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Thermostable ligase mediated DNA amplification system for the detection of genetic diseases

  • US 5,830,711 A
  • Filed: 06/05/1995
  • Issued: 11/03/1998
  • Est. Priority Date: 05/03/1990
  • Status: Expired due to Term
First Claim
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1. A method for distinguishing a first nucleotide sequence which differs by at least a single base from a second nucleotide sequence comprising:

  • providing a sample potentially 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 but not to the second 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 thermocyclable 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, and the thermocyclable ligase to form an amplification mixture;

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

    -85°

    C., wherein the oligonucleotides of the first oligonucleotide set, when hybridized to the first nucleotide sequence, ligate to one another to amplify linearly a sequence of nucleotides complementary to the first nucleotide sequence and, when hybridized to the second nucleotide sequence, do not ligate together and individually separate from the second nucleotide sequence during the denaturation treatment; and

    detecting the presence of the first nucleotide sequence in the sample by detecting the presence of ligated oligonucleotides of the first oligonucleotide set.

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