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Thermostable ligase-mediated DNA amplifications system for the detection of genetic disease

  • US 5,494,810 A
  • Filed: 11/22/1994
  • Issued: 02/27/1996
  • Est. Priority Date: 05/03/1990
  • Status: Expired due to Term
First Claim
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1. A method for amplifying a first nucleotide sequence and a second nucleotide sequence which are complementary and together form separate strands of a double stranded DNA molecule, said method comprising:

  • providing a sample containing the first nucleotide sequence and the second nucleotide sequence;

    providing a first oligonucleotide set of at least two oligonucleotides suitable for ligation together at a first ligation junction and for hybridization without mismatch at the first ligation junction to the first nucleotide sequence, wherein the at least two oligonucleotides hybridize adjacent to one another on the first nucleotide sequence and have a hybridization temperature of about 50°

    C. to 85°

    C.;

    providing a second oligonucleotide set of at least two oligonucleotides suitable for ligation together at a second ligation junction and for hybridization without mismatch at the second ligation junction to the second nucleotide sequence, wherein the at least two oligonucleotides of the second oligonucleotide set hybridize adjacent to one another on the second nucleotide sequence and have a hybridization temperature of about 50°

    to 85°

    C.;

    providing a thermostable ligase which does not become irreversibly denatured and lose its catalytic activity when subjected to temperatures ranging from about 50°

    C. to 105°

    C.;

    blending the sample, the at least two oligonucleotides of the first set, the at least two oligonucleotides of the second set, and the thermostable ligase to form an amplification mixture; and

    subjecting the amplification mixture to a series of cycles comprising a denaturation treatment, wherein the ligated first oligonucleotide set is separated from the first nucleotide sequence and the litigated second oligonucleotide set is separated from the second nucleotide sequence, and a thermal hybridization treatment at a temperature of 50°

    -85°

    C., wherein the first oligonucleotide set hybridizes to the first nucleotide sequence and its oligonucleotides ligate to one another while the second oligonucleotide set hybridizes to the second nucleotide sequence and its oligonucleotides ligate to one another, to amplify exponentially the first and second nucleotide sequences in the DNA, wherein the at least two oligonucleotides of the second oligonucleotide set are complementary to the at least two oligonucleotides of the first oligonucleotide set with an oligonucleotide from the first oligonucleotide set complementing an oligonucleotide from the second oligonucleotide set with a single base overhang.

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