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Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis

  • US 5,952,174 A
  • Filed: 09/15/1997
  • Issued: 09/14/1999
  • Est. Priority Date: 02/07/1994
  • Status: Expired due to Term
First Claim
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1. A method for determining the identity of a nucleotide present at a preselected single nucleotide site in a single-stranded target nucleic acid molecule, said method employing a set of oligonucleotides having at least two members, a first and a second oligonucleotide, that hybridize to said target molecule, and comprising the steps:

  • (A) incubating said target molecule in the presence of said set of oligonucleotides, wherein said first oligonucleotide of said set is a primer oligonucleotide that hybridizes to a first region of said target molecule, such that a 3'"'"' terminus of said hybridized first oligonucleotide is immediately adjacent to the preselected site; and

    wherein said second oligonucleotide of said set hybridizes to a second region of said target molecule, such that the 5'"'"' terminus of said hybridized second oligonucleotide is separated from the 3'"'"' terminus of said first hybridized oligonucleotide by a single nucleotide gap at the position of said preselected site;

    (B) incubating said hybridized molecules, in the presence of a polymerase, and a nucleoside triphosphate mixture composed of dideoxynucleoside triphosphate species and a deoxynucleoside triphosphate species, such that regardless of the identity of the nucleotide of said preselected site, a template-dependent, polymerase-mediated extension reaction will occur, causing a nucleoside triphosphate species of said nucleoside triphosphate mixture, complementary to that of the nucleotide of the preselected site, to become incorporated onto the 3'"'"' terminus of said hybridized first oligonucleotide; and

    to thereby fill the gap between said hybridized first and second oligonucleotides and cause said oligonucleotides to abut;

    (C) incubating said hybridized molecules in the presence of a ligase under conditions sufficient to permit said ligase to ligate together abutting hybridized first and second oligonucleotides to thereby form a ligation product if the deoxynucleoside triphosphate species of said nucleoside triphosphate mixture has been incorporated onto the 3'"'"' terminus of said hybridized first oligonucleotide; and

    (D) detecting whether any ligation product is formed.

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