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Quantative method for early detection of mutant alleles and diagnostic kits for carrying out the method

  • US 5,512,441 A
  • Filed: 11/15/1994
  • Issued: 04/30/1996
  • Est. Priority Date: 11/15/1994
  • Status: Expired due to Fees
First Claim
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1. A process for detection in a nucleic acid test sample taken from the genome of an organism of a mutant nucleotide sequence in a specific region of the genome, wherein said region can contain said mutant nucleotide sequence or a wild-type nucleotide sequence, wherein the test sample is suspected of containing a first genomic strand of nucleic acid having said region with the mutant nucleotide sequence together with a second genomic strand of nucleic acid having said region with the wild-type nucleotide sequence, wherein the first genomic strand, if present in the test sample, is present or is caused to be present in the form of a first genomic duplex consisting of the first genomic strand and a first complementary strand, and the second genomic strand is present or is caused to be present in the test sample in the form of a second genomic duplex consisting of the second genomic strand and a second complementary strand, the process comprising:

  • (i) a first amplification step comprising amplifying material in the first and second genomic duplexes present in the test sample in a first polymerase chain reaction in which upstream and downstream long tail primers, DNA polymerase, four ,different nucleotide triphosphates and a buffer are used in a repetitive series of reaction steps involving template denaturation, primer annealing and extension of annealed primers to form first and second synthesized nucleic acid duplexes, said first synthesized nucleic acid duplexes having said region with the mutant nucleotide sequence, and said second synthesized nucleic acid duplexes having said region with the wild-type nucleotide sequence, said upstream and downstream long tail primers being selected such that synthesized strands formed in the first polymerase chain reaction using the upstream and downstream long tail primers can anneal with Upstream and downstream short tail primers which do not anneal with any nucleic acid strands in the first or second genomic duplexes, said long tail upstream primers also being selected such that the second synthesized duplexes have a restriction site which is not present in said first synthesized duplexes due to the presence in said first synthesized duplexes of said region with the mutant nucleotide sequence, said restriction site being cleavable with a first restriction enzyme,ii) a first digestion step comprising treating at least a portion of the test sample containing the first and second synthesized duplexes with said first restriction enzyme whereby selectively to cleave said second synthesized duplexes while leaving said first synthesized duplexes uncleaved,iii) a second amplification step comprising amplifying material in the uncleaved first synthesized duplexes in a second polymerase chain reaction in which the upstream and downstream short tail primers are used selectively further to amplify nucleic acid strands not cleaved in step (ii) whereby to form further synthesized duplexes, said upstream and downstream short tail primers being selected such that they anneal with nucleic acid strands synthesized in said first amplification step but do not anneal with any strands of the first or second genomic duplexes whereby the upstream and downstream short tail primers can be used in the second amplification step selectively to amplify material in duplexes synthesized in the first amplification step but cannot amplify material in the first or second genomic duplexes, each of said upstream short tail primers being labelled with a first substance that binds tightly with a second substance such that upstream ends of the further synthesized duplexes bind to a supporting surface coated with the second substance, each of said downstream short tail primers being labelled with a detectable marker;

    iv) a binding step comprising causing contact between the test sample and the supporting surface coated with said second substance whereby further synthesized duplexes labelled with the first substance bind thereto;

    v) a second digestion step wherein the test sample is again treated with the first restriction enzyme selectively to cleave synthesized duplexes containing nucleic acid strands having said region with the wild type sequence; and

    vi) a detection step comprising washing to remove unbound duplexes and assaying for the detectable marker to detect the presence of the mutant nucleotide sequence on uncleaved duplexes bound to the supporting surface.

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