WO1998012352A1 - Procedes de detection des fragments de restriction polymorphes (rfpl) amplifies et clives - Google Patents

Procedes de detection des fragments de restriction polymorphes (rfpl) amplifies et clives Download PDF

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Publication number
WO1998012352A1
WO1998012352A1 PCT/US1997/016467 US9716467W WO9812352A1 WO 1998012352 A1 WO1998012352 A1 WO 1998012352A1 US 9716467 W US9716467 W US 9716467W WO 9812352 A1 WO9812352 A1 WO 9812352A1
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WO
WIPO (PCT)
Prior art keywords
sequence
primer
restriction site
strand
detectable label
Prior art date
Application number
PCT/US1997/016467
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English (en)
Inventor
Frederick Ausubel
Ronald W. Davis
Daphne Preuss
Original Assignee
The General Hospital Corporation
Board Of Trustees Of The Leland Stanford Junior University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from US08/715,484 external-priority patent/US6004783A/en
Application filed by The General Hospital Corporation, Board Of Trustees Of The Leland Stanford Junior University filed Critical The General Hospital Corporation
Priority to EP19970942525 priority Critical patent/EP0948645A4/fr
Priority to CA002266750A priority patent/CA2266750A1/fr
Publication of WO1998012352A1 publication Critical patent/WO1998012352A1/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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
    • C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
    • 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

Definitions

  • This invention relates to the generation and detection of genetic polymorphisms .
  • RFLP restriction fragment length polymorphic
  • PCR-based approach involves the use of single short PCR primers of arbitrary sequence called RAPD primers (for random amplified polymorphic DNA; Reiter et al., Proc. Natl. Acad. Sci. USA 89: 1477-1481 , 1992; Williams et al., Theoret. Appl. Genet. 82:489-498, 1991).
  • RAPD primers for random amplified polymorphic DNA
  • SSLPs for simple sequence length polymorphism
  • the method employing SSLPs is based on amplification across tandem repeats of one or a few nucleotides known as "microsatellites.” Microsatellites occur frequently and randomly in most eukaryotic genomes and display a high degree of polymorphism due to variation in the numbers of repeated units.
  • a third category of PCR-based markers are called AFLPs (for amplified fragment length polymo ⁇ hisms).
  • DNA from two polymo ⁇ hic strains are cleaved with one or two restriction endonucleases, and adapters are ligated to the ends of the cleaved fragments.
  • the fragments are then amplified using primers complementary to the adapter(s).
  • the primers contain short stretches of random nucleotides at their 3' ends, which result in limiting the number of amplified fragments generated.
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a specific binding pair, the second primer being tagged with a detectable label; (b) digesting the PCR product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) contacting the reaction product of step (b) with the second member of the specific binding pair, immobilized on a solid support; and (d) measuring the level of the detectable label bound to the solid support, the presence of the detectable label bound to the solid support being an indication of the absence of the polymo ⁇ hic restriction site in the nucleic acid.
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a specific binding pair, the second primer being tagged with a first detectable label; (b) digesting the PCR product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) annealing and ligating to the single-stranded ends generated in the reaction of step (b) an oligonucleotide tagged with a second detectable label; (d) contacting the reaction product of step (c) with the second member of the specific binding pair, immobilized on a solid support; and (e) determining the levels of the first and second detectable labels bound to the solid support, the presence of only the first detect
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the method involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) digesting a portion of the reaction of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site, while leaving another portion of the reaction of step (a) undigested; (c) denaturing the digested and undigested portions from step (b); (d) contacting the product of step (c) with an oligonucleotide complementary to a sequence in the strand of the product of step (c) containing the detectable label, the sequence being between the polymo ⁇ hic restriction and the sequence complementary to the second primer, the oligonucleotide being
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, the second primer being tagged with a second detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) denaturing the reaction product of step (b); (d) contacting the product of step (c) with a first and a second oligonucleotide, the first oligonucleotide being complementary to a first sequence in the strand of the product of step (c) containing the first detectable label, the first sequence being between the polymo ⁇ hic restriction site and the sequence corresponding to the first primer, the first oligonucleo
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, the second primer being tagged with a second detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) denaturing the reaction product of step (b); (d) contacting the product of step (c) with a first and a second oligonucleotide, the first oligonucleotide being complementary to a first sequence in the strand of the product of step (c) containing the first detectable label, the first sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer, the first oligonucleot
  • the invention method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, the second primer being tagged with a second detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) denaturing the reaction product of step (b); (d) contacting the product of step (c) with a first and a second oligonucleotide, the first oligonucleotide being complementary to a first sequence in the strand of the product of step (c) containing the first detectable label, the first sequence being between the polymo ⁇ hic restriction site and the sequence corresponding to the first primer, the first oligonucleotide being
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, the second primer being tagged with a second detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) denaturing the reaction product of step (b); (d) contacting the product of step (c) with a first and a second oligonucleotide, the first oligonucleotide being complementary to a first sequence in the strand of the product of step (c) containing the first detectable label, the first sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer, the first oligonucleot
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) contacting the reaction product of step (b) with the second member of the first specific binding pair, immobilized on a first solid support; (d) denaturing the reaction product of step (c) not bound to the first solid support; (e) contacting the product of step (d) with an oligonucleotide complementary to a sequence in the strand of the product of step (d) containing the detectable label, the sequence being between the polymo
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) annealing and ligating to the single-stranded ends generated in the reaction of step (b) a first oligonucleotide tagged with the first member of a first specific binding pair; (d) contacting the reaction product of step (c) with the second member of the first specific binding pair, immobilized on a first solid support; (e) denaturing the reaction product of step (d) not bound to the first solid support; (f) contacting the product of step
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) contacting the reaction product of step (b) with the second member of the first specific binding pair, immobilized on a first solid support; (d) denaturing the reaction product from step (c) not bound to the first solid support; (e) contacting the product of step (d) with an oligonucleotide complementary to a sequence in the strand of the product of step (d) containing the detectable label, the sequence being between the
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) digesting the reaction product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (c) annealing and ligating to the single-stranded ends generated in the reaction of step (b) a first oligonucleotide tagged with the first member of a first specific binding pair; (d) contacting the reaction product of step (c) with the second member of the first specific binding pair, immobilized on a first solid support; (e) denaturing the reaction product of step (d) not bound to the first solid support; (f) contacting the product of step
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) digesting the product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d)
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) digesting the PCR product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d)
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step
  • step (a) by PCR using a third primer and the second primer, the third primer containing the first sequence, the third primer being tagged with a detectable label;
  • step (c) digesting a portion of the reaction of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site, while leaving another portion of the reaction of step (b) undigested;
  • step (d) denaturing the digested and undigested portions from step (c);
  • step (e) contacting the product of step (d) with an oligonucleotide complementary to a second sequence in the strand of the product of step (d) containing the detectable label, the second sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer, the oligonucleotide being tagged with a first member of a specific binding pair;
  • step (e) contacting the reaction product of step (e) with the second member of the specific binding pair, immobilized on a solid support; and (
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of:
  • step (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid;
  • step (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with a first detectable label, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a second detectable label;
  • step (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site;
  • step (e) contacting the product of step (d) with a first and a second oligonucle
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with a first detectable label, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a second detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) denaturing the
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with a first detectable label, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a second detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) denaturing the
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving (a) amplifying the nucleic acid by PCR using a first and second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with a first detectable label, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a second detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) denaturing the reaction product of step (
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a first specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) contacting the
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third primer and the second primer, the third primer containing the first sequence, the third primer being tagged with a detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) annealing and ligating to the single-stranded ends generated in the reaction of step (c) a first oligonucleotide tagged with the first member of a first specific binding pair; (e) contacting the reaction product of step (d) with the second member of the
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a first specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d)
  • step (e) denaturing the reaction product from step (d) not bound to the first solid support
  • step (f) contacting the product of step (e) with an oligonucleotide complementary to a third sequence in the strand of the product of step (e) containing the detectable label, the third sequence being between the polymo ⁇ hic restriction site and the sequence corresponding to or complementary to the second primer, the oligonucleotide being immobilized on a second solid support; and (g) determining the ratio of the level of the detectable label bound to the first solid support to the level of the detectable label bound to the second solid support, a ratio of 0:1 being an indication of a homozygote containing the polymo ⁇ hic restriction site, in a case where the total amount of the reaction product from step (d) not bound to the first solid support was used in steps (e) and (f); a ratio of 1 :0 being an indication of a homozygote lacking the polymo ⁇ hic restriction site, in a case where the total amount of the reaction product from step (d) not bound to the first solid support was used in steps
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the method involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) amplifying the product of step (a) by PCR using a third primer and the second primer, the third primer containing the first sequence, the third primer being tagged with a detectable label; (c) digesting the reaction product of step (b) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site; (d) annealing and ligating to the single- stranded ends generated in the reaction of step (c) a first oligonucleotide tagged with the first member of a first specific binding pair; (e) contacting the reaction product of step (d)
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing one or more sets of a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a specific binding pair, the second primer being tagged with a detectable label.
  • the kit further contains the second member of the specific binding pair, immobilized on a solid support.
  • the kit further contains an oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with a second detectable label.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) an oligonucleotide complementary to a sequence in the strand of the nucleic acid complementary to the second primer, the sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer, the oligonucleotide being tagged with a first member of a specific binding pair; and (c) the second member of the specific binding pair, immobilized on a solid support.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, the second primer being tagged with a second detectable label; (b) a first oligonucleotide, the first oligonucleotide being complementary to a first sequence in the strand of the nucleic acid complementary to the second primer, the first sequence being between the polymo ⁇ hic restriction site and either the sequence corresponding to the first primer or the sequence complementary to the second primer, the first oligonucleotide being tagged with the first member of a first specific binding pair; (c) a second oligonucleotide, the second oligonucleotide being complementary to a second sequence in the strand of the nucleic acid complementary to the first primer, the second sequence
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label; (b) the second member of the first specific binding pair, immobilized on a first solid support; (c) an oligonucleotide complementary to a first sequence in the strand of the nucleic acid containing the sequence corresponding to the second primer, the first sequence being between the polymo ⁇ hic restriction site and the sequence corresponding to the second primer, the oligonucleotide being tagged with the first member of a second specific binding pair; and (d) the second member of the second specific binding pair, immobilized on a second solid support.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) a first oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with the first member of a first specific binding pair; (c) the second member of the first specific binding pair, immobilized on a first solid support; (d) a second oligonucleotide complementary to a sequence in the strand of the nucleic acid complementary to the second primer, the sequence being between the polymo ⁇ hic restriction site and either the sequence corresponding to the first primer or the sequence
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label; (b) the second member of the first specific binding pair, immobilized on a first solid support; and (c) an oligonucleotide complementary to a first sequence in the strand of the nucleic acid containing the sequence corresponding to the second primer, the first sequence being between the polymo ⁇ hic restriction site and the sequence corresponding to the second primer, the oligonucleotide being immobilized on a second solid support.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled; (b) a first oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with the first member of a first specific binding pair; (c) the second member of the first specific binding pair, immobilized on a first solid support; and (d) a second oligonucleotide complementary to a sequence in the strand of the nucleic acid complementary to the second primer, the sequence being between the polymo ⁇ hic restriction site and either the sequence corresponding to the first primer or the
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label.
  • the kit further contains the second member of the specific binding pair, immobilized on a solid support.
  • the kit further contains an oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with a second detectable label.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) a third primer containing the first sequence, the third primer being tagged with a detectable label; (c) an oligonucleotide complementary to a second sequence in the strand of the nucleic acid containing the sequence complementary to the second primer, the second sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer, the oligonucleotide being tagged with a first member of a specific binding pair; and (d) the second member of the specific binding pair, immobilized on a solid support.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with a first detectable label, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a second detectable label; (c) a first oligonucleotide, the first oligonucleotide being complementary to a third sequence in the strand of the nucleic acid complementary to the second primer, the third sequence being between the polymo ⁇ hic restriction site and either the sequence complementary to the
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a first specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) the second member of the first specific binding pair, immobilized on a first solid support; (d) an oligonucleotide complementary to a third sequence in the strand of the nucleic acid corresponding to the second primer, the sequence being between the polymo
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) a third primer containing the first sequence, the third primer being tagged with a detectable label; (c) a first oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with the first member of a first specific binding pair; (d) the second member of the first specific binding pair, immobilized on a first solid support; (e) a second oligonucleotide complementary to a second sequence in the strand of the nucleic acid corresponding to the first primer, the kit containing: (a)
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid, the second primer containing a second sequence not complementary to or present in the nucleic acid; (b) a third and a fourth primer, the third primer containing the first sequence or a sequence complementary to the first sequence, the third primer being tagged with the first member of a first specific binding pair, the fourth primer containing the second sequence or a sequence complementary to the second sequence, the fourth primer being tagged with a detectable label; (c) the second member of the first specific binding pair, immobilized on a first solid support; and (d) an oligonucleotide complementary to a third sequence in the strand of the nucleic acid corresponding to the second primer, the third sequence being between
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: (a) a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer containing a first sequence not complementary to or present in the nucleic acid; (b) a third primer containing the first sequence, the third primer being tagged with a detectable label; (c) a first oligonucleotide complementary to the single-stranded ends generated in the nucleic acid upon digestion of the nucleic acid with the restriction enzyme corresponding to the polymo ⁇ hic restriction site, the oligonucleotide being tagged with the first member of a first specific binding pair; (d) the second member of the first specific binding pair, immobilized on a first solid support; and (e) a second oligonucleotide complementary to a second sequence in the strand of the nucleic acid corresponding to the first
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) amplifying the nucleic acid by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, whereby the resultant PCR product is of a defined size readily resolved by gel electrophoresis; (b) digesting the PCR product of step (a) with the restriction endonuclease corresponding to the polymo ⁇ hic restriction site, the digestion products being differentially sized; (c) separating the reaction products of step (b) by gel electrophoresis; and (d) detecting the separated reaction products, the presence of only uncleaved products being an indication of a homozygote lacking the polymo ⁇ hic restriction site, the presence of only cleaved products being an indication of a homozygote containing the polymo ⁇ hic restriction site, and the presence of both clea
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid, the kit containing: a first and a second primer flanking the polymo ⁇ hic restriction site and capable of generating a PCR product of a defined size that is readily resolved by gel electrophoresis.
  • the first and/or the second primers are detectably labeled.
  • the PCR product generated is between 100 and 1000 base pairs in length.
  • the invention features a method for identifying a polymo ⁇ hic restriction site in a nucleic acid, involving the steps of: (a) digesting DNA isolated from a first sample with a first restriction endonuclease; (b) ligating to each of the ends of the reaction products of step (a) a first adaptor; (c) digesting the products of step (b) with a second restriction endonuclease; (d) ligating to each of the ends of the reaction products generated in step (c) a second adaptor; (e) amplifying the reaction products of step (d) by PCR using a first primer complementary to the first adaptor and a second primer complementary to the second adaptor, the second primer being tagged with a first member of a specific binding pair (preferably, biotin); (f) in a separate set of reactions, digesting DNA isolated from a second sample with the first restriction endonuclease; (g) ligating to each of the ends of the ends of the
  • DNA adaptor and a third DNA adaptor, the first and third DNA adaptors having regions complementary to the ends generated by a first restriction endonuclease ends but differing in overall sequence and the second DNA adaptor having a region complementary to the ends generated by a second restriction endonuclease, the second restriction endonuclease site corresponding to the polymo ⁇ hic restriction site; and (b) a first primer, a second primer, and a third primer, the first primer being complementary to the first DNA adaptor, the second primer being complementary to the second DNA adaptor and being tagged with a first member of a specific binding pair, and the third primer being complementary to the third DNA adaptor.
  • This kit may further contain the second member of the specific binding pair immobilized on a solid support.
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the first primer is tagged with a detectable label
  • the amplifying generates a PCR product containing a first strand tagged with the detectable label and an unlabeled second strand.
  • the PCR product is then digested with a restriction endonuclease corresponding to the polymo ⁇ hic restriction site to generate a digestion product, which is denatured to generate a denatured product.
  • the denatured product is contacted with a first probe, which contains a sequence that hybridizes to a first sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the first probe is also immobilized on a first binding element.
  • the first binding element is monitored for the presence of the detectable label, and detection of the detectable label on the first binding element indicates the absence of the polymo ⁇ hic restriction site in the nucleic acid, and a failure to detect the detectable label on the first binding element indicates the presence of the polymo ⁇ hic restriction site in the nucleic acid.
  • the first binding element is a region on a solid support, such as a glass plate or a microchip.
  • This method can also include contacting the denatured product with a second, a third, or a fourth probe.
  • the second probe which is immobilized on a second binding element, contains a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that corresponds to the first primer.
  • the third probe which is immobilized on a third binding element, contains a sequence that hybridizes to a third sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand corresponding to the second primer.
  • the fourth probe which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the second, third, or fourth binding element can be monitored for the presence of the detectable label.
  • the first, second, third, and fourth binding elements can each be distinct regions on a solid support, such as a glass plate or a microchip.
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the first primer is tagged with a first detectable label and the second primer is tagged with a second detectable label.
  • the amplifying generates a PCR product containing a first strand tagged with the first detectable label and a second strand tagged with the second detectable label.
  • the first and second detectable labels can be identical or distinct.
  • the PCR product is treated with a restriction endonuclease corresponding to the polymo ⁇ hic restriction site to generate a digestion product, which is denatured to generate a denatured product.
  • the denatured product is contacted with a first and a second probe.
  • the first probe which is immobilized on a first binding element, contains a sequence that hybridizes to a first sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the second probe which is immobilized on a second binding element, contains a sequence that hybridizes to a second sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the first and second binding elements can each be distinct regions on a solid support, such as a glass plate or a microchip.
  • the first binding element is monitored for the presence of the first detectable label and the second binding element is monitored for the presence of the second detectable label. Detection of the first detectable label on the first binding element and detection of the second detectable label on the second binding element indicates the absence of the polymo ⁇ hic restriction site in the nucleic acid, and a failure to detect the first detectable label on the first binding element and a failure to detect the second detectable label on the second binding element indicates the presence of the polymo ⁇ hic restriction site in the nucleic acid.
  • This method can also include contacting the denatured product with a third or a fourth probe.
  • the third probe which is immobilized on a third binding element, contains a sequence that hybridizes to a third sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand corresponding to the first primer.
  • the fourth probe which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand corresponding to the second primer.
  • the third or fourth binding element can be monitored for the presence of the first or second detectable label.
  • the first, second, third, and fourth binding elements can be each distinct regions on a solid support, such as a glass plate or a microchip.
  • the invention features a method for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site. The amplifying generates a PCR product containing a first strand containing a sequence corresponding to the first primer and a second strand containing a sequence corresponding to the second primer.
  • the PCR product is treated with a restriction endonuclease corresponding to the polymo ⁇ hic restriction site to generate a digestion product, which is denatured to generate a denatured product.
  • the denatured product is contacted with an oligonucleotide to generate a first reaction product.
  • the oligonucleotide contains a 3' portion that hybridizes to a first region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence corresponding to the first primer.
  • the oligonucleotide is blocked so that it cannot serve as a primer for DNA polymerase.
  • the oligonucleotide contains a
  • the first reaction product is treated with a DNA polymerase to extend the unblocked, primed 3' end to generate a second reaction product, which is amplified by
  • the first and the second detectable labels in this method can be identical or distinct.
  • the second PCR product is denatured to generate a second denatured product, which is contacted with a first and a second probe.
  • the first probe which is immobilized on a first binding element, contains a sequence that hybridizes to a first sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the second probe which is immobilized on a second binding element, contains a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the first and second binding elements can each be distinct regions on a solid support, such as a glass support or a microchip.
  • the first binding element is monitored for the presence of the second detectable label and the second binding element is monitored for the presence of the first detectable label. Detection of the second detectable label on the first binding element and detection of the first detectable label on the second binding element indicates a heterozygote, detection of the second detectable label on the first binding element and a failure to detect the first detectable label on the second binding element indicates a homozygote containing the polymo ⁇ hic restriction site, and detection of the first detectable label on the second binding element and a failure to detect the second detectable label on the first binding element indicates a homozygote lacking the polymo ⁇ hic restriction site.
  • This method can also include contacting the second denatured product with a third or a fourth probe.
  • the third probe which is immobilized on a third binding element, contains a sequence that hybridizes to a third sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand corresponding to the first primer.
  • the fourth probe which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand corresponding to the second primer.
  • the third or fourth binding element can be monitored for the presence of the first or second detectable label.
  • the first, second, third, and fourth binding elements can each be distinct regions on a solid support, such as a glass plate or a microchip.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • the kit can contain one or more sets of a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the first primer is tagged with a detectable label, so that amplifying the nucleic acid by PCR with the first and second primers generates a PCR product containing a first strand tagged with the detectable label and a second strand.
  • the kit also can include one or more first probes, each of which containing a sequence that hybridizes to a first sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • Each of the first probes is immobilized on a first binding element.
  • This kit can also contain one or more sets of a second, third, or fourth probe.
  • Each of the second probes, which is immobilized on a second binding element contains a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that corresponds to the first primer.
  • Each of the third probes, which is immobilized on a third binding element contains a sequence that hybridizes to a third sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand co ⁇ esponding to the second primer.
  • Each of the fourth probes which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the first binding element in this kit can be a region on a solid support, such as a glass plate or a microchip.
  • the first, second, third, and fourth binding elements can each be distinct regions on a solid support, such as a glass plate or a microchip.
  • One or more second primers in this kit can each contain a second detectable label.
  • the kit can further contain a second probe, which is immobilized on a second binding element, and contains a sequence that hybridizes to a second sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the kit can also contain one or more sets of a third or a fourth probe.
  • Each of the third probes, which are immobilized on a third binding element contain a sequence that hybridizes to a third sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand co ⁇ esponding to the first primer.
  • each of the fourth probes which are immobilized on a fourth binding element, contain a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand corresponding to the second primer.
  • the kit can further contain one or more second probes, which are immobilized on a second binding element, that each contain a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that co ⁇ esponds to the first primer.
  • the invention features a kit for detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • This kit can contain one or more sets of a first and a second PCR primer flanking the polymo ⁇ hic restriction site.
  • the first primer can be tagged with a first detectable label, so that amplifying the nucleic acid by PCR using the first and second primers generates a PCR product containing a first strand tagged with the first detectable label and a second strand.
  • the first primer can be unlabeled.
  • This kit can also include one or more oligonucleotides containing a 3' portion that hybridizes to a first region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence co ⁇ esponding to the first primer.
  • the oligonucleotide is blocked so that it cannot serve as a primer for DNA polymerase.
  • the oligonucleotide contains a 5' portion that does not hybridize to a second region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence that is complementary to the second primer.
  • This kit can also include one or more third primers, each of which that hybridizes to a sequence that is complementary to the 5' portion of the oligonucleotide. The third primer can be tagged with a second detectable label.
  • kits are also included in this kit.
  • Each of the first probes which are immobilized on a first solid support, contain a sequence that hybridizes to a first sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • Each of the second probes which are immobilized on a second solid support, contain a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the first and the second detectable labels in this kit can be distinct or identical.
  • the first and second binding elements can each be distinct regions on a solid support, such as a glass support or a microchip.
  • This kit can further contain one or more sets of a third or a fourth probe.
  • Each of the third probes, which are immobilized on a third binding element contain a sequence that hybridizes to a third sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand co ⁇ esponding to the first primer.
  • Each of the fourth probes, which are immobilized on a fourth binding element contain a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand co ⁇ esponding to the second primer.
  • the one or more sets of the first, second, third, and fourth binding elements in this kit can each be distinct regions on a solid support, such as a glass plate or a microchip.
  • the detectable label is selected from the group consisting of digoxigenin, fluorescent labels (e.g., fluorescein and rhodamine), enzymes (e.g., horseradish peroxidase and alkaline phosphatase), biotin (which can be detected by anti-biotin specific antibodies or enzyme-conjugated avidin derivatives), radioactive labels (e.g., 2 P and 125 I), colorimetric reagents, and chemiluminescent reagents.
  • fluorescent labels e.g., fluorescein and rhodamine
  • enzymes e.g., horseradish peroxidase and alkaline phosphatase
  • biotin which can be detected by anti-biotin specific antibodies or enzyme-conjugated avidin derivatives
  • radioactive labels e.g., 2 P and 125 I
  • colorimetric reagents e.g., 2 P and 125 I
  • chemiluminescent reagents
  • the specific binding pairs are selected from the group consisting of avidin-biotin, streptavidin- biotin, hybridizing nucleic acid pairs, interacting protein pairs, antibody-antigen pairs, reagents containing chemically reactive groups (e.g., reactive amino groups), and nucleic acid sequence-nucleic acid binding protein pairs.
  • the solid supports used in the methods of the invention are selected from the group consisting of agarose, acrylamide, and polystyrene beads; polystyrene microtiter plates (for use in, e.g., ELISA); silicon, gold, or glass chips (e.g., microchips), slides, or plates; and nylon and nitrocellulose membranes (for use in, e.g., dot or slot blot assays).
  • heterozygote refers to an individual with different alleles at co ⁇ esponding loci on homologous chromosomes. Accordingly, the term “heterozygous,” as used herein, describes an individual or strain having different allelic genes at one or more paired loci on homologous chromosomes.
  • homozygote refers to an individual with the same allele at co ⁇ esponding loci on homologous chromosomes. Accordingly, the term “homozygous,” as used herein, describes an individual or a strain having identical allelic genes at one or more paired loci on homologous chromosomes.
  • co ⁇ esponding as used herein to describe a nucleic acid strand, e.g., a nucleic acid strand co ⁇ esponding to a particular PCR primer, is meant to indicate that the strand contains the sequence of the particular PCR primer.
  • the term When used to compare a polymo ⁇ hic restriction site to a restriction endonuclease site, the term again indicates that the two sequences are identical.
  • An advantage of certain detection methods of the present invention over many other methods used to detect genetic polymo ⁇ hisms is that gel electrophoresis is not required in the analysis.
  • the methods of the present invention are readily adaptable for automation, allowing large numbers of samples to be processed in relatively short periods of time, at lower costs.
  • detection of an a ⁇ ay of samples is carried out simultaneously on a solid support, such as a glass slide or a microchip, further reducing processing time and cost. Detection of signals on a ⁇ ays can be carried out quantitatively or qualitatively.
  • Those detection methods of the invention utilizing gel electrophoresis are also advantageous because they provide a rapid and inexpensive approach to the identification of large numbers of PCR-based genetic and RFLP markers.
  • the method of the invention useful for cloning genetic polymo ⁇ hisms also represents an improvement over cu ⁇ ent methods. Because the process of selecting out a tagged (e.g., biotinylated) DNA having a polymo ⁇ hism involves a specific hybridization step, candidate DNA from any source may be utilized. For example, DNA from random clones, CDNA libraries, YAC libraries, or any other DNA collection may be screened; pure preparations of genomic DNAs are not required. Moreover, like other methods of the invention, this cloning procedure is rapid and inexpensive.
  • All methods of the invention are useful in clinical diagnostic testing, genomic mapping, positional cloning of genes defined by mutation (such as those that cause inherited disease in humans or resistance to pathogens in crop plants), DNA finge ⁇ rinting (e.g., for forensic analysis and paternity testing), crop and livestock breeding programs, and other related applications.
  • the detection methods of the invention are useful for bacterial typing utilizing known conserved polymo ⁇ hic sequences diagnostic of the bacterium.
  • this approach is useful for distinguishing one bacterium from another (e.g., for the identification of Salmonella in a food sample); polymo ⁇ hism-containing sequences prefe ⁇ ed for this approach include those present in conserved ribosomal RNA genes.
  • this approach is useful for screening bacteria (e.g., clinical isolates) for antibiotic resistance; in this case, known polymo ⁇ hic restriction sites within the antibiotic resistance marker are utilized.
  • the instant methods of bacterial typing decrease false positive results frequently obtained using cu ⁇ ent PCR-based techniques.
  • Fig. 1 is a schematic of a RFLP detection method involving the use of a first PCR primer tagged with a detectable label (X) and a second PCR primer tagged with the first member of a specific binding pair (Y).
  • the products are digested with the restriction endonuclease (R) co ⁇ esponding to the polymo ⁇ hic restriction site, contacted with the second member of the specific binding pair immobilized on a solid support, and the level of the detectable label (X) bound to the solid support is determined.
  • R restriction endonuclease
  • Fig. 2 is a schematic of a RFLP detection method involving the use of a first PCR primer tagged with a first detectable label (X) and a second PCR primer tagged with the first member of a specific binding pair (Y).
  • the products are digested with the restriction endonuclease (R) co ⁇ esponding to the polymo ⁇ hic restriction site, and an oligonucleotide tagged with a second detectable label (Z) is annealed and ligated to the single-stranded ends generated in the digestion.
  • R restriction endonuclease
  • Z second detectable label
  • Fig. 3 is a schematic of a RFLP detection method involving the use of a first
  • PI detectable label
  • P2 second unlabeled PCR primer
  • R restriction endonuclease
  • Both digested and undigested reactions are then denatured and contacted with an oligonucleotide tagged with the first member of a specific binding pair, the oligonucleotide being complementary to the PI strand and located to the right of the restriction site (R) near to, but not overlapping, primer P2.
  • the reactions are then contacted with the second member of the specific binding pair immobilized on a solid support, and the levels of PI in digested versus undigested reactions are compared.
  • Fig. 4 is a schematic of a RFLP detection method involving the use of a first PCR primer tagged with a first detectable label (PI) and a second PCR primer tagged with a second detectable label (P2).
  • the products are digested with the restriction endonuclease (R) co ⁇ esponding to the polymo ⁇ hic restriction site, denatured, and contacted with a first oligonucleotide complementary to the PI strand and located to the right of the restriction site (R) near to, but not overlapping primer P2, and a second oligonucleotide complementary to the P2 strand and located to the right of the restriction site (R) near to, but not overlapping the sequence complementary to primer P2.
  • R restriction endonuclease
  • Both the first and second oligonucleotides are tagged with the first member of a specific binding pair (Y).
  • the reactions are then contacted with the second member of the specific binding pair immobilized on a solid support, and the ratio of PI to P2 bound to the solid support is determined.
  • Fig. 5 is a schematic of a RFLP detection method involving the use of a first
  • PCR primer tagged with a detectable label X
  • Y first specific binding pair
  • the products are digested with the restriction enzyme (R) co ⁇ esponding to the polymo ⁇ hic restriction site, and are contacted with the second member of the first specific binding pair immobilized on a first solid support.
  • Fig. 6 is a schematic of a RFLP detection method involving the use of a first unlabeled PCR primer and a second PCR primer tagged with a detectable label (X).
  • the products are digested with the restriction enzyme (R) co ⁇ esponding to the polymo ⁇ hic restriction site, and contacted with an oligonucleotide complementary to the single-stranded ends generated in the digestion, the oligonucleotide being tagged with the first member of a specific binding pair.
  • R restriction enzyme
  • the products are then contacted with the second member of the first specific binding pair, bound to a first solid support.
  • the filtrate is then bound to a solid support with the anchor sequence (or contacted with an oligonucleotide complementary to the X strand, the oligonucleotide being tagged with the first member of a second specific binding pair, and then contacted with the second member of the second specific binding pair immobilized on a second solid support), and the levels of the detectable label bound to the first solid support and the anchor sequence (or second solid support) are determined.
  • Fig. 7 is a schematic of a RFLP detection method involving the use of PCR primers flanking the polymo ⁇ hic restriction site (the "Alu I" site). Following PCR amplification, the reaction products are digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site (Alu I), and the fragments are run on an agarose gel. The separated fragments are detected as an indication of the presence or absence of the polymo ⁇ hic marker.
  • Fig. 8 is a schematic of a typical gel analysis according the method described in
  • Figs. 9A-9E are schematics of a method for cloning polymo ⁇ hic restriction fragments.
  • Fig. 10 is a schematic of a non-gel based method for detection of CAPS markers.
  • Fig. 11 is a schematic of parallel processing of 100 CAPS markers on a microchip containing an a ⁇ ay of oligonucleotide probes.
  • Fig. 12 is a schematic of a method for detecting a cleaved end of a CAPS marker.
  • Fig. 13 is a schematic of a method for distinguishing heterozygous CAPS alleles from homozygous CAPS alleles, involving the use of the method for detecting a cleaved end of a CAPS marker shown in Fig. 12.
  • the present invention provides several methods for detecting Cleaved Amplified Polymo ⁇ hic Sequences (CAPS; Konieczny et al., The Plant Journal 4(2):403-410, 1993).
  • CAPS Cleaved Amplified Polymo ⁇ hic Sequences
  • a nucleic acid containing a polymo ⁇ hic restriction site is amplified using primers flanking the restriction site. The resulting
  • PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and the digested products are analyzed by gel electrophoresis.
  • the detection methods of the present invention vary greatly from one another in detail, however they share three central features: (1) the nucleic acid containing the polymo ⁇ hic restriction site is amplified by PCR using differently labeled primers flanking the polymo ⁇ hic restriction site, (2) the resulting PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site (which will cleave the DNA of some individuals but not cleave the DNA of others, depending on the presence of the polymo ⁇ hism), and (3) the resulting digestion products are analyzed by detection of the labels they contain, and/or labels attached to oligonucleotides complementary to the digestion products, in order to determine the identity of the polymo ⁇ hic restriction site.
  • the methods of the invention allow rapid and efficient analyses of a large number of samples.
  • the nucleic acid sample containing the polymo ⁇ hic restriction site being analyzed can be obtained from any source, e.g., a tissue homogenate, blood, amniotic fluid, chorionic villus samples, and a bacterial culture; and can be obtained from these sources using standard methods. Only a minute quantity of nucleic acid is required, and can be DNA or RNA (in the case of RNA, a reverse transcription step is required before the PCR step).
  • PCR methods used in the methods of the present invention are ca ⁇ ied out using standard methods (see, e.g., Ausubel et al., Cu ⁇ ent Protocols in Molecular Biology, John Wiley and Sons, New York, 1989; Erlich, PCR Technology, Stockton Press, New York, 1989; Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Harcourt Brace Javanovich, New York, 1990; Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989). Restriction enzyme digestion is also carried out by standard methods using any of a number of available restriction endonucleases (see, e.g., Ausubel et al., supra; New England Biolabs,
  • the primers and oligonucleotides used in the methods of the present invention are preferably DNA, and can be synthesized using standard techniques and, when appropriate, detectably labeled using standard methods (Ausubel et al., supra).
  • Detectable labels that can be used to tag the primers and oligonucleotides used in the methods of the invention include, but are not limited to, digoxigenin, fluorescent labels (e.g., fluorescein and rhodamine), enzymes (e.g., horseradish peroxidase and alkaline phosphatase), biotin (which can be detected by anti-biotin specific antibodies or enzyme-conjugated avidin derivatives), radioactive labels (e.g., 32 P and 125 I), colorimetric reagents, and chemiluminescent reagents.
  • the labels used in the methods of the invention are detected using standard methods.
  • the specific binding pairs useful in the methods of the invention include, but are not limited to, avidin-biotin, streptavidin-biotin, hybridizing nucleic acid pairs, interacting protein pairs, antibody-antigen pairs, reagents containing chemically reactive groups (e.g., reactive amino groups), and nucleic acid sequence-nucleic acid binding protein pairs.
  • the solid supports useful in the methods of the invention include, but are not limited to, agarose, acrylamide, and polystyrene beads; polystyrene microtiter plates (for use in, e.g., ELISA); and nylon and nitrocellulose membranes (for use in, e.g., dot or slot blot assays).
  • Some methods of the invention employ solid supports containing a ⁇ ays of nucleic acid probes.
  • solid supports made of materials such as glass (e.g., glass plates), silicon or silicon-glass (e.g., microchips), or gold (e.g., gold plates) can be used.
  • Methods for attaching nucleic acid probes to precise regions on such solid surfaces e.g., photolithographic methods, are well known in the art, and can be used to make solid supports for use in the invention.
  • kits which contain the reagents required for carrying out the assays.
  • the kits can contain reagents for carrying out the analysis of a single polymo ⁇ hic restriction site (for use in, e.g., diagnostic methods) or multiple polymo ⁇ hic restriction sites (for use in, e.g., genomic mapping).
  • multiple sets of the appropriate primers and oligonucleotides are provided in the kit.
  • the kits may contain the enzymes used in the methods, and the reagents for detecting the labels, e.g., the substrates for enzyme labels, etc.
  • the kits can also contain solid substrates for used in carrying out the method of the invention.
  • the kits can contain solid substrates, such as glass plates or silicon or glass microchips, containing a ⁇ ays of nucleic acid probes.
  • the invention provides methods and kits for generating and detecting the presence or absence of a polymo ⁇ hic restriction site in a nucleic acid.
  • Examples I-IX and XII-XIV describe eight variations of the methods of the invention.
  • Example X describes a prefe ⁇ ed use for the methods of the invention.
  • Example XI describes a prefe ⁇ ed method for cloning polymo ⁇ hic restriction fragments.
  • the following examples are meant to illustrate, but not limit, the methods of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters of molecular biology which are obvious to those skilled in the art are within the spirit and scope of the present invention.
  • the nucleic acid containing the polymo ⁇ hism is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a specific binding pair, the second primer being tagged with a detectable label.
  • the resulting PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site and the digested products are contacted with the second member of the specific binding pair, immobilized on a solid support. The level of the detectable label bound to the solid support is then measured.
  • the presence of the detectable label bound to the solid support is an indication of the absence of the polymo ⁇ hic restriction site in the nucleic acid, while the absence of the detectable label bound to the solid support is an indication of the presence of the polymo ⁇ hic restriction site in the nucleic acid.
  • An embodiment of this method is shown in Fig. 1.
  • Example II This method is identical to that described in Example I, with the added step of annealing and ligating to the single-stranded ends generated in the digestion reaction, an oligonucleotide tagged with a second detectable label. After applying the reaction to the second member of the specific binding pair, the levels of both the first and the second detectable labels bound to the solid support are determined.
  • the presence of only the first detectable label bound to the solid support is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • the presence of only the second detectable label bound to the solid support is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • the presence of both the first and the second detectable labels bound to the solid support is an indication of a heterozygote.
  • An embodiment of this method is shown in Fig. 2.
  • the cleaved ends of the CAPS products can be labeled by using a method described in further detail below in Example XIV. Briefly, in this method, the denatured product is contacted with an oligonucleotide to generate a first reaction product.
  • the oligonucleotide contains a 3' portion that hybridizes to a first region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymorphic restriction site containing a sequence co ⁇ esponding to the first primer.
  • the 3' end of the oligonucleotide is blocked by, e.g., a di-deoxynucleotide, so that it cannot serve as a primer for DNA polymerase.
  • the oligonucleotide contains a 5' portion that does not hybridize to a second region in the first strand that flanks the polymorphic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence that is complementary to the second primer.
  • the use of such an oligonucleotide to label a cleaved end of a CAPS marker is illustrated in Fig. 12. This method can also be applied to the CAPS detection techniques of, for example, Examples VI and VIII.
  • the nucleic acid is amplified using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled.
  • a portion of the PCR reaction is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, while another portion is left undigested. Both the digested and undigested portions are then denatured, and contacted with an oligonucleotide tagged with the first member of a specific binding pair.
  • the oligonucleotide is complementary to a sequence in the strand of the PCR product containing the detectable label, the sequence being between the polymo ⁇ hic restriction site and the sequence complementary to the second primer.
  • the reaction is then contacted with the second member of the specific binding pair, immobilized on a solid support, and the ratio of the levels of the detectable label bound to the solid support between undigested and digested samples is determined.
  • a ratio of 1 :0 between equivalent portions of undigested and digested samples is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 1: 1 between equivalent portions of undigested and digested samples is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 2: 1 between equivalent portions of undigested and digested samples is an indication of a heterozygote. While the sample volumes used for detection and comparison need not be equivalent, the appropriate calculations must be carried out to account for this adjustment prior to determining the ratio of detectable label in digested and undigested samples. An embodiment of this method is shown in Fig. 3.
  • the nucleic acid is amplified by PCR using a first primer and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a first detectable label, and the second primer being tagged with a second detectable label.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, denatured, and contacting with a first and a second oligonucleotide.
  • the first oligonucleotide is complementary to a first sequence in the strand of the PCR product containing the first detectable label, the first sequence being between the polymo ⁇ hic restriction site and the sequence co ⁇ esponding to the first primer.
  • the first oligonucleotide is tagged with the first member of a first specific binding pair.
  • the second oligonucleotide is complementary to a second sequence in the strand of the PCR product containing the second detectable label.
  • the second sequence is on the same side of the polymo ⁇ hic restriction site as the first sequence, and is not contained within, or complementary to, either the first or the second primer.
  • the second oligonucleotide is tagged with the first member of a second specific binding pair.
  • a first portion of the reaction is then contacted with the second member of the first specific binding pair, immobilized on a first solid support, while a second portion of the reaction is contacted with the second member of the second specific binding pair, immobilized on a second solid support.
  • the ratio of the levels of the first and second detectable labels bound to the first and second solid supports is then determined.
  • a ratio of 1 :0 between equivalent amounts of the first and second portions is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 1 :1 between equivalent amounts of the first and second portions is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 2: 1 between equivalent amounts of the first and second portions is an indication of a heterozygote.
  • the ratios differ, as follows.
  • the ratio of the levels of the first and second detectable labels bound to the first and second solid supports is 0: 1 between equivalent amounts of the first and second portions in the case of a homozygote containing the polymo ⁇ hic restriction site.
  • the ratio is 1 : 1 between equivalent amounts of the first and second portions in the case of a homozygote lacking the polymo ⁇ hic restriction site, and the ratio is 1 :2 between equivalent amounts of the first and second portions in the case of a heterozygote.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and the reaction is then contacted with the second member of the first specific binding pair, immobilized on a first solid support.
  • the material not bound to the first solid support is denatured and contacted with an oligonucleotide complementary to a sequence in the strand of the PCR product containing the detectable label.
  • the sequence is between the polymo ⁇ hic restriction site and the sequence co ⁇ esponding to the second primer, and the oligonucleotide is tagged with the first member of a second specific binding pair.
  • the reaction is then contacted with the second member of the second specific binding pair, immobilized on a second solid support, and the ratio of the level of the detectable label bound to the first solid support compared to the level of the detectable label bound to the second solid support is determined.
  • a ratio of 0: 1 is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 1 :0 is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 1 : 1 is an indication of a heterozygote.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and a first oligonucleotide tagged with the first member of a first specific binding pair is annealed and ligated to the single-stranded ends generated in the digestion reaction.
  • the reaction is then contacted with the second member of the first specific binding pair, immobilized on a first solid support.
  • the material not bound to the first solid support is denatured, and contacted with a second oligonucleotide complementary to a sequence in the strand of the PCR product containing the detectable label, the sequence being between the polymo ⁇ hic restriction site and either the sequence co ⁇ esponding to the first primer or the sequence complementary to the second primer.
  • the second oligonucleotide is tagged with the first member of a second specific binding pair.
  • the reaction is then contacted with the second member of the second specific binding pair, immobilized on a second solid support, and the ratio of the level of the detectable label bound to the first solid support compared to the level of the detectable label bound to the second solid support is determined.
  • a ratio of 1 :0 is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 0:1 is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 1 : 1 is an indication of a heterozygote.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with the first member of a first specific binding pair, the second primer being tagged with a detectable label.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and contacted with the second member of the first specific binding pair, immobilized on a first solid support.
  • the material not bound to the first solid support is denatured and contacted with an oligonucleotide complementary to a sequence in the strand of the PCR product containing the detectable label.
  • the sequence is between the polymo ⁇ hic restriction site and the sequence co ⁇ esponding to the second primer, and the oligonucleotide is immobilized on a second solid support (e.g., a nylon or nitrocellulose membrane).
  • the ratio of the level of detectable label bound to the first solid support to the level of detectable label bound to the second solid support is then determined.
  • a ratio of 0: 1 is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 1 :0 is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 1 : 1 is an indication of a heterozygote.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, the first primer being tagged with a detectable label, the second primer being unlabeled.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and a first oligonucleotide tagged with the first member of a first specific binding pair is annealed and ligated to the single-stranded ends generated in the digestion reaction.
  • the reaction is contacted with the second member of the first specific binding pair, immobilized on a first solid support.
  • the material not bound to the first solid support is denatured, and contacted with a second oligonucleotide complementary to a sequence in the strand of the PCR product containing the detectable label.
  • the sequence is between the polymo ⁇ hic restriction site and either the sequence co ⁇ esponding to the first primer or the sequence complementary to the second primer, and the second oligonucleotide is immobilized on a second solid support (e.g., a nylon or nitrocellulose membrane).
  • the ratio of the level of the detectable label bound to the first solid support to the level of the detectable label bound to the second solid support is then determined.
  • a ratio of 1 :0 is an indication of a homozygote containing the polymo ⁇ hic restriction site
  • a ratio of 0: 1 is an indication of a homozygote lacking the polymo ⁇ hic restriction site
  • a ratio of 1 : 1 is an indication of a heterozygote.
  • PCR primers containing nucleic acid tags on their 5' ends can also be used in the methods of the invention. These primers can be used in pairs, or in combination with un-tagged primers, in the initial cycles of PCR, followed by the addition of a "universal primer(s)" complementary to the nucleic acid tags in the first primers, and contain detectable labels (e.g., biotin, fluorescent, or ELISA tags).
  • detectable labels e.g., biotin, fluorescent, or ELISA tags.
  • the use of nucleic acid tagged primers in the early rounds of PCR is a cost-effective measure, as only one set of primers, the universal primers, which can be used in the analysis of many different polymorphic sites, need to be detectably labeled.
  • the sets of primers specific for individual polymo ⁇ hic restriction sites do not have to be tagged with detectable labels, but rather need only to be complementary to the universal primers in their 5* ends.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site, and, as shown in Fig. 7, the digestion products are run on a gel (preferably an agarose gel).
  • the first and second primers are preferably designed to generate a PCR product that is easily resolvable on an agarose gel (e.g., preferably larger than 100 base pairs and smaller than 1000 base pairs), and the polymo ⁇ hic restriction site is preferably located at an asymmetric position within the amplified fragment.
  • primers are designed to produce PCR amplified products of 300 base pairs, and cleavage at the RFLP site yields products of 200 base pairs and 100 base pairs.
  • sets of primer pairs are provided that detect a number of RFLP markers.
  • Each set of primers may be provided, for example, in one of the wells of a 96-well microtiter plate, and PCR reactions run independently. Following restriction digestion, the reaction products are transfe ⁇ ed to an agarose gel and separated by electrophoresis. A typical result of this method is shown in Fig. 8.
  • Detection of the amplified and cleaved products after electrophoretic separation can be ca ⁇ ied out by standard methods of DNA staining (e.g., ethidium bromide staining) or blotting (e.g., Southern blotting).
  • DNA staining e.g., ethidium bromide staining
  • blotting e.g., Southern blotting
  • one or both of the PCR primers can be detectably labeled, and the labels can be detected as described above.
  • RFLP subtraction provides a large number of polymo ⁇ hic genetic markers, while the methods of the present invention provide efficient methods for their analysis.
  • RFLP subtraction results in the purification of fragments that are present in one population (the tracer) but absent in another (the driver). Purification is achieved by removing all of the fragments in the tracer DNA that have counte ⁇ arts in the driver DNA using subtractive hybridization (Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Harcourt Brace Javanovich, New York, 1990).
  • the tracer is a size fraction of digested DNA from one strain and the driver is the same size fraction from a polymo ⁇ hic strain.
  • the products obtained after removing the common sequences are RFLPs; they are sized tracer fragments whose driver counte ⁇ arts are not found in the same size fraction.
  • RFLP subtraction preparation of the driver and tracer, subtractive hybridization, and removal of non-hybridizing sequences from the tracer.
  • genomic DNA from two different strains is digested with a restriction endonuclease, and the ends of the restriction fragments from each strain are capped with different oligonucleotide adapters.
  • the low molecular weight fragments are then purified from a slice of an agarose gel and amplified using one of the adapter strands as a PCR primer.
  • a biotinylated primer can be used to amplify the driver so that driver DNA can be removed following the subtractive hybridizations by binding to avidin coated beads.
  • Figure 9 shows a prefe ⁇ ed method for cloning polymo ⁇ hic restriction fragments.
  • the object of this method is to clone restriction fragments from organism B (generated by restriction endonuclease A) that do not contain cleavage sites for restriction endonuclease B, and which correspond to restriction fragments in organism A (generated by restriction endonuclease A) that do contain at least one restriction site for restriction endonuclease B.
  • These polymo ⁇ hic restriction fragments are useful as CAPS markers for the detection methods described above.
  • Fig. 9A genomic DNA isolated from polymo ⁇ hic individuals A and B is separately digested with restriction enzyme A, which preferably leaves so-called sticky ends.
  • restriction enzyme A which preferably leaves so-called sticky ends.
  • a different oligonucleotide adaptor (#3) is ligated to the restriction fragments from individual B.
  • Fig. 9B the restriction fragments from Fig. 9A are cleaved with restriction endonuclease B, which again preferably leaves sticky ends.
  • an oligonucleotide adaptor (#2), with complementary sticky ends for enzyme B, is ligated to the restriction fragments generated by cleavage with enzyme B.
  • the DNA fragments from individual A are amplified using the PCR with an oligonucleotide primer complementary to adaptor #1 and with a biotinylated oligonucleotide primer complementary to adaptor #2.
  • Fig. 9D the amplified products originating from individual A are mixed with the non-amplified fragments of Fig. 9B from individual B.
  • the mixed DNA fragments are then heat denatured, annealed, and adsorbed onto an avidin-coated solid support (e.g., beads).
  • the avidin coated support containing the adsorbed fragments is thoroughly washed.
  • the adsorbed fragments may be eluted, re-amplified with the same primers as above, adsorbed onto a fresh avidin-containing support, and thoroughly washed. This step can be repeated as many times as is necessary or desired.
  • the fragments adsorbed to the avidin-coated beads are eluted and amplified using PCR with primers complementary to adaptor #3.
  • the amplified products should co ⁇ espond to the desired restriction fragments described above.
  • amplified fragments are cloned and then tested individually using the Southern DNA blot hybridization method for their ability to display the desired RFLP.
  • Example XII the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site, with the first primer being tagged with a detectable label.
  • the amplification generates a PCR product containing a first strand tagged with the detectable label and a second, unlabeled strand.
  • the PCR product is digested with the restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site and the digestion product is denatured.
  • the denatured product is contacted with a first probe that (1) contains a sequence that hybridizes to a first sequence in the first strand of the PCR product, and (2) is immobilized on a first binding element.
  • the first sequence is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the first binding element is monitored for the presence of the detectable label.
  • this method can employ the use of a second, a third, or a fourth probe.
  • the second probe contains a sequence that hybridizes to a second sequence which is in the first strand and is between the polymo ⁇ hic restriction site and the sequence in the first strand that co ⁇ esponds to the first primer.
  • the third probe contains a sequence that hybridizes to a third sequence which is in the second strand and is between the polymo ⁇ hic restriction site and the sequence in the second strand co ⁇ esponding to the second primer.
  • the fourth probe contains a sequence that hybridizes to a fourth sequence which is in the second strand and is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the second, third, and fourth probes are immobilized on a second, third, and fourth binding element, respectively.
  • the second binding element can be monitored for the presence of the detectable label as a positive control, while the third or fourth binding elements can be monitored for the presence of the detectable label as negative controls.
  • the first, second, third, and fourth binding elements in this and in other methods of the invention, can be present on a solid support having similar sets of binding elements for testing different nucleic acids (see, for example, Fig. 1 1).
  • binding elements for example, the first, second, third, and fourth binding elements, used in this method of the invention can be present as distinct regions on a single solid support.
  • they can be specific sets of nucleic acids bound to distinct regions on a glass plate or on a microchip, such as a glass, silicon, or glass- silicon microchip (see above).
  • a nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the first primer is tagged with a first detectable label and the second primer is tagged with a second detectable label.
  • the amplification thus generates a PCR product containing a first strand tagged with the first detectable label and a second strand tagged with the second detectable label.
  • the first and second labels can be identical or distinct.
  • the PCR product is treated with a restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site to generate a digestion product, which is denatured to generate a denatured product.
  • the denatured product is contacted with a first and a second probe.
  • the first probe which is immobilized on a first binding element, contains a sequence that hybridizes to a first sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the second probe which is immobilized on a second binding element, contains a sequence that hybridizes to a second sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the first binding element is monitored for the presence of the first detectable label and the second binding element is monitored for the presence of the second detectable label. Detection of the first detectable label on the first binding element and detection of the second detectable label on the second binding element indicates the absence of the polymo ⁇ hic restriction site in the nucleic acid, while a failure to detect the first detectable label on the first binding element and a failure to detect the second detectable label on the second binding element indicates the presence of the polymo ⁇ hic restriction site in the nucleic acid.
  • this method can involve the use of a third and fourth probe.
  • the third probe which is immobilized on a third binding element, contains a sequence that hybridizes to a third sequence which is in the first strand and that is between the polymo ⁇ hic restriction site and the sequence in the first strand co ⁇ esponding to the first primer.
  • the fourth probe which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence which is in the second strand and that is between the polymo ⁇ hic restriction site and the sequence in the second strand co ⁇ esponding to the second primer.
  • the third or fourth binding elements can be monitored for the presence of the first or second detectable labels as controls.
  • the third binding element can be monitored for the presence of the first detectable label and the fourth binding element can be monitored for the presence of the second detectable label.
  • the first and second, or the first, second, third, and fourth binding elements can be present as distinct regions on a solid support, such as glass (e.g., a glass plate) or a microchip (e.g., a silicon or a silicon-glass microchip).
  • a solid support such as glass (e.g., a glass plate) or a microchip (e.g., a silicon or a silicon-glass microchip). Embodiments of this method are illustrated in Figs. 10 and 11.
  • the nucleic acid is amplified by PCR using a first and a second primer flanking the polymo ⁇ hic restriction site.
  • the amplification generates a PCR product containing a first strand containing a sequence co ⁇ esponding to the first primer and a second strand containing a sequence co ⁇ esponding to the second primer.
  • the PCR product is treated with a restriction endonuclease co ⁇ esponding to the polymo ⁇ hic restriction site to generate a digestion product, which is denatured to generate a denatured product.
  • the denatured product is contacted with an oligonucleotide to generate a first reaction product.
  • the oligonucleotide contains a 3' portion that hybridizes to a first region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence co ⁇ esponding to the first primer.
  • the 3' end of the oligonucleotide is blocked by, e.g., a di-deoxynucleotide, so that it cannot serve as a primer for DNA polymerase.
  • the oligonucleotide contains a 5' portion that does not hybridize to a second region in the first strand that flanks the polymo ⁇ hic restriction site on the side of the polymo ⁇ hic restriction site containing a sequence that is complementary to the second primer.
  • the use of such an oligonucleotide to label a cleaved end of a CAPS marker is illustrated in Fig. 12.
  • the first reaction product is treated with a DNA polymerase to extend the unblocked, primed 3' end to generate a second reaction product, which is amplified by PCR using the first primer, tagged with a first detectable label, and a third primer, which hybridizes to a sequence that is complementary to the 5' portion of the oligonucleotide, to generate a second PCR product.
  • the third primer is tagged with a second detectable label.
  • the first and second detectable labels can be identical or distinct.
  • the second PCR product is denatured to generate a second denatured product, which is contacted with a first and a second probe.
  • the first probe which is immobilized on a first binding element, contains a sequence that hybridizes to a first sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand that is complementary to the first primer.
  • the second probe which is immobilized on a second binding element, contains a sequence that hybridizes to a second sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand that is complementary to the second primer.
  • the first binding element is monitored for the presence of the second detectable label and the second binding element is monitored for the presence of the first detectable label. Detection of the second detectable label on the first binding element and detection of the first detectable label on the second binding element indicates a heterozygote, detection of the second detectable label on the first binding element and a failure to detect the first detectable label on the second binding element indicates a homozygote containing the polymo ⁇ hic restriction site, and detection of the first detectable label on the second binding element and a failure to detect the second detectable label on the first binding element indicates a homozygote lacking the polymo ⁇ hic restriction site.
  • this method can employ a third or a fourth probe.
  • the third probe which is immobilized on a third binding element, contains a sequence that hybridizes to a third sequence in the first strand that is between the polymo ⁇ hic restriction site and the sequence in the first strand co ⁇ esponding to the first primer.
  • the fourth probe which is immobilized on a fourth binding element, contains a sequence that hybridizes to a fourth sequence in the second strand that is between the polymo ⁇ hic restriction site and the sequence in the second strand co ⁇ esponding to the second primer.
  • the third or fourth binding elements can be monitored for the presence of the first or second detectable labels as controls.
  • the third binding element can be monitored for the presence of the first detectable label.
  • the first and second, or the first, second, third, and fourth binding elements can be present as distinct regions on a solid support, such as a glass (e.g., a glass plate) or silicon (e.g., a microchip) support.
  • a solid support such as a glass (e.g., a glass plate) or silicon (e.g., a microchip) support. This embodiment is illustrated in Fig. 13.
  • oligonucleotides as described above provides several advantages. For example, because there can be a significant amount of overlap between the oligonucleotide and the cleaved product, highly stringent conditions can be used in the annealing reaction, leading to increased specificity.
  • the 5' end of the oligonucleotide can be the same for many CAPS markers, as it is by design not homologous to any amplified sequences co ⁇ esponding to a CAPS marker for an organism of interest.

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  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention a pour objet des procédés de détection de sites de restriction polymorphes dans des acides nucléiques, ainsi que les nécessaires de mise en application de ces procédés.
PCT/US1997/016467 1996-09-18 1997-09-17 Procedes de detection des fragments de restriction polymorphes (rfpl) amplifies et clives WO1998012352A1 (fr)

Priority Applications (2)

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EP19970942525 EP0948645A4 (fr) 1996-09-18 1997-09-17 Procedes de detection des fragments de restriction polymorphes (rfpl) amplifies et clives
CA002266750A CA2266750A1 (fr) 1996-09-18 1997-09-17 Procedes de detection des fragments de restriction polymorphes (rfpl) amplifies et clives

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US08/715,484 1996-09-18
US08/715,484 US6004783A (en) 1994-03-18 1996-09-18 Cleaved amplified RFLP detection methods

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000028081A2 (fr) * 1998-11-09 2000-05-18 Methexis N.V. Analyse d'amplicon restreint
EP1025266A1 (fr) * 1998-03-27 2000-08-09 JOHNSON & JOHNSON RESEARCH PTY LIMITED Methodes de diagnostic a base d'acide nucleique catalyseur
WO2000050632A2 (fr) * 1999-02-22 2000-08-31 Lynx Therapeutics, Inc. Fragments d'adn polymorphes et leurs utilisations
WO2002016645A2 (fr) * 2000-08-21 2002-02-28 Lynx Therapeutics, Inc. Fragments d'adn polymorphiques et utilisations de ces derniers
WO2002050305A1 (fr) * 2000-12-20 2002-06-27 Murdoch Childrens Research Institute Procede permettant de detecter si un organisme est homozygote ou heterozygote a l'aide d'amorces marquees et du polymorphisme de longueur des fragments de restriction (rflp)
EP1337666A1 (fr) * 2000-11-01 2003-08-27 The General Hospital Corporation Typage de souches bacteriennes
AU2002215696B2 (en) * 2000-12-20 2007-07-19 Murdoch Childrens Research Institute Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP
EP2006393A2 (fr) * 2006-03-14 2008-12-24 Oryzon Genomics, S.A. Procede d'analyse d'acides nucleiques

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US4925785A (en) * 1986-03-07 1990-05-15 Biotechnica Diagnostics, Inc. Nucleic acid hybridization assays
US5523225A (en) * 1993-09-13 1996-06-04 Regents Of The University Of Colorado DNA sequence encoding human cystathionine β-synthase
US5629158A (en) * 1989-03-22 1997-05-13 Cemu Bitecknik Ab Solid phase diagnosis of medical conditions

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WO1995025538A1 (fr) * 1994-03-18 1995-09-28 The General Hospital Corporation Methodes de detection de polymorphismes amplifies et clives des sites de restriction

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US4775619A (en) * 1984-10-16 1988-10-04 Chiron Corporation Polynucleotide determination with selectable cleavage sites
US4925785A (en) * 1986-03-07 1990-05-15 Biotechnica Diagnostics, Inc. Nucleic acid hybridization assays
US5629158A (en) * 1989-03-22 1997-05-13 Cemu Bitecknik Ab Solid phase diagnosis of medical conditions
US5523225A (en) * 1993-09-13 1996-06-04 Regents Of The University Of Colorado DNA sequence encoding human cystathionine β-synthase

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Cited By (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1025266A4 (fr) * 1998-03-27 2002-10-29 Johnson & Johnson Res Pty Ltd Methodes de diagnostic a base d'acide nucleique catalyseur
EP1025266A1 (fr) * 1998-03-27 2000-08-09 JOHNSON & JOHNSON RESEARCH PTY LIMITED Methodes de diagnostic a base d'acide nucleique catalyseur
WO2000028081A3 (fr) * 1998-11-09 2000-08-03 Methexis N V Analyse d'amplicon restreint
WO2000028081A2 (fr) * 1998-11-09 2000-05-18 Methexis N.V. Analyse d'amplicon restreint
JP2002537774A (ja) * 1999-02-22 2002-11-12 リンクス セラピューティクス，インコーポレイテッド 多型ｄｎａフラグメントおよびその使用
AU779231B2 (en) * 1999-02-22 2005-01-13 Lynx Therapeutics, Inc. Polymorphic DNA fragments and uses thereof
JP4669614B2 (ja) * 1999-02-22 2011-04-13 ソレクサ・インコーポレイテッド 多型ｄｎａフラグメントおよびその使用
WO2000050632A3 (fr) * 1999-02-22 2001-03-29 Lynx Therapeutics Inc Fragments d'adn polymorphes et leurs utilisations
WO2000050632A2 (fr) * 1999-02-22 2000-08-31 Lynx Therapeutics, Inc. Fragments d'adn polymorphes et leurs utilisations
WO2002016645A3 (fr) * 2000-08-21 2003-04-03 Lynx Therapeutics Inc Fragments d'adn polymorphiques et utilisations de ces derniers
WO2002016645A2 (fr) * 2000-08-21 2002-02-28 Lynx Therapeutics, Inc. Fragments d'adn polymorphiques et utilisations de ces derniers
EP1337666A1 (fr) * 2000-11-01 2003-08-27 The General Hospital Corporation Typage de souches bacteriennes
EP1337666A4 (fr) * 2000-11-01 2005-01-19 Gen Hospital Corp Typage de souches bacteriennes
AU2002215696B2 (en) * 2000-12-20 2007-07-19 Murdoch Childrens Research Institute Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP
US7459271B2 (en) 2000-12-20 2008-12-02 Murdoch Childrens Research Institute Method for detecting whether an organism is homozygous or heterozygous using labelled primers and RFLP
WO2002050305A1 (fr) * 2000-12-20 2002-06-27 Murdoch Childrens Research Institute Procede permettant de detecter si un organisme est homozygote ou heterozygote a l'aide d'amorces marquees et du polymorphisme de longueur des fragments de restriction (rflp)
EP2006393A2 (fr) * 2006-03-14 2008-12-24 Oryzon Genomics, S.A. Procede d'analyse d'acides nucleiques
EP2006393A4 (fr) * 2006-03-14 2009-11-18 Oryzon Genomics Sa Procede d'analyse d'acides nucleiques

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EP0948645A4 (fr) 2002-11-05
CA2266750A1 (fr) 1998-03-26
EP0948645A1 (fr) 1999-10-13

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