Classifications

• • 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
  • C12Q1/6813—Hybridisation assays
  • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism

Definitions

• the present invention is in the field of recombinant DNA technology. More specifically, the invention is directed to molecules and methods suitable for identifying one or more single nucleotide polymorphisms in a single reaction in the genome of a plant, animal, or microorganism, and using such sites to analyze identity, ancestry or genetic traits.
• the capacity to genotype an animal, plant or microbe is of fundamental importance to forensic science, medicine and epidemiology and public health, and to the breeding and exhibition of animals. Such a capacity is needed, for example, to determine the identity of the causative agent of an infectious disease, to determine whether two individuals are related, or to map genes within an organism's genome.
• RFLPs restriction fragment length polymorphisms
• the DNA sequence variations are in regions of the genome that are characterized by short tandem repeats ("STRs") that include tandem di- or tri-nucleotide repeated motifs of nucleotides. These tandem repeats are also referred to as "variable number tandem repeat" (“VNTR”) polymorphisms. VNTRs have been used in identity and paternity analysis (Weber, J.L., U.S. Patent 5,075,217; Armour, J.A.L. et al. FEBS Lett. 307:113- 115 (1992); Jones, L. et al. Eur. T. Haematol. 39:144-147 (1987); Horn, G.T. et al..
• a third class of DNA sequence variation results from single nucleotide polymorphisms ("SNPs") that exist between individuals of the same species.
• SNPs single nucleotide polymorphisms
• Such polymorphisms are far more frequent than STRs and NNTRs.
• such polymorphisms comprise mutations that are the determinative characteristic in a genetic disease. Indeed, such mutations may affect a single nucleotide in a protein-encoding gene in a manner sufficient to actually cause the disease (i.e. hemophilia, sickle-cell anemia, etc.).
• these SNPs are in noncoding regions of a genome.
• the present invention provides such an improved method. Indeed, the present invention provides methods and gene sequences that permit the genetic analysis of identity and parentage, and the diagnosis of disease by discerning the variation of multiple single nucleotide polymorphisms.
• the present invention is directed to molecules that comprise single nucleotide polymorphisms (SNPs) that are present in all life forms.
• SNPs single nucleotide polymorphisms
• the invention is directed to methods for (i) identifying one or more novel single nucleotide polymorphisms (ii) methods for the repeated analysis and testing of these SNPs in different samples and (iii) methods for exploiting the existence of such sites in the genetic analysis of animals, plants, and microbes.
• the invention provides one or more interrogation nucleic acid (or nucleic acid analog) primer molecules having a polynucleotide sequence complementary to one or more nucleotide sequences of a genomic DNA segment of any organism, the genomic segment being located immediately 3'-distal to a single nucleotide polymorphic site, X, of a single nucleotide polymorphic allele of the mammal; and wherein template-dependent extension of the nucleic acid (or nucleic acid analog) primer molecule by a single nucleotide (or nucleotide analog) extends the primer molecule by a single nucleotide, (or analog) the single nucleotide (or analog) being complementary to the nucleotide, X, of the single nucleotide polymorphic allele.
• the invention concerns an embodiment wherein the template-dependent extension of the primer is conducted in the presence of one or more dideoxynucleotide triphosphate derivatives (or analogs) selected from the group consisting of ddATP, ddTTP, ddCTP and ddGTP (or other chain terminating base analogs), but in the absence of dATP, dTTP, dCTP and dGTP.
• one or more dideoxynucleotide triphosphate derivatives selected from the group consisting of ddATP, ddTTP, ddCTP and ddGTP (or other chain terminating base analogs)
• the invention further provides a method for identifying one or more single nucleotide polymorphic sites in a single reaction which comprises the steps: (A) hybridizing one or more of distinguishable interrogation oligonucleotide (or oligonucleotide analog) primers to one or more target nucleic acid molecules wherein each oligonucleotide primer is complementary to a specific and unique region of each target nucleic acid molecule such that the 3' end of each primer is immediately proximal to a specific and unique target nucleotide of interest;
• each interrogation oligonucleotide (or analog) with a template-dependent polymerase wherein said extension occurs in the presence of one or more non-extendible nucleotide (or nucleotide analog) species;
• each nucleotide (or analog) of interest by determining, for each interrogation primer employed, the identity of the non-extendible nucleotide (or nucleotide analog) incorporated into such primer, said identified non-extendible nucleotide (or nucleotide analog) being complementary to said primer's target nucleotide;
• Figure 1 illustrates the preferred method for cloning random genomic fragments. Genomic DNA is size fractionated, and then introduced into a plasmid vector, in order to obtain random clones. PCR primers are designed, and used to sequence the inserted genomic sequences.
• Figure 2 illustrates the data generated by the preferred method for identifying new polymorphic sequences which is cycle sequencing of a random genomic fragment.
• Figure 3 illustrates the RFLP method for screening random clones for polymorphic sequences.
• Figure 4 shows a graph of the probability that two individuals will have identical genotypes with given panels of genetic markers.
• Figure 5 shows a graph of the probability that given panels of 20 genetic markers will exclude a random alleged father in a paternity suit in which the mother is not in question.
• Figure 6 illustrates the preferred method for genotyping SNPs. The seven steps illustrate how GBA can be performed starting with a biological sample.
• the particular gene sequences of interest to the present invention comprise "single nucleotide polymorphisms.”
• a "polymorphism” is a variation in the DNA sequence of some members of a species. The genomes of animals and plants naturally undergo spontaneous mutation in the course of their continuing evolution (Gusella, J.F., Ann. Rev. Biochem. 55:831-854 (1986)). The majority of such mutations create polymorphisms. The mutated sequence and the initial sequence co-exist in the species' population. In some instances, such co-existence is in stable or quasi-stable equilibrium. In other instances, the mutation confers a survival or evolutionary advantage to the species, and accordingly, it may eventually (i.e. over evolutionary time) be incorporated into the genome of every member of that species.
• a polymorphism is thus said to be "allelic,” in that, due to the existence of the polymorphism, some members of a species may have the unmutated sequence (i.e., the original "allele") whereas other members may have a mutated sequence (i.e., the variant or mutant "allele"). In the simplest case, only one mutated sequence may exist, and the polymorphism is said to be diallelic. The occurrence of alternative mutations can give rise to triallelic polymorphisms, etc. An allele may be referred to by the nucleotide(s) that comprise the mutation.
• the present invention is directed to a particular class of allelic polymorphisms, and to their use in genotyping plants, animals, or microbes.
• allelic polymorphisms are referred to herein as "single nucleotide polymorphisms," or “SNPs.”
• Single nucleotide polymorphisms are defined by the following attributes.
• a central attribute of such a polymorphism is that it contains a polymorphic site, "X,” which is the site of variation between allelic sequences.
• a second characteristic of a SNP is that its polymorphic site "X" is frequently preceded by and followed by "invariant" sequences of the allele.
• the polymorphic site of the SNP is thus said to lie “immediately” 3' to a "5'-proximal” invariant sequence, and “immediately” 5' to a "3'-distal” invariant sequence. Such sequences flank the polymorphic site.
• the term "single" of single nucleotide polymorphisms refers to the number of nucleotides of the polymorphism (i.e. one nucleotide); it is unrelated to the number of polymorphisms present in the target DNA (which may range from one to many).
• a sequence is said to be an "invariant" sequence of an allele if the sequence does not vary in the population of the species, and if mapped, would map to a "corresponding" sequence of the same allele in the genome of every member of the species population. It should be noted that two or more SNP's may be very close in proximity to each other. Two sequences are said to be “corresponding” sequences if they are analogs of one another obtained from different sources. The gene sequences that encode hemoglobin in two humans illustrate “corresponding" allelic sequences. The definition of "corresponding alleles” provided herein is intended to clarify, but not to alter, the meaning of that term as understood by those of ordinary skill in the art.
• Each row of Table 1 shows the identity of the nucleotide of the polymorphic site of "corresponding” equine alleles, as well as the invariant 5'-proximal and 3'-distal sequences that are also attributes of that SNP.
• "Corresponding alleles” are illustrated in Table 2 with regard to human alleles.
• Each row of Table 2 shows the identity of the nucleotide of the polymorphic site of "corresponding” human alleles, as well as the invariant 5'-proximal and 3'-distal sequences that are also attributes of that SNP.
• each SNP can be defined in terms of either the plus strand or the minus strand. Thus, for every SNP, one strand will contain an immediately 5'-proximal invariant sequence and the other strand will contain an immediately 3'-distal invariant sequence.
• each SNP's polymorphic site, "X" is a single nucleotide
• each strand of the double-stranded DNA of the SNP will contain both an immediately 5'-proximal invariant sequence and an immediately 3'-distal invariant sequence.
• SNPs of the present invention involve a substitution of one nucleotide for another at the SNP's polymorphic site
• SNPs can also be more complex, and may comprise a deletion of a nucleotide from, or an insertion of a nucleotide into, one of two corresponding sequences.
• a particular gene sequence may contain an A in a particular polymorphic site in some animals, whereas in other animals a single or multiple base deletion might be present at that site.
• the preferred SNPs of the present invention have both an invariant proximal sequence and invariant distal sequence, SNPs may have only an invariant proximal or only an invariant distal sequence.
• Nucleic acid molecules having a sequence complementary to that of an immediately 3'-distal invariant sequence of a SNP can, if extended in a "template-dependent” manner, form an extension product that would contain the SNP's polymorphic site.
• a preferred example of such a nucleic acid molecule is a nucleic acid molecule whose sequence is the same as that of a 5'-proximal invariant sequence of the SNP.
• “Template-dependent” extension refers to the capacity of a polymerase to mediate the extension of a primer such that the extended sequence is complementary to the sequence of a nucleic acid template.
• a “primer” is a single-stranded oligonucleotide (or oligonucleotide analog) or a single-stranded polynucleotide (or polynucleotide analog) that is capable of being extended by the covalent addition of a nucleotide (or nucleotide analog) in a "template-dependent" extension reaction.
• the primer In order to possess such a capability, the primer must have a 3'-hydroxyl (or other chemical group suitable for polymerase mediated extension) terminus, and be hybridized to a second nucleic acid molecule (i.e. the "template").
• a primer is composed of: (1) a unique sequence of 8 bases or longer complementary to a specific region of the target molecule such that the 3' end of the primer is immediately proximal to a target nucleotide of interests, and (2) a 5' tail composed of a neutral component of a specific and unique length, physical, or chemical characteristic.
• the complementary region of the primer is about 20 bases, however, primers of shorter or greater length may suffice.
• the complementary region of the primer is from about 12 bases to about 20 bases.
• the neutral component of the 5' tail is any non-specific, non- hybridizing polymer or chemical group such as polyT, abasic residues, etc.
• a “polymerase” is an enzyme that is capable of incorporating nucleoside triphosphates (or appropriate analog) to extend a 3'-hydroxyl group of a nucleic acid molecule, if that molecule has hybridized to a suitable template nucleic acid molecule.
• Polymerase enzymes are discussed in Watson, J.D., In: Molecular Biology of the Gene, 3rd Ed., W.A. Benjamin, Inc., Menlo Park, CA (1977), which reference is incorporated herein by reference, and similar texts.
• Other polymerases such as the large proteolytic fragment of the DNA polymerase I of the bacterium E. coli. commonly known as "Klenow" polymerase, E.
• Nucleic acids having the same sequence as that of the immediately 3' distal invariant sequence of a SNP can be ligated in a template dependent fashion to a primer that has the same sequence as that of the immediately 5' proximal sequence that has been extended by one nucleotide in a template dependent fashion.
• the single nucleotide polymorphic sites of the present invention can be used to analyze the DNA of any plant, animal, or microbe.
• Such sites are suitable for analyzing the genome of mammals, including humans, non- human primates, domestic animals (such as dogs, cats, etc.), farm animals (such as cattle, sheep, etc.) and other economically important animals. They may, however, be used with regard to other types of animals, plants, and microorganisms.
• SNPs have several salient advantages for use in genetic analysis over STRs and VNTRs.
• SNPs occur at greater frequency (approximately 10-100 fold greater), and with greater uniformity than STRs and VNTRs.
• the greater frequency of SNPs means that they can be more readily identified than the other classes of polymorphisms.
• the greater uniformity of their distribution permits the identification of SNPs "nearer" to a particular trait of interest.
• any polymorphism that is linked to the particular locus can be used to predict the probability that an individual will be exhibiting that trait.
• NNTR analysis will be of very limited value. Similarly, if the locus is far from any detectable
• RFLP an RFLP analysis would not be accurate.
• SNPs of the present invention are present approximately once every 300 bases in the mammalian genome, and exhibit uniformity of distribution, a SNP can, statistically, be found within 150 bases of any particular genetic lesion or mutation. Indeed, the particular mutation may itself be an SNP. Thus, where such a locus has been sequenced, the variation in that locus' nucleotide is determinative of the trait in question.
• SNPs are more stable than other classes of polymorphisms.
• Their spontaneous mutation rate is approximately 10 " , approximately 1,000 times less frequent than VNTRs.
• VNTR-type polymorphisms are characterized by high mutation rates.
• SNPs have the further advantage that their allelic frequency can be inferred from the study of relatively few representative samples. These attributes of SNPs permit a much higher degree of genetic resolution of identity, paternity exclusion, and analysis of an animal's predisposition for a particular genetic trait than is possible with either RFLP or VNTR polymorphisms.
• SNPs reflect the highest possible definition of genetic information - nucleotide position and base identity. Despite providing such a high degree of definition, SNPs can be detected more readily than either
• VNTR-type polymorphisms are most easily detected through size fractionation methods that can discern a variation in the number of the repeats.
• RFLPs are most easily detected by size fractionation methods following restriction digestion.
• SNPs can be characterized using any of a variety of methods. Such methods include the direct or indirect sequencing of the site, the use of restriction enzymes where the respective alleles of the site create or destroy a restriction site, the use of allele-specific hybridization probes, the use of antibodies that are specific for the proteins encoded by the different alleles of the polymorphism, or by other biochemical interpretation.
• GAA Genetic Bit Analysis
• GBA is a method of polymorphic site interrogation in which the nucleotide sequence information surrounding the site of variation in a target DNA sequence is used to design an oligonucleotide primer that is complementary to the region immediately adjacent to, but not including, the variable nucleotide in the target DNA.
• the target DNA template is selected from the biological sample and hybridized to the interrogating primer.
• This primer is extended by a single labeled dideoxynucleotide (or analog) using a DNA polymerase in the presence of one or more chain terminating nucleoside triphosphate precursors (or suitable analogs).
• Cohen, D. et al. (PCT Application WO91/02087) describes another related method of genotyping wherein dideoxynucleotides are used to extend a single primer by a single nucleotide in order to determine the sequence at a desired locus.
• Dale et al. (PCT Application WO90/09455) discloses a method for sequencing a "variable site" using a primer in conjunction with a single dideoxynucleotide species. The method of Dale et al., further discloses the use of multiple primers and the use of a separation element.
• PCT Application W095/17676 describes an apparatus for the separation, concentration and detection of such target molecules in a liquid sample.
• Cheeseman, P.C. U.S. Patent No. 5,302,509 describes a related method of determining the sequence of a single stranded DNA molecule. The method of Cheeseman employs fluorescently labeled 3'-blocked nucleotide triphosphates with each base having a different fluorescent label.
• Wallace et al. (PCT Application W089/ 10414) describes multiple PCR procedures which can be used to simultaneously amplify multiple regions of a target by using allele specific primers. By using allele specific primers, amplification can only occur if a particular allele is present in a sample.
• Such deoxynucleotide misincorporation events may be due to the Km of the DNA polymerase for the mispaired deoxy- substrate being comparable, in some sequence contexts, to the relatively poor Km of even a correctly base paired dideoxy- substrate (Kornberg, A., et al.. In: DNA Replication, 2nd Edition, W.H. Freeman and Co., (1992); New York; Tabor, S. et al. Proc. Natl. Acad. Sci. fU.SA.) 26:4076-4080 (1989)). This effect would contribute to the background noise in the polymorphic site interrogation.
• the method of the present invention permits or greatly facilitates the determination of the nucleotides present at multiple SNPs.
• a preferred method for discovering polymorphic sites involves comparative sequencing of genomic DNA fragments from a number of haploid genomes.
• such sequencing is performed by preparing a random genomic library that contains 0.5 - 3 Kb fragments of DNA derived from one member of a species. Sequences of these recombinants are then used to facilitate PCR sequencing of a number of randomly selected individuals of that species at the same genomic loci. From such genomic libraries (typically of approximately 50,000 clones), several hundred (200-500) individual clones are purified, and the sequences of the termini of their inserts are determined. Only a small amount of terminal sequence data (100-200 bases) need be obtained to permit PCR amplification of the cloned region.
• the purpose of the sequencing is to obtain enough sequence information to permit the synthesis of primers suitable for mediating the amplification of the equivalent fragments from genomic DNA samples of other members of the species.
• sequence determinations are performed using cycle sequencing methodology.
• the primers are used to amplify DNA from a panel of randomly selected members of the target species.
• the number of members in the panel determines the lowest frequency of the polymorphisms that are to be isolated. Thus, if six members are evaluated, a polymorphism that exists at a frequency of, for example, 0.01 might not be identified.
• a sampling of six members would be expected to identify only those polymorphisms that occur at a frequency of greater than about 0.08 (i.e. 1.0 total frequency divided by 6 members divided by 2 alleles per genome). Thus, if one desires the identification of less frequent polymorphisms, a greater number of panel members must be evaluated.
• Cycle sequence analysis (Mullis, K. et al.. Cold Spring Harbor Symp. Quant. Biol. 51:263-273 (1986); Erlich H. et al, European Patent Application 50,424; European Patent Application 84,796, European Patent Application 258,017, European Patent Application 237,362; Mullis, K., European Patent Application 201,184; Mullis K. et al. U.S. Patent No. 4,683,202; Erlich, H., U.S. Patent No. 4,582,788; and Saiki, R. et al. U.S. Patent No. 4,683,194)) is facilitated through the use of automated DNA sequencing instruments and software (Applied Biosystems, Inc.).
• FIG. 2 illustrates the preferred method for identifying new polymorphic sequences which is cycle sequencing of a random genomic fragment.
• the PCR fragments from the animal is electroeluted from acrylamide gels and sequenced using repetitive cycles of thermostable Taq DNA polymerase in the presence of a mixture of dNTPs and fluorescently or chemically labeled ddNTPs.
• the products are then separated and analyzed using an automated DNA sequencing instrument of Applied Biosystems, Inc.
• the data is analyzed using ABI software. Differences between sequences of different animals are identified by the software and confirmed by inspecting the relevant portion of the chromatograms on the computer screen. Differences are presented as "DNA Polymorphisms" only if the data is available for both strands and present in more than one haploid example among the five horses tested.
• the top panel shows an "A” homozygote, the middle panel an "AT” heterozygote and the bottom panel a "T” homozygote.
• the DNA sequence polymorphisms are identified by comparing the restriction endonuclease cleavage profiles generated by a panel of several restriction enzymes on products of the PCR reaction from the genomic templates of unrelated members. Most preferably, each of the restriction endonucleases used will have four base recognition sequences, and will therefore allow a desirable number of cuts in the amplified products.
• the restriction digestion patterns obtained from the genomic DNAs are preferably compared directly to the patterns obtained from PCR products generated using the corresponding plasmid templates. Such a comparison provides an internal control which indicates that the amplified sequences from the genomic and plasmid DNAs derive from equivalent loci. This control also allows identification of primers that fortuitously amplify repeated sequences, or multicopy loci, since these will generate many more fragments from the genomic DNA templates than from the plasmid templates.
• any of a variety of methods can be used to identify the polymorphic site, "X," of the single nucleotide polymorphisms of the present invention.
• the preferred method of such identification involves directly ascertaining the sequence of the polymorphic site for each polymorphism being analyzed. This approach is thus markedly different from the RFLP method which analyzes patterns of bands rather than the specific sequence of a polymorphism.
• the detection of polymorphic sites in a sample of DNA may be facilitated through the use of DNA amplification methods. Such methods specifically increase the concentration of sequences that span the polymorphic site, or include that site and sequences located either distal or proximal to it. Such amplified molecules can be readily detected by gel electrophoresis or other means. The most preferred method of achieving such amplification employs
• PCR using primer pairs that are capable of hybridizing to the proximal sequences that define a polymorphism in its double-stranded form.
• LCR Ligase Chain Reaction
• LCR can be performed with oligonucleotides having the proximal and distal sequences of the same strand of a polymorphic site.
• either oligonucleotide will be designed to include the actual polymorphic site of the polymorphism.
• the reaction conditions are selected such that the oligonucleotides can be ligated together only if the target molecule either contains or lacks the specific nucleotide that is complementary to the polymorphic site present on the oligonucleotide.
• the oligonucleotides will not include the polymorphic site, such that when they hybridize to the target molecule, a "gap" is created (see, Segev, D., PCT Application WO90/01069). This gap is then "filled” with complementary dNTPs (as mediated by DNA polymerase), or by an additional pair of oligonucleotides. Thus, at the end of each cycle, each single strand has a complement capable of serving as a target during the next cycle and exponential amplification of the desired sequence is obtained.
• OLA Oligonucleotide Ligation Assay
• nucleic acid amplification procedures such as transcription-based amplification systems (Malek, L.T. et al.. U.S. Patent 5,130,238; Davey, C. et al.. European Patent Application 329,822; Schuster et a U.S. Patent 5,169,766; Miller, H.I. et al. PCT Application WO89/06700; Kwoh, D. et al.. Proc. Natl. Acad. Sci. (U.S.A.) 86:1173 (1989); Gingeras, T.R. ejt al., PCT Application WO88/ 10315)), or isothermal amplification methods (Walker, G.T. et al. Proc. Natl. Acad. Sci. (U.S.A.) 89:392-396 (1992)) may also be used.
• transcription-based amplification systems Malek, L.T. et al.. U.S
• the direct analysis of the sequence of SNPs in the present invention can be accomplished using either the "dideoxy-mediated chain termination method,” also known as the “Sanger Method” (Sanger, F., et al.. J. Molec. Biol. 94:441 (1975)) or the “chemical degradation method,” “also known as the “Maxam-Gilbert method” (Maxa , A.M., et al. Proc. Natl. Acad. Sci. (U.S.A.) 74:560 (1977), both references herein incorporated by reference).
• Methods for sequencing DNA using either the dideoxy-mediated method or the Maxam-Gilbert method are widely known to those of ordinary skill in the art.
• nucleic acid sample contains double-stranded DNA (or RNA), or where a double-stranded nucleic acid amplification protocol (such as PCR) has been employed, it is generally desirable to conduct such sequence analysis after treating the double-stranded molecules so as to obtain a preparation that is enriched for, and preferably predominantly, only one of the two strands.
• a thermo stable polymerase is used and the reaction is heated and cooled one or more times. This allows the double stranded template to separate from its complimentary strand and subsequently anneal to the interrogation primer(s) during the cooling step. Competition for hybridization by the other template strand can be compensated for by repeated cycling of the melting-cooling conditions.
• Single-stranded DNA molecules may also be produced using the single-stranded DNA bacteriophage M13 (Messing, J. et al.. Meth. Enzymol. 101:20 (1983); see also, Sambrook, J., et al.. (In: Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989)).
• the method entails phosphorolating the 5'- terminus of one strand of a double-stranded amplification product, and then permitting a 5'->3' exonuclease (such as T7 exonuclease) to preferentially degrade the phosphorylated strand.
• a 5'->3' exonuclease such as T7 exonuclease
• Other methods have also exploited the nuclease resistant properties of phosphorothioate derivatives in order to generate single-stranded DNA molecules (Benkovic et al.. U.S. Patent No. 4,521,509; Sayers, J.R. et al.. Nucl. Acids Res. 16:791-802 (1988); Eckstein, F. et al..
• the phosphorothioate derivative is included in the primer.
• the nucleotide derivative may be incorporated into any position of the primer, but will preferably be incorporated at the 5'- terminus of the primer, most preferably adjacent to one another.
• the primer molecules will have a complementary region approximately 25 nucleotides in length, and contain from about 4% to about 100%, and more preferably from about 4% to about 40%, and most preferably about 16%, phosphorothioate residues (as compared to total residues).
• the nucleotides may be incorporated into any position of the primer, and may be adjacent to one another, or interspersed across all or part of the primer.
• the present invention can be used in concert with an amplification protocol, for example, PCR.
• an amplification protocol for example, PCR.
• the primers may require adjustment, especially of the annealing temperature, in order to optimize the reaction.
• nucleotide derivatives into DNA or RNA can be accomplished enzymatically, using a DNA polymerase (Vosberg, H.P. et al.. Biochemistry 16: 3633-3640 (1977); Burgers, P.M.J. et al.. T. Biol. Chem. 254:6889-6893 (1979); Kunkel, T.A., In: Nucleic Acids and Molecular Biology. Vol. 2, 124-135 (Eckstein, F. et al.. eds.), Springer-Verlag, Berlin, (1988); Olsen, D.B. et al.. Proc. Natl. Acad. Sci.
• a DNA polymerase Vosberg, H.P. et al.. Biochemistry 16: 3633-3640 (1977); Burgers, P.M.J. et al.. T. Biol. Chem. 254:6889-6893 (1979); Kunkel, T.A., In: Nucleic Acid
• phosphorothioate nucleotide derivatives can be incorporated synthetically into an oligonucleotide (Zon, G. et al., Anti-Cane. Drug Des. 6:539-568 (1991)).
• the primer molecules are permitted to hybridize to a complementary target nucleic acid molecule, and are then extended, preferably via a polymerase, to form an extension product.
• the presence of the phosphorothioate nucleotides in the primers renders the extension product resistant to nuclease attack.
• the amplification products containing phosphorothioate or other suitable nucleotide derivatives are substantially resistant to "elimination” (i.e., degradation) by "5'-» 3"' exonucleases such as T7 exonuclease or exonuclease, and thus a 5' ⁇ 3' exonuclease will be substantially incapable of further degrading a nucleic acid molecule once it has encountered a phosphorothioate residue.
• the incubation of the extension product and its template - the target - in the presence of a 5' ⁇ 3' exonuclease results in the destruction of the template strand, and thereby achieves the preferential production of the desired single strand.
• the preferred method of determining the identity of the polymorphic site of a polymorphism involves nucleic acid hybridization. Although such hybridization can be performed on a solid-phase (see, Saiki, R.K. et al., Proc. Natl. Acad. Sci. (U.S.A.) 86:6230-6234 (1989); Gilham et al. T. Amer. Chem. Soc 86:4982 (1964) and Kremsky et al.. Nucl. Acids Res. 15:3131-3139 (1987)), it is preferable to hybridize in solution (Berk, A.J., et al.. Cell 12:721-732 (1977); Hood, L.E., et al..
• biotinylated ddNTPs are preferably prepared by reacting the four respective (3- aminopropyn-l-yl)nucleoside triphosphates with sulfosuccinimidyl 6- (biotinamido)hexanoate.
• sulfosuccinimidyl 6- biotinamido
• 3-aminopropyn-l-yl) nucleoside 5'- triphosphates are prepared as described by Hobbs, F.W. (T. Org. Chem. 54:3420-3422 (1989)) and by Hobbs, F.W. et al.. (U.S. Patent No. 5,047,519).
• the identity of the nucleotide(s) of the polymorphic sites of the present invention can be determined, for example, using a variation of the oligonucleotide-based diagnostic assay of nucleic acid sequence variation disclosed by Goelet, P. et al. (PCT Application W092/15712, herein incorporated by reference).
• the present invention comprises an improvement over the method for analyzing SNPs described in U.S. Patent Application Serial No. 08/216,538 (herein incorporated by reference), in that it permits or facilitates the simultaneous or nearly simultaneous analysis of multiple SNPs.
• the present invention preferably employs one or more purified interrogation oligonucleotides having defined sequences that can hybridize to the target molecule in solution.
• interrogation oligonucleotides generally refers to oligonucleotide primers whose sequences are complementary to an immediate proximal or distal sequence of one or more single nucleotide polymorphisms.
• one or more interrogation oligonucleotide primers having sequences that are complementary to specific regions of the target molecule are prepared using the above-described methods.
• the primers have approximately 12 to 20 bases which are complementary to a specific region of the target molecule.
• the oligonucleotide primers hybridizes the target molecule such that the 3' end of each primer is immediately proximal to a target nucleotide of interest (such as a SNP).
• a target nucleotide of interest such as a SNP
• the oligonucleotide primer contains a 5' tail composed of a neutral component (e.g., poly T, abasic residues, or other non- specific, non-hybridizing polymer or chemical label).
• the neutral component is assigned a specific and unique length.
• the primers may or may not contain a primer-specific label. However, in the most preferred embodiment, the oligonucleotide primers do contain a primer-specific label.
• the interrogation primers are then incubated in the presence of the target DNA molecule (preferably a genomic DNA molecule) having one or more single nucleotide polymorphisms where the immediately 3' distal sequence for each SNP is complementary to that of the interrogation primer, a DNA polymerase and a chain terminating nucleotide (or nucleotide analog) triphosphate derivative.
• the target DNA molecule preferably a genomic DNA molecule
• a DNA polymerase a chain terminating nucleotide (or nucleotide analog) triphosphate derivative
• dATP dGTP, dCTP, dTTP
• chain terminating nucleotide triphosphate derivatives e.g., ddATP, ddGTP, ddCTP, ddTTP, etc.
• ddATP ddGTP, ddCTP, ddTTP, etc.
• unincorporated nucleotide triphosphate(s) in the reaction is immaterial to the reaction, such unincorporated nucleotides may be separated by a number of means.
• the identity of the incorporated nucleotide is determined by and is complementary to, the nucleotide of the polymorphic site of the polymorphism.
• the non-extendible nucleotide may be labeled, preferably with 32 P or a florescent molecule.
• Other labels suitable for the present invention include, but are not limited to, biotin, iminobiotin, hapten, an antigen, a cofactor, dintrophenol, lipoic acid, an olefinic compound, a detectable polypeptide, a molecule that is electron dense, an enzyme capable of depositing an insoluble reaction product.
• Florescent molecules suitable for the present invention include, but are not limited to, fluorescein, rhodamine, texas red, FAM, JOE, TAMRA, ROX, HEX, TET, Cy3, Cy3.5, Cy5, Cy 5.5, IRD40, IRD41 and BODIPY.
• Electron dense indicator molecules suitable for the present invention include, but are not limited to, ferritin, hemocyanin and colloidal gold.
• the detectable polypeptide may be indirectly detectable by specifically complexing the detectable polypeptide with a second polypeptide covalently linked to an indicator molecule.
• the detectable polypeptide is preferably selected from the group consisting of avidin and strepavidin
• the second polypeptide is preferably selected from the group consisting of biotin and iminobiotin.
• the resultant extended primers are separated for analysis on a suitable matrix.
• Any of a number of methods can be used to separate the extended primers for analysis. Such methods include, but are not limited to: mass specrrometry, (oligonucleotide array hybridization) flow cytometry, HPLC, FPLC, size exclusion chromatography, affinity chromatography, gel electrophoresis, etc.
• the extended primers are separated under denaturing conditions; however, denaturing conditions are not required for effective separation.
• non-extendible nucleotide refers to a synthetic or naturally occurring nucleotide analog that is capable of being incorporated by a template dependent polymerase.
• Synthetic or naturally occurring nucleotide analogs suitable for use in the present invention include, but are not limited to, acyclic ribose nucleotide analogs, substituted ribose nucleotide analogs, and modified ribose nucleotide analogs.
• Synthetic nucleotide analogs are preferably selected from the group consisting of fructose based nucleotide analogs, chemically modified purines that retain the ability to specifically base pair with naturally occurring nucleotides, chemically modified pyrimidines that retain the ability to specifically base pair with naturally occurring nucleotides, and any compound that retains the ability to specifically base pair with naturally occurring nucleotides.
• the extended primers are separated on a denaturing, size separating matrix such as a standard sequencing gel having an appropriate acrylamide concentration.
• This embodiment employs interrogation primers containing a 5' tail having a specific and unique length. The extended primers are differentially separated based upon the specific and unique length of the 5' tail. While the preferred embodiment employs labeled chain terminating nucleotide(s) (or nucleotide analog(s)), the present embodiment is also directed towards differentially labeled interrogation primers.
• One sub-embodiment employs differentially labeled chain terminating nucleotides (i.e., dideoxynucleotides).
• An alternate sub-embodiment employs one or more chain terminating nucleotides wherein only a single chain terminating nucleotide is labeled.
• Another preferred embodiment employs interrogation primers containing unique and specific sequences capable of hybridizing to complimentary sequences arrayed on a solid phase.
• the interrogation primers are separate by exposing them to arrayed capture primers and identifying each single nucleotide polymorphism through detection of the labeled base in the context of its location on the solid phase capture primer array.
• the resultant extended primers are separated using suitable affinity separation methods.
• affinity separation methods are generally drawn to receptor-ligand methods (e.g., avidin-strepavidin, etc.), monoclonal antibody methods, etc.
• the 5' terminus of each primer is coupled to a unique ligand for which there is a corresponding unique receptor.
• the receptors are preferably immobilized to a solid surface (i.e., a bead, a column, a dipstick, a microtiter plate, etc.).
• the ligand-labeled primers are separated by exposing the reaction mixture to the corresponding receptors. It is then possible to determine the identity of each single nucleotide polymorphism using the methods described above.
• Another embodiment employs primers coupled (covalently or otherwise) to uniquely sized moieties (e.g., BSA, lysozyme, ovalbumin, etc.).
• the uniquely sized primers are separated by passing the primers through a suitable size exclusion chromatography column and the identity of each single nucleotide polymorphism is identified using the methods described above.
• Labels suitable for use in the present invention include, but are not limited to: enzymes ( ⁇ -galactosidase, lucif erase, etc.), radioactive isotopes (i.e., 32 P, 13 C, 3 H, etc.), fluorescent moieties (i.e., fluorescein, rhodamine, etc.), chromophores.
• the primers can be either directly labeled or coupled with a distinct ligand which may be either labeled or unlabeled.
• the ligand molecule may be coupled to the oligonucleotide primer by covalent coupling, ionic interactions, non-specific adsorption, or specific but non- covalent ligand-receptor interactions.
• ligand refers generally to a given protein or chemical compound to which there is a corresponding distinct receptor.
• Ligands suitable for use in the present invention include, but are not limited to, a hapten, an antigen, a cofactor, biotin, iminobiotin, dinitrophenol, lipoic acid, an olefinic compound, an oligonucleotide, protein nucleic acid (“PNA”) sequences designed to hybridize specifically to a complementary oligonucleotide, and PNA sequences that functions as a receptor.
• Additional ligands suitable for use in the present invention include, but are not limited to, an antibody, an enzyme, a polypeptide, strepavidin and avidin.
• the ligand is capable of forming a complex by binding with a detectably labeled polypeptide.
• the detectable label suitable for use in the present invention includes, but is not limited to, an antibody, an enzyme capable of depositing insoluble reaction products, strepavidin and avidin.
• the detectably labeled polypeptide is selected from randomly generated polypeptide libraries.
• receptor refers generally to a given protein or chemical compound to which there is a corresponding ligand.
• Receptors suitable for use in the present invention include, but are not limited to, an antigen, a cofactor, biotin, iminobiotin, dinitrophenol, lipoic acid, an olefinic compound, an oligonucleotide, PNA sequences designed to hybridize specifically to a complementary oligonucleotide, and PNA sequences that functions as a ligand.
• the receptor is capable of forming a complex by binding with a detectably labeled polypeptide.
• the detectable label suitable for use in the present invention includes, but is not limited to, an antibody, an enzyme capable of depositing insoluble reaction products, strepavidin and avidin.
• the detectably labeled polypeptide is selected from randomly generated polypeptide libraries.
• the receptor may be coupled to the matrix. Suitable methods for coupling the receptor to the matrix include, but are not limited to, covalent coupling, ionic interactions, non-specific adsorption, specific but non-covalent ligand-receptor interactions.
• the ligand-receptor suitable for use in the present invention includes, but is not limited to, complementary hybridizing nucleic acids, complementary hybridizing PNAs, and other complementary synthetic nucleic acid analogs.
• the present invention can also be used to identify SNPs without separation of the primers.
• at least one added chain terminating nucleotide triphosphate derivative is uniquely labeled, such that the addition of a nucleotide to an interrogation primer can be detected (either by the labeling of the oligonucleotide, or the failure of the oligonucleotide to become labeled).
• the incorporation of such label is indicative that one of the SNPs is a T.
• the identification of the primers through the primer specific label and the incorporated nucleotides enables the genotyping of the target molecule.
• the nucleotide of the polymorphic site is thus determined by assaying which of the set of labeled nucleotides has been incorporated into the 3' terminus of the oligonucleotide by the primer-dependent polymerase.
• the non-extendible nucleotide or nucleotide analog may be identified by any of a number of physical or chemical method. However, the preferred physical or chemical means are selected from the group consisting of polarization spectroscopy, mass spectroscopy, infra-red spectroscopy, ultraviolet spectroscopy, visible spectroscopy or NMR spectroscopy.
• the present method is directed at methods to identify multiple SNPs in a single reaction
• the present invention can also confirm the identity of multiple SNPs in a single reaction.
• the identity of each SNP for both the plus and minus strand of the target nucleic acid are determined as previously described. The sequence is confirmed where the plus and minus strand for each SNP analyzed are complementary.
• the identity of the nucleotide of the polymorphic site is determined using a polymerase/ligase mediated process.
• multiple oligonucleotide primers are simultaneously employed for the detection of multiple SNPs in the same reaction.
• an oligonucleotide primer is employed that is complementary to an immediately 3' distal invariant sequence of a SNP.
• a second oligonucleotide complementary to the 5'- proximal sequence of the polymorphism being analyzed, but incapable of hybridizing to the oligonucleotide primer is used.
• oligonucleotides are incubated in the presence of DNA containing the single nucleotide polymorphism that is to be analyzed, and at least one 2', 5'-deoxynucleotide triphosphate.
• the incubation reaction further includes a DNA polymerase and a DNA ligase.
• Both oligonucleotides are thus capable of hybridizing to the same strand of the single nucleotide polymorphism being analyzed. Sequence considerations cause the two oligonucleotides to hybridize to the proximal and distal sequences of the SNP that flank the polymorphic site (X) of the polymorphism; the hybridized oligonucleotides are thus separated by a "gap" of a single nucleotide at the precise position of the polymorphic site.
• a polymerase and a 2', 5'-deoxynucleotide triphosphate complementary to (X) permits ligation of the primer extended with the complementary 2', 5'-deoxynucleotide triphosphate to the hybridized oligo complementary to the distal sequence, a 2', 5'-deoxy- nucleotide triphosphate that is complementary to the nucleotide of the polymorphic site permits the creation of a ligatable substrate.
• the identity of the polymorphic site that was opposite the "gap" can then be determined by any of several means.
• the 2', 5'-deoxynucleotide triphosphate of the reaction is labeled, and its detection thus reveals the identity of the complementary nucleotide of the polymorphic site.
• Several different 2', 5'-deoxynucleotide triphosphates may be present, each differentially labeled.
• separate reactions can be conducted, each with a different 2', 5'-deoxynucleotide triphosphate.
• the 2', 5'-deoxynucleotide triphosphates are unlabeled, and the second, soluble oligonucleotide is labeled. Separate reactions are conducted, each using a different unlabeled 2', 5'- deoxynucleotide triphosphate. While the above-described embodiment details a polymerase/ligase mediated method for the detection of a single polymorphic site, it is generally understood that the method can employ the simultaneous use of multiple unique oligonucleotide primers for the detection of multiple polymorphic sites.
• the sensitivity of nucleic acid hybridization detection assays may be increased by altering the manner in which detection is reported or signaled to the observer.
• assay sensitivity can be increased through the use of detectably labeled reagents.
• Kourilsky et ah (U.S. Patent 4,581,333) describe the use of enzyme labels to increase sensitivity in a detection assay.
• Fluorescent labels Albarella et al.. EP 144914
• chemical labels Sheldon HI et al.. U.S. Patent 4,582,789; Albarella et aL U.S. Patent 4,563,417)
• modified bases Miyoshi et al., EP 119448
• the utility of the polymorphic sites of the present invention stems from the ability to use such sites to predict the statistical probability that two individuals will have the same alleles for any given polymorphisms.
• SNPs can be used for any of a variety of purposes. Where a particular individual has been previously tested, such testing can be used as a "fingerprint" which can be used to determine the identity of a particular individual. Where a putative parent or both parents of an individual have been tested, the methods of the present invention may be used to determine the likelihood that a particular animal is or is not the progeny of such parent or parents. Thus, the detection and analysis of SNPs can be used to exclude paternity of a male for a particular individual (such as a father's paternity of a particular child), or to assess the probability that a particular individual is the progeny of a selected female (such as a particular child and a selected mother).
• the present invention permits the construction of a genetic map of a target species.
• the particular array of polymorphisms identified by the methods of the present invention can be correlated with a particular trait, in order to predict the predisposition of a particular animal (or plant) to such genetic disease, condition, or trait.
• the term “trait” is intended to encompass “genetic disease,” “condition,” or “characteristics.”
• the term, “genetic disease” denotes a pathological state caused by a mutation, regardless of whether that state can be detected or is asymptomatic.
• a “condition” denotes a predisposition to a characteristic (such as asthma, weak bones, blindness, ulcers, cancers, heart or cardiovascular illnesses, skeleto-muscular defects, etc.).
• a “characteristic” is an attribute that imparts economic value to a plant or animal. Examples of characteristics include longevity, speed, endurance, rate of aging, fertility, etc.
• the most useful measurements for determining the power of an identification and paternity testing system are: (i) the "probability of identity” (p(ID)) and (ii) the "probability of exclusion” (p(exc)).
• the p(ID) calculates the likelihood that two random individuals will have the same genotype with respect to a given polymorphic marker.
• the p(exc) calculates the likelihood, with respect to a given polymo ⁇ hic marker, that a random male will have a genotype incompatible with him being the father in an average paternity case in which the identity of the mother is not in question.
• a desirable test will preferably measure multiple unlinked loci in parallel. Cumulative probabilities of identity or non-identity, and cumulative probabilities of paternity exclusion are determined for these multi-locus tests by multiplying the probabilities provided by each locus. The statistical measurements of greatest interest are: (i) the cumulative probability of non-identity (cum p(non ⁇ D)), and (ii) the cumulative probability of paternity exclusion (cum p(exc)).
• the probability of parentage exclusion (representing the probability that a random male will have a genotype, with respect to a given locus, that makes him incompatible as the sire in an average paternity case where the identity of the mother is not in question) is given by the equation:
• the cumulative probability of non-exclusion (representing the value obtained when n loci are used) is thus: cum p(non-exc) - K p(non-exc l )p ⁇ non-exc 2 )p(non-exc 3 )....p(non-exc n )
• cum p(exc) 1 - cum p(non-exc)
• p ⁇ exc pq(l-pq) + qr ⁇ l-qr) + pr(l-pr) + 3pqr(l-pqr)
• Figures 3 and 4 show how the cum p(non ⁇ D) and the cum p(exc) increase with both the number and type of genetic loci used. It can be seen that greater discriminatory power is achieved with fewer markers when using three allele systems.
• the choice between whether to use loci with 2, 3 or more alleles is, however, largely influenced by the above-described biochemical considerations.
• a polymorphic analysis test may be designed to score for any number of alleles at a given locus. If allelic scoring is to be performed using gel electrophoresis, each allele should be easily resolvable by gel electrophoresis. Since the length variations in multiple allelic families are often small, human DNA tests using multiple allelic families include statistical corrections for mistaken identification of alleles. Furthermore, although the appearance of a rare allele from a multiple allelic system may be highly informative, the rarity of these alleles makes accurate measurements of their frequency in the population extremely difficult. To correct for errors in these frequency estimates when using rare alleles, the statistical analysis of this data must include a measure of the cumulative effects of uncertainty in these frequency estimates. The use of these multiple allelic systems also increases the likelihood that new or rare alleles in the population will be discovered during the course of large population screening. The integrity of previously collected genetic data would be empirically revised to reflect the discovery of a new allele.
• loci with many alleles could potentially offer some short-term advantages (because fewer loci would need to be screened)
• polymorphisms detected in a set of individuals of the same species can be analyzed to determine whether the presence or absence of a particular polymorphism correlates with a particular trait.
• a set of polymo ⁇ hisms i.e. a "polymorphic array”
• a mutually exclusive characteristic for example, with respect to horses, brittle bones vs. non-brittle bones; maturity onset blindness vs. no blindness; predisposition to asthma, cardiovascular disease, etc. vs. no such predisposition).
• the alleles of each polymorphism of the set are then reviewed to determine whether the presence or absence of a particular allele is associated with the particular trait of interest. Any such correlation defines a genetic map of the individual's species. Alleles that do not segregate randomly with respect to a trait can be used to predict the probability that a particular animal will express that characteristic. For example, if a particular polymorphic allele is present in only 20% of the members of a species that exhibit a cardiovascular condition, then a particular member of that species containing that allele would have a 20% probability of exhibiting such a cardiovascular condition. As indicated, the predictive power of the analysis is increased by the extent of linkage between a particular polymo ⁇ hic allele and a particular characteristic.
• the predictive power of the analysis can be increased by simultaneously analyzing the alleles of multiple polymo ⁇ hic loci of a particular trait.
• a second polymo ⁇ hic allele was found to also be present in 20% of members exhibiting the cardiovascular condition, however, all of the evaluated members that exhibited such a cardiovascular condition had a particular combination of alleles for these first and second polymorphisms, then a particular member containing both such alleles would have a very high probability of exhibiting the cardiovascular condition.
• the detection of multiple polymo ⁇ hic sites permits one to define the frequency with which such sites independently segregate in a population. If, for example, two polymo ⁇ hic sites segregate randomly, then they are either on separate chromosomes, or are distant to one another on the same chromosome. Conversely, two polymo ⁇ hic sites that are co-inherited at significant frequency are linked to one another on the same chromosome. An analysis of the frequency of segregation thus permits the establishment of a genetic map of markers.
• the present invention provides a means for mapping the genomes of plants and animals. The resolution of a genetic map is proportional to the number of markers that it contains.
• the methods of the present invention can be used to isolate a large number of polymo ⁇ hic sites, they can be used to create a map having any desired degree of resolution.
• the sequencing of the polymo ⁇ hic sites greatly increases their utility in gene mapping.
• Such sequences can be used to design oligonucleotide primers and probes that can be employed to "walk” down the chromosome and thereby identify new marker sites (Bender, W. et al., T. Supra. Molec. Struc. 10(Supp.):32 (1979); Chinault, A.C. et al. Gene 5:111-126 (1979); Clarke, L. et al.. Nature 287:504-509 (1980)).
• the resolution of the map can be further increased by combining polymorphic analyses with data on the phenotype of other attributes of the plant or animal whose genome is being mapped.
• polymorphic analyses with data on the phenotype of other attributes of the plant or animal whose genome is being mapped.
• biochemical data can be used to increase the resolution of the genetic map.
• a biochemical determination (such as a serotype, isoform, etc.) is studied in order to determine whether it co- segregates with any polymo ⁇ hic site.
• Such maps can be used to identify new gene sequences, to identify the causal mutations of disease, for example.
• the identification of the SNPs of the present invention permits one to use complimentary oligonucleotides as primers in PCR or other reactions to isolate and sequence novel gene sequences located on either side of the SNP.
• the present invention includes such novel gene sequences.
• the genomic sequences that can be clonally isolated through the use of such primers can be transcribed into RNA, and expressed as protein.
• the present invention also includes such protein, as well as antibodies and other binding molecules capable of binding to such protein.
• LOD scoring methodology has been developed to permit the use of RFLPs to both track the inheritance of genetic traits, and to construct a genetic map of a species (Lander, S. et al., Proc. Natl. Acad. Sci. (U.S.A.) 83:7353-7357 (1986); Lander, S. et al. Proc. Natl. Acad.
• the polymorphisms of the present invention comprise direct genomic sequence information and can therefore be typed by a number of methods.
• the analysis must be gel- based, and entail obtaining an electrophoretic profile of the DNA of the target animal.
• an analysis of the polymo ⁇ hisms may be performed using spectrophotometric methods, and can readily be automated to facilitate the analysis of large numbers of target animals.
• Primer #1 has a 5 base T-tail
• primer #2 has a 10 base T-tail
• primer #3 has a 15 base T-tail.
• the amplification may be mediated using primers that contain 4 phosphorothioate-nucleotide derivatives, as taught by Nikiforov, T. (U.S.
• a second round of PCR may be performed using "asymmetric" primer concentrations.
• the products of the first reaction are diluted 1/1000 in a second reaction.
• One of the second round primers is used at the standard concentration of 2 M while the other is used at 0.08 M. Under these conditions, single stranded molecules are synthesized during the reaction.
• the primer mixture is hybridized to the single stranded target template and a single base extension reaction using DNA polymerase and the four modified non-extendible nucleotides is allowed to occur.
• For each reaction tube only one modified non-extendible nucleotide is labeled, preferably with 2 P or a florescent molecule.
• the resultant extended primers are then separated for analysis on a standard 12% sequencing gel. Thus, it is possible to determine the identity of the SNP corresponding to each primer based upon its electrophoretic mobility and the identity of the labeled non- extendible nucleotide.
• the resultant extended primers are subsequently separated over a suitable size exclusion column (e.g., sephadex, sepharose, etc.) and the eluate is analyzed (e.g., with a scintillation counter) to determine the identity of the inco ⁇ orated nucleotide.
• a suitable size exclusion column e.g., sephadex, sepharose, etc.
• the eluate is analyzed (e.g., with a scintillation counter) to determine the identity of the inco ⁇ orated nucleotide.
• a peptide or protein affinity ligand is covalently coupled to the interrogation oligonucleotide using, for example, the methods disclosed by Chu et al., (Nucleic Acids Res. 1£: 3671-3691 (1988)), herein inco ⁇ orated by reference.
• the affinity ligand-interrogation primer complex is then hybridized to the target nucleic acid molecule and the single base extension reaction, described above, is allowed to occur in the presence of four differentially labeled dideoxynucleotide species (ddA, ddT, ddC, and ddG). Where desired, fewer species of dideoxynucleotides may be employed.
• the corresponding monoclonal antibody to the peptide or protein is immobilized to a microtiter plate (Nunc). Each monoclonal antibody is immobilized to the microtiter plate at room temperature in a buffered solution. The plate is then washed with a TNTw solution three times to remove any excess unbound proteins.
• each dideoxynucleotide triphosphate is preferably uniquely labeled.
• ddATP could be labeled with 32 P, ddGTP labeled with 3 H, ddCTP labeled with 35 S, and ddTTP labeled with 125 I.
• Table 3 illustrates the hypothetical results obtained from hybridization with 6 oligonucleotides hybridized to a preparation containing nucleic acids of interest, and the result of a single base extension reaction.
• the unincorporated ddNTP's may be separated from the extended probes using any of a variety of means (e.g., suitable spin column (i.e., CentriSep spin columns), etc.).
• the labeled inco ⁇ orated dideoxynucleotide triphosphates are subsequently detected using a scintillation counter. As each isotope has a distinct emission spectra, the scintillation counter can determine the identity of multiple single nucleotide polymo ⁇ hisms without the need for purification procedures.
• Primer 1, 2, 3 and 4 contain in addition to sequences complimentary to the template DNA, unique sequences for subsequent hybridization to capture oligonucleotides on a solid surface.
• a single stranded DNA template is probed with the four interrogation primers and the single base extension reaction using DNA polymerase and the four non-extendible nucleotides is allowed to occur.
• each non-extendible nucleotide is uniquely and distinctly labeled.
• the resultant extended primers are subsequently applied to the surface of an oligonucleotide array.
• the array consists of four separate and spatially distinct capture oligonucleotides, each of which is complimentary to a unique sequence on one of the interrogation primers. Each interrogation primer is effectively separated by hybridization to its corresponding surface bound capture primer. The identity of the labeled nucleotides is then determined by suitable methods.

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