probe assays where the probe is labeled before the elongation step, since separating a labeled oligonucleotide probe from a labeled, but short primer-elongation product is more difficult than separating the same labeled, primer- elongation product from the unincorporated, labeled nucleotide substrates.
  • detection can proceed in a conventional fashion, either on the solid phase or otherwise. It should be apparent that the binding system used in forms (1) and (2) of the present method should be independant of the binding system used to attach detectable label to the modified nucleotides during the detection step.
  • Detectably labeled does not mean that the detectable signal must be present at the time of incorporation.
  • the fluorescent substrates described below require activation.
  • Detectably labeled does not necessarily mean that the nucleotide substrates carry a reporter such that there is not only the ability to detect the label, but also to identify the nucleotide. If only one nucleotide is present in the reaction, then detection of incorporation is sufficient for identification.
  • Prober et. al EP-A 252683
  • DyeDeoxy terminators a trademark of Applied Biosystems, Inc., Foster City, California
  • each of the modified nucleotides have a similar mobility shift when run on a sequencing gel.
  • four chain-terminating nucleotides that are distinguishably labeled are present in each reaction. The need for four different labels is eliminated if the number of reactions per sample are increased ( Figure 6) .
  • chain-terminating nucleotides may be present in the initial reaction, but only one must be detectably labeled.
  • the chain-elongating dNTP substrates are not a component of the reaction of the present invention.
  • the chain-terminating nucleotides described in the present invention are labeled with a fluorescent signal generator (reporter) .
  • a suitable fluorescent reporter is one that can be detected in its unprotected form at or below the level of detection that can be quickly achieved with 32 P, i.e., about 10 ⁇ 14 moles.
  • Specific desirable characteristics may include a large coefficient of extinction in the region of excitation, a high quantum yield, an optimal excitation or emission wavelength (preferably above 350 nm) , and photostability.
  • fluorescent dyes that are efficiently excited by an argon laser are desirable because of the low cost of this laser.
  • the reporter is a fluroescent dye chosen from the group consisting of xanthenes (e.g., fluoresceins, eosins, erythrosins) , rhodamines (e.g., tetramethylrhodamine, Texas Red®), benzamidizoles, ethidiums, propidiums, anthracyclines, mithramycins, acridines, actino ycins, merocyanines, coumarins (e.g., 4-methyl-7-methoxycoumarin) , pyrenes, chrysenes, stilbenes, anthracenes, naphthalenes (e.g., dansyl, 5-dimethylamino-l-naphthalenesulfonyl) , salicyclic acids, benz-2-oxa-l-diazoles (also known as benza fluroescent dye chosen from the group
  • an analyte strand contains a nucleotide position of interest (N) , the identity of which is to be determined by the assay, is defined as the first base of the analyte nucleic acid strand which is beyond the 5' end of the target nucleotide sequence in the 3' to 5' direction.
  • a probe polynucleotide is produced as a reagent having a binding region complementary to the target nucleotide sequence (TNS) .
  • the probe polynucleotide consists only of that complementary sequence; in other embodiments, the probe is extended in the 5' direction in a manner that does not interfere with the recognition and complementary base pairing to the target nucleotide sequence.
  • the diagram in Figure la illustrates the double stranded nucleic acid region which forms when the probe binds to analyte strand An by complementary base pairing to the target nucleotide sequence TNS.
  • the 3' end of the probe will be utilized as a primer and elongated opposite the analyte strand An which serves as a template for nucleotide incorporation.
  • the nucleotide incorporated (N*) will be complementary to the nucleotide position of interest (N) .
  • the * symbol is used to illustrate a detectable label attached to the nucleotide.
  • the enzyme, primer and nucleic acid analyte are chosen together such that a nucleotide complementary to the target nucleotide of interest is incorporated.
  • a reverse transcriptase e.g. the Klenow fragment of £. poll DNA Polymerase I or TAQ polymerase
  • a eukaryotic DNA polymerase e.g. the Klenow fragment of £. poll DNA Polymerase I or TAQ polymerase
  • a eukaryotic DNA polymerase may be used, with the probe being DNA or RNA.
  • Figures lc-e are examples to illustrate the more schematic drawings of Figures la&b.
  • the target nucleotide of interest is a T.
  • the complementary nucleotide. A* will be covalently attached to the primer ( Figure Id) .
  • the nucleotide of interest was a C, then the complementary nucleotide that is incorporated will be a G* ( Figure le) .
  • the nucleic acid sample being analyzed contains molecules of several types, then several different nucleotides may be incorporated and covalently attached to the primer (e.g.
  • Figures lf-h are very similar to Figures lc-e except that the opposite nucleic acid strand is utilized as template thus the oligonucleotide probe is chosen to correspond to a different TNS.
  • Figures 2 & 3 illustrate in schematic form the sequence of events that comprise preferred embodiments for carrying out the present invention.
  • the immobilized strand is used as the template (An)
  • the eluted or non-immobilized strand is used as template.
  • the polymerase chain reaction PCR
  • the removal of the unincorporated nucleotides and primers is essential, and can be performed by binding the double stranded PCR product to a solid support, e.g. by a biotin (B) - streptavidin complex, and rinsing away the unbound material.
  • the two strands are then denatured (e.g.
  • the immobilized template strand is.rinsed, while in Figure 3 the soluble, eluted strand is used as template after neutralizing the NaOH solution.
  • the probe oligonucleotide is then hybridized to the template strand and the hybridized probe is elongated by addition of a single, chain terminating nucleotide.
  • the enzyme utilized in the reaction is a DNA polymerase such as reverse transcriptase and all four chain terminating nucleotides may be present, although only one must be detectably labeled. The unincorporated nucleotides are removed from the reaction by washing.
  • the template strand was not previously immobilized, so the probe oligonucleotide can now be captured onto solid support for efficient washing.
  • the nature of the label present on the elongated primer may be measured directly after efficient removal of the unincorporated substrate. That is, the primer may still be bound to the solid support, either directly as shown in Figure 3 or indirectly through the hybrid formed with the analyte strand ( Figure 2 without the final denaturation step) .
  • the labeled primer is released from the beads after heating in the presence of formamide and EDTA. The magnetic beads do not interfere with standard gel electrophoresis although they are loaded into the sample well along with the sample.
  • Figure 4 illustrates that the nucleotide of interest (N) can even be located at the end of a nucleic acid strand. This is different from that of multiple nucleotides during the reaction.
  • Figure 5 illustrates that the assay is useful for detecting insertions and deletions as well as the point mutations illustrated in Figures lc-h.
  • two template nucleic acid strands are drawn, with the upper strand differing from the lower strand by the presence of two T's (note that one strand may be considered to have a deletion, or the other strand may be considered to have an insertion) .
  • Figures 5b&c illustrate one possible choice of probe and the resulting difference in the nucleotide incorporated when the two different strands are used as the template, An.
  • Figure 6 illustrates how the number of assays must increase if all four chain terminating nucleotides are not detectably labeled and distinguishable one from another. The number of reactions required to identify the nucleotide of interest in a given.sample is dependent upon how many of the different, possible substrates are detectably labeled and distinguishable.
  • nucleotide substrates are present in reaction and all four are detectably labeled and distinguishable from each other (e.g., ddATP*, ddTTP*, ddCTP*, ddGTP* are provided as substrate)
  • ddATP*, ddTTP*, ddCTP*, ddGTP* are provided as substrate
  • Method 3 All possible nucleotide substrates are present in each reaction but perhaps only one label is available for substrate labeling (e.g., the same as when radioactively labeled ddNTP's are utilized).
  • 1st Reaction provide substrates ddATP*, ddGTP, ddCTP, ddTTP.
  • 2nd Reaction provide substrates ddATP*, ddGTP*, ddCTP, ddTTP.
  • 3rd Reaction provide substrates ddATP, ddGTP, ddCTP, ddTTP.
  • nucleotide position of interest is that of the lower strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene as described by publication in the GenBank database, accession M12130 for the locus RO:Musrpolii2.
  • a 602 nucleotide portion of this sequence from nucleotide 4915 to 5517 is illustrated in its double stranded form in Figure 8, with the nucleotide position of interest for this example being at position 5430 on the lower strand (occupied by a bold-faced T in the sequence of the Wildtype allele which is shown in this Figure 8) .
  • the target nucleotide sequence is chosen as the 21 nucleotide sequence (3'TAACGACAACAGCCCGTCGTC5') that immediately flanks the nucleotide of interest such that the nucleotide position of interest is the next contiguous nucleotide in the 3' to 5' direction on that nucleic acid strand (see Figure lc) .
  • the oligonucleotide probe consisted of the 21 nucleotide sequence 5 1 ATTGCTGTTGTCGGGCAGCAG 3' (probe A of Figures 8 and 9), and is perfectly complementary to the target nucleotide sequence defined above. It is synthesized on an Applied Biosystems DNA synthesizer and further purified by HPLC to consist of a single oligonucleotide species, 21 nucleotides in length. (methods as described in Oligonucleotide Synthesis, A Practical Approach ed. M.J. Gait, IRL Press 1984) .
  • the claimed method will be illustrated using three different amplified DNAs in three separate, but similar reactions.
  • the three starting biological materials used for the amplification process are each known to contain the mouse RNA polymerase II gene.
  • the three samples are designated Wildtype, Mutant, and Heterozygote. They are known to differ at nucleotide position 5430 (as shown in bold faced type in Figure 9), with the Wildtype allele containing an A-T base pair, the Mutant allele containing a G-C base pair, and the Heterozygous sample containing an equal mixture of these two alleles.
  • the starting biological materials are obtained from J. Corden and are as described in Bartolomei and Corden, Molec. and Cell. Biol.
  • the Wildtype and Mutant alleles are provided as bacterial strains containing the recombinant plasmids pE26-4 and pE26-7 respectively.
  • the biological sample designated as Heterozygous is obtained as a cell line A21.
  • DNA of each of the recombinant plasmids is prepared by standard molecular biology procedures (described in Sambrook et al.. Molecular Cloning: A Laboratory Manual 1989), and genomic DNA is prepared from the A21 cell line as described in Corsaro and Pearson, Somatic Cell Genet. 7:603-616, 1981.
  • the target nucleotide sequence and the nucleotide position of interest are within a 602 base pair segment of the RNA polymerase II gene.
  • the copy number of this segment is increased using exponential amplification, using DNA of each of the three biological starting materials described above.
  • the oligonucleotide primers used for PCR amplification of the region of interest in the RNA polymerase II gene are designated PCR amplification Primer 1
  • each primer for PCR amplification, thirty-three picomoles of each primer (one primer biotinylated at the 5' end) is mixed with approximately 1 ug of genomic DNA (or 0.1 ug of plasmid DNA) in a 50 ⁇ l reaction mixture containing
  • the mixture is incubated at 95°C for 2 min to separate the DNA strands and cooled on ice; 2.5 units of TAQ polymerase (AmpliTaq, Perkin Elmer Cetus) is added, and the reaction mixture is overlaid with approximately 35 ⁇ l of mineral oil.
  • TAQ polymerase AmpliTaq, Perkin Elmer Cetus
  • the amplification conditions varied slightly in the course of the experiments, but are usually performed in a Perkin-Elmer/Cetus thermal cycler using an initial cycle consisting of 4 min 94°C, 45 sec 55°C, 5 min 68°C, followed by 35 cycles with the same parameters except the denaturation at 94°C is 1 min.
  • 10-15 ⁇ l aliquots of the amplified fragment are run on a 1.5% agarose gel and visualized by ethidium bromide staining using standard procedures to ensure that an amplified fragment of the expected size is produced. Utilizing these primers and any of the three DNAs described above, a 602 bp PCR amplification product of double stranded DNA is consistently obtained. Aliquots of the remainder of the PCR amplification sample are then utilized in the method of this invention.
  • the double- stranded PCR amplification product contains a biotin moiety due to the biotin originally presnet on PCR amplification Primer 2.
  • the separation is done by binding the biotinylated PCR amplification product to a streptavidin-coated solid support and rinsing away the non-biotinylated, PCR amplification Primer 1 and the unincorporated nucleotides.
  • the tube containing magnetic beads and DNA is placed near a magnet to draw the beads to one side of the tube. After approximately four minutes of magnetization, the supernatant is removed.
  • the beads (with DNA bound) are then washed three times with TE buffer (10 mM Tris pH8, 1 mM EDTA) using magnetization for removal of the supernatant which contains dNTP's and non-biotinylated PCR amplification primer. Care is taken that the beads did not dry between washes. 5. After the final wash, 16 ⁇ l of sterile distilled water is added to the bead-bound DNA.
  • the double-stranded DNA is denatured by addition of 4 ⁇ l of 0.5M NaOH, 2 mM EDTA solution, and incubated at room temperature for 5 min. Afterwards, the sample is magnetized and the supernatant removed. (The supernatant may be kept if the non-bead bound strand is to be used as template - e.g. Example 5) .
  • the bead- bound DNA pellet is gently resuspended in 100 ⁇ l TE buffer to neutralize any NaOH remaining.
  • Formation of the anal ⁇ f.e-probe hybrid and enzvmatic extension of the probe with a chain terminating nucleotide complementary to the nucleotide position of interest 7.
  • the TE buffer is removed following magnetization, and 7 ⁇ l of the following solution is added: 2 ⁇ l sterile water 1 ⁇ l 125 ⁇ M ddTTP (unlabeled) 1 ⁇ l 125 ⁇ M ddCTP (unlabeled)
  • the labeling reaction is at 42°C for 10 minutes, and then the reaction is placed on ice and 100 ⁇ l of TE is added. 11. The sample is again magnetized for 4 minutes and the supernatant removed, followed by 3 washes of 100 ⁇ l TE buffer (magnetization between each wash) to remove unincorporated nucleotides.
  • the sample from step 12 is diluted 1:16 fold further in FE containing crystal violet, for easier visualization in loading the sample and to get the sample in a reasonable concentration for detection by slab gel electrophoresis on the Genesis 2000 DNA analysis system (methods as described by the instrument documentation, with a few parameters described in more detail below) .
  • control primer is added as a mobility standard, but is later found to be an unnecessary component and is omitted in later electrophoresis runs.
  • the first three lanes shown in Figure 10a are results from running such samples from each of the three reactions.
  • the position of the peak corresponding to the elongated probe is designated WT, Mutant, and Het for the three reactions of this example.
  • the peak at position S corresponds to the control primer that is added at the time of electrophoresis.
  • the fluorescently labeled chain terminators used in the examples of this patent are either purchased from duPont NEN Biotechnology Systems (Boston, MA) , or obtained as a kind gift from Dr. Douglas Amorese of that firm. The nature and detection of these SF-ddNTPs are described in Prober et al. (1988) Science 238, 336-341.
  • the chain terminators are distinguished by a ratio of the measured fluorescence from two photomultiplier tubes (PMT) . Each PMT value is displayed as either a red or green mark on the output computer monitor, with a sample forming a peak as it passes by the excitory laser.
  • Figure 7b An example of the type of data collected using standard gel electrophoresis methods on the Genesis is shown in Figure 7b for SF-ddGTP-505 and SF-ddTTP-526.
  • Figure 7c illustrates the type of measurement made when the fluorescent substrate SF-ddGTP-505 or SF-ddCTP-519 is loaded via syringe into the capillary mounted onto the Genesis 2000 detection system. The multiple peaks represent the same sample being pushed several times in front of the laser beam.
  • the green/red ratio for a particular fluorescent substrate is different from that of Figure 7b, the nucleotides can be distinguished in this new detection system at concentrations similar to that of gel electrophoresis as illustrated in Figure 7a.
  • the PMT ratio green/red peak height
  • the three reaction samples of this example i.e. the WT, Mutant, and Het peak
  • the WT, Mutant, and Het peak are rerun at lower dilution (since the voltage of two of them are originally too high as shown in Figure 10a) .
  • the resulting sample peaks are displayed in Figure 10b with a smaller display window for easier measurement.
  • the measured green/red ratios are as follows:
  • nucleotide of interest as the nucleotide complementary to the chain terminating nucleotide which is added:
  • the nucleotide at the position of interest is the nucleotide complementary to the nucleotide that is incorporated.
  • the reaction performed on the Wildtype allele indicates that it does contain a thymidine (T) on the lower strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene, for the nucleotide incorporated is SF-ddATP-512.
  • the reaction performed on the Mutant allele indicates that it does contain a cytosine (C) on the lower strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene, for the nucleotide incorporated is SF-ddGTP-505.
  • the reaction performed on DNA originating from the A21 cell line contains an equal number of thymidine (T) and cytosine (C) residues at the position of interest, for an approximately equal number of SF-ddATP-512 and SF-ddGTP- 505 are incorporated onto the probe.
  • the nucleotide position of interest is that of the upper strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene as described by'publication in the GenBank database, accession M12130 for the locus RO:Musrpolii2.
  • a 602 nucleotide portion of this sequence from nucleotide 4915 to 5517 is illustrated in its double stranded form in Figure 8, with the nucleotide position of interest for this example being at position 5430 on the upper strand (occupied by a bold-faceted A in the sequence of the Wildtype allele which is shown in this Figure 8) .
  • the target nucleotide sequence is chosen as the 21 nucleotide sequence (5 ⁇ TGTAGAGGGCAAGCGGATCC3 1 ) that immediately flanks the nucleotide of interest such that the nucleotide position of interest is the next contiguous nucleotide in the 3' to 5' direction on that nucleic acid strand (see also Figure If) .
  • the oligonucleotide probe consisted of the 21 nucleotide sequence 5 1 GGATCCGCTTGCCCTCTACAT 3' (probe B of Figures 8 and 9), and is perfectly complementary to the target nucleotide sequence defined above. Synthesis and purification is as described in Example 1.
  • Example 1 Starting biological sample: In this example, the claimed method will be illustrated using two of the same starting biological samples as described in Example 1: that of the Wildtype and Mutant. They are prepared as described in Example 1.
  • the region of interest is amplified from the Wildtype and Mutant samples using methods as described in Example 1 with PCR amplification Primer 1 and PCR amplification Primer 2, except in this Example 2, the PCR amplification Primer 1 is biotinylated at the 5' end and the PCR amplification Primer 2 is not.
  • Preparation of the analvte -strand is described in Example 1 with PCR amplification Primer 1 and PCR amplification Primer 2, except in this Example 2, the PCR amplification Primer 1 is biotinylated at the 5' end and the PCR amplification Primer 2 is not.
  • Example 1 steps 1-6 the complementary strand which is bound to the solid support, for this strand contains the biotin from the PCR amplification reaction.
  • the invention is practiced as in the steps of Example 1 on the Wildtype and Mutant analyte strands with the following exceptions: a) The two unlabeled nucleotide substrates in step 7 are ddGTP and ddATP . b) The two fluorescently labeled nucleotide substrates in step 9 are 1 ⁇ l of 30 uM SF-ddCTP-519 and 1 ⁇ l of 125 ⁇ M SF-ddTTP-526. The results shown in Figure 11 illustrate that the Wildtype and Mutant allele have green/red ratios of 0.35 and 0.7 respectively when probe B is used. The calibration graph of Figure 7a shows that this corresponds to the incorporation of SF-ddTTP-526 and SF-ddCTP-519 for the Wildtype and Mutant allele.
  • THE CONCLUSION for the two samples of this example are as expected (see Figure 9) :
  • the reaction performed on the Wildtype allele indicates that it does contain a adenine (A) on the upper strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene, for the nucleotide incorporated is SF-ddTTP-526.
  • the reaction performed on the Mutant allele indicates that it does contain a guanine (G) on the upper strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene, for the nucleotide incorporated is SF-ddCTP-519.
  • EXAMP E 3 Aim The reaction performed on the Wildtype allele indicates that it does contain a adenine (A) on the upper strand at nucleotide position 5430 of the mouse RNA polymerase II largest subunit gene, for the nucleotide incorporated is SF-ddTTP-526.
  • the lower panel of Figure 12 illustrates a significant level of misincorporation of SF-ddTTP-526 as a complementary base for the guanine (G) present on the Mutant analyte strand for the primer extension of probe B (refer to Figure 9) .
  • EXAMPLE 4 Aims: 1. To illustrate that the use of all four detectably labeled nucleotides in a single reaction gives essentially the same PMT green/red ratio as compared to Example 2 when the correct base is present. 2. To illustrate that a lowering of the concentration of ddNTP substrate by 5 fold may improve the ability to rinse away unincorporated ddNTP's without affecting the ability to measure the sample.
  • Example 2 except the unlabeled nucleotides are omitted from step 7 and all four ddNTPs are present in step 9 at 1/5 the concentration.
  • the results shown for Mutant and Wildtype amplified alleles are shown in the two panels of Figure 12.
  • In these lanes as in other lanes of the experiment, there is essentially no peak of unincorporated nucleotides present in the sample.
  • nucleic acid strand that is not bound to a solid support as the analyte strand.
  • nucleotide position of interest is that of the lower strand, occupied by a circled G on Figure 14.
  • the nucleotide sequence shown is a portion of the Wildtype AJL. gene of maize (Schwarz-Sommer et al., EMBO J. 6:287-294 (1987) .
  • the target nucleotide sequence is chosen as the 21 nucleotide sequence (3'GACGAACTCCTAGCTCATCAC5') that immediately flanks the nucleotide of interest such that the nucleotide position of interest is the next contiguous nucleotide in the 3' to 5' direction on that nucleic acid strand.
  • the oligonucleotide probe consists of the 21 nucleotide sequence 5' CTGCTTGAGGATCGAGTAGTG 3* (Primer C of Figure 14), and is perfectly complementary to the target nucleotide sequence defined above.
  • Primer C is biotinylated at the 5' end during primer synthesis and is HPLC purified by methods described in Example 1.
  • PCR amplification primers A & B homologous to a section of the maize Al gene are designed, synthesized, and used to amplify the genomic fragment from total maize DNA containing the mutant a-dt allele of maize by methods described in Example 1.
  • the Primer B is the PCR amplification primer containing biotin at the 5* end.
  • Preparation of the analyte strand is performed as in steps 1-6 of Example 1, except that the non-bead bound strand from step 6 will be used as the analyte strand below.
  • the sample is incubated 42°C 10 minutes.
  • Dynabeads prepared as in step 1 are added and followed by a 37°C incubation for 15 minutes with intermitent shaking. This is to promote binding of the nested, biotinylated primer (containing fluorescent label from the primer extension reaction) .
  • the sample is magnetized for 4 minutes and unincorporated nucleotides present in the supernatant are removed.
  • the final bead pellet is washed 3 times with 100 ⁇ l TE (magnetizing each time to remove the buffer) .
  • the final bead pellet is resuspended in 5 ⁇ l of
  • EXAMPLE 6 Aim: 1. To illustrate the use of another primer dependent DNA polymerase in practicing this invention.
  • the method of this invention is capable of distinguishing a heterozygous DNA sample with one reaction (in this case, an equal number of cytosine and guanine nucleotides at the nucleotide position of interest) .
  • Genomic DNA is prepared from maize leaf material as described in Example 5, but the exact nature of the nucleotide of interest in the sample is unknown until after the method of this invention is performed. (Standard DNA sequence analysis of this region of the DNA later confirmed that the biological material is heterozygous at the nucleotide position of interest with both cytosine (C) and guanine (G) being present in essentially equal amounts (data not shown) .
  • step 7 the following are the additions made to the neutralized, non-bead bound strand: 8.0 ⁇ l of 5X Sequenase buffer (200 mM Tris pH 7,
  • step 9 1.5 ⁇ l of 100 mM Dithiothreitol is added (in addition to the fluorescent substrates SF- ddCTP-519 and SF-ddGTP-505) , and 1 ⁇ l of 13 units/ ⁇ l
  • step 10 the labeling reaction is at 37°C for 10 minutes.
  • Example 6 The results of Example 6 are shown in Figure 16. It is seen that a single peak of incorporation appears, suggesting that the Sequenase II enzyme can also be used for the practice of this invention with no significant 3'-5' exonuclease activity.
  • Example 6 it is determined that the nucleotide position of interest in the sample is occupied by an approximately equal number of cytosine (C) and guanine (G) residues.
  • EXAMPLE 7 This example illustrates identification of a nucleotide of interest at a defined location in samples of a 700 bp DNA element, Cinl, from the analysis is carried out in a single reaction Northern Flint Line of Zea mays in a single reaction.
  • the Cinl element is repetitive in Zea mays.
  • the oligonucletodie probe is a 21 nucleotide sequence perfectly complementary to the target nucleotide sequence.
  • the target nucleotide sequence is a 21 nucleotide sequence that immediately flanks the nucleotide of interest at position 500. Synthesis of these sequences is as shown in Example 1.
  • the region of interest containing the nucleotide of interest on each Cinl template is amplified as in Example 1 using amplification primers 1 and 2.
  • Primer 1 is biotinylated at the 5' end.
  • reaction buffer contains:
  • the labeling reaction takes place at 42°C for 10 minutes, is placed on ice and 100 ⁇ l of TE buffer is added.
  • the chain-terminating nucleotide which extended the probe at the position complementary to the nucleotide of interest is detected and determined as shown in Example 1.
  • the nucleotide of interest at position 500 is identified as the nucleotide complementary to the chain- terminating nucleotide which extended the probe in the labeling reaction.
  • the presence and nature of a polymorphism can be determined by comparing the samples tested.
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO:16:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • xi SEQUENCE DESCRIPTION: SEQ ID NO: 19:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
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  • xi SEQUENCE DESCRIPTION: SEQ ID NO:22:
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • CTCGGCTCTC AGGACCATAA TCATCCTTGA TAAAGTGAGG AAGAGTCCGA TGCTTGAATC 60
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)
  • MOLECULE TYPE DNA (genomic)

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Abstract

On décrit un procédé d'identification d'un nucléotide se trouvant en un point défini sur une séquence d'acide nucléique. Une sonde d'oligonucléotide est fusionnée à une séquence nucléotidique cible d'un échantillon d'acide nucléique en un point immédiatement adjacent et à l'extrémité 3' par rapport au nucléotide à étudier. La sonde est alors étendue dans la direction du nucléotide à étudier dans un milieu de réaction contenant au moins un triphosphate nucléotidique de terminaison de chaîne (ATP, GTP, TTP, et CTP). Le nucléotide à étudier est complémentaire au nucléotide marqué incorporé dans l'amorce par la réaction d'extension.
PCT/US1992/001691 1991-03-13 1992-03-12 Procede d'identification d'un nucleotide present dans un acide nucleique en une position definie WO1992016657A1 (fr)

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