Method of detecting nucleotide polymorphism
First Claim
1. A method of detecting a single nucleotide polymorphism contained in a nucleic acid sample, comprising the steps of (a) applying a wild-type primer and one or two types of variant primer either simultaneously or separately together with DNA polymerase to a chromosome or fragment thereof including a specific single nucleotide polymorphism site contained in a sample, and (b) detecting the nucleotide polymorphism contained in the nucleic acid sample according to whether or not the primers are elongated, wherein the second nucleotide from the 3′
- end of the primer corresponds to the respective nucleotides anticipated in the nucleotide polymorphism site.
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Abstract
A method of detecting variation or polymorphism in a nucleic acid sequence, which is useful particularly in diagnosing a hereditary disease, analyzing nucleotide polymorphism, etc., and primers to be used therein. More particularly speaking, this method comprises treating a primer for wild type and one or two primers for variant with DNA polymerase either simultaneously or separately, and detecting the nucleotide polymorphism contained in the nucleic acid sample by judging whether or not the primers are extended, or whether or not the primers are amplified with a reverse primer. In this method, the 3′-end second bases of the primer for wild type and one or two primers for variant correspond to respective nucleotides anticipated in the nucleotide polymorphism site. Further, at least one of the bases from the 3′-end third position to the 5′-end is substituted by a base not complementary to the base in the chain hybridized with the primer in the chromosome or fragment. Furthermore, the not complementary base as described above differs from primer to primer.
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Citations
35 Claims
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1. A method of detecting a single nucleotide polymorphism contained in a nucleic acid sample, comprising the steps of
(a) applying a wild-type primer and one or two types of variant primer either simultaneously or separately together with DNA polymerase to a chromosome or fragment thereof including a specific single nucleotide polymorphism site contained in a sample, and (b) detecting the nucleotide polymorphism contained in the nucleic acid sample according to whether or not the primers are elongated, wherein the second nucleotide from the 3′ - end of the primer corresponds to the respective nucleotides anticipated in the nucleotide polymorphism site.
- View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)
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14. A method of detecting a single nucleotide polymorphism contained in a nucleic acid sample, comprising the steps of
(a) applying a wild-type primer and one or two types of variant primer either simultaneously or separately together with DNA polymerase to a chromosome or fragment thereof including a specific single nucleotide polymorphism site contained in a sample, and (b) detecting the nucleotide polymorphism contained in the nucleic acid sample according to whether or not the chromosome or fragment containing the specific single nucleotide polymorphism site is amplified, and wherein the second nucleotide from the 3′ - end of the primer corresponds to the respective nucleotides anticipated in the nucleotide polymorphism site.
- View Dependent Claims (15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25)
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26. A single nucleotide polymorphism detection reagent kit comprising a wild-type primer, one or two types of variant primer, DNA polymerase and 4 types of deoxynucleoside triphosphate (dNTP), wherein the second base from the 3′
- end of each primer corresponds to the respective nucleotides anticipated in the nucleotide polymorphism site.
- View Dependent Claims (27, 28, 29, 30)
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31. A single nucleotide polymorphism detection reagent kit comprising a wild-type primer, one or two types of variant primer, DNA polymerase, 4 types of deoxynucleoside triphosphate (dNTP) and a detection probe, wherein the second base from the 3′
- end of each primer corresponds to the respective nucleotides anticipated in the nucleotide polymorphism site.
- View Dependent Claims (32, 33, 34, 35)
Specification