Ligase/polymerase-mediated genetic bit analysis of single nucleotide polymorphisms and its use in genetic analysis
First Claim
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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Abstract
A method is provided for determining the identity of a nucleotide at a preselected site in a nucleic acid molecule. The method involves the incorporation of a nucleoside triphosphate that is complementary to the nucleotide present at the preselected site onto the terminus of a primer molecule, and their subsequent ligation to a second oligonucleotide. The reaction is monitored by detecting a specific label attached to the reaction'"'"'s solid phase or by detection in solution.
283 Citations
13 Claims
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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:
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(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. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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Specification