Primer extension methods for detecting nucleic acids
First Claim
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1. A method for identifying a single nucleotide in a nucleic acid sample, the method comprising the steps of:
- (a) annealing an oligonucleotide primer to a nucleic acid sample under conditions that promote exact complementary hybridization between said primer and a portion of a nucleic acid in said sample;
(b) extending said primer by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;
(c) separating said extended primer from said portion;
(d) repeating steps (a) through (c) at least once; and
(e) identifying said base incorporated into said extended primer in step (b), thereby to identify said single nucleotide.
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Abstract
Methods are provided for selective nucleic acid sequence detection in single base primer extension reactions of high sensitivity. These methods are useful for detecting small amounts of mutant nucleic acid in a heterogeneous biological sample. These methods are particularly useful for identifying individuals with gene mutations indicative of early colorectal cancer.
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Citations
24 Claims
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1. A method for identifying a single nucleotide in a nucleic acid sample, the method comprising the steps of:
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(a) annealing an oligonucleotide primer to a nucleic acid sample under conditions that promote exact complementary hybridization between said primer and a portion of a nucleic acid in said sample;
(b) extending said primer by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;
(c) separating said extended primer from said portion;
(d) repeating steps (a) through (c) at least once; and
(e) identifying said base incorporated into said extended primer in step (b), thereby to identify said single nucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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13. A method for identifying a single nucleotide in a nucleic acid sample, the method comprising the steps of:
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(a) annealing an oligonucleotide primer to a nucleic acid sample, wherein said primer is a segmented primer comprising two or more probes, wherein none of said probes alone is capable of serving as a primer for template-dependent extension but wherein, when said two or more probes anneal to said nucleic acid sample adjacent to each other, they are capable of priming template-dependent extension;
(b) extending said primer by a single base;
(c) separating said extended primer from said portion;
(d) repeating steps (a) through (c) at least once; and
(e) identifying said base incorporated into said extended primer in step (b), thereby to identify said single nucleotide. - View Dependent Claims (14, 15)
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16. A method for detecting the presence of a known single nucleotide polymorphic variant, said method comprising the steps of:
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(a) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a single nucleotide polymorphic variant;
(b) extending said primer by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;
(c) separating said primer from said nucleic acid;
(d) repeating steps (a) through (c) at least once; and
(e) identifying said single base, thereby to identify the polymorphic variant. - View Dependent Claims (17, 18)
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19. A method for identifying a single nucleotide polymorphic variant in a population, said method comprising the steps of:
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(a) obtaining a sample comprising nucleic acid from each member of a plurality of individuals in a population;
(b) annealing an oligonucleotide primer to a portion of said nucleic acid in each sample, said primer being complementary to a portion of the nucleic acid immediately upstream of a suspected single nucleotide polymorphic variant;
(c) extending said primer in each sample by a single base, wherein said extension reaction is performed in an extension solution comprising at least two different nucleotides;
(d) separating said primer from said nucleic acid in each sample;
(e) repeating steps (b) through (d) at least once;
(f) identifying said single base in each sample; and
(g) identifying a single nucleotide polymorphic variant as the presence of more than one base identified in step (f). - View Dependent Claims (20)
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21. A method for detecting the presence of a target nucleotide at a genetic locus of interest, said method comprising the steps of:
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(a) obtaining a sample comprising heterogeneous nucleic acid;
(b) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a target nucleotide at a genetic locus of interest;
(c) extending said primer in the presence of a labeled nucleotide that is complementary to said target nucleotide;
(d) further extending said primer in the presence of a terminator nucleotide, thereby to generate an extension product;
(e) separating said primer from said nucleic acid;
(f) repeating steps (b) through (e) at least once; and
(g) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target nucleotide is present at said genetic locus. - View Dependent Claims (22, 23)
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24. A method for detecting the presence of a target single nucleotide polymorphic variant at a genetic locus of interest, said method comprising the steps of:
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(a) obtaining a sample comprising heterogeneous nucleic acid;
(b) annealing an oligonucleotide primer to a portion of a nucleic acid suspected to be immediately upstream of a target single nucleotide polymorphic variant position;
(c) extending said primer in the presence of a labeled nucleotide that is complementary to a nucleotide suspected to be present at said target position;
(d) further extending said primer in the presence of an unlabeled terminator nucleotide, thereby to generate an extension product;
(e) separating said primer from said nucleic acid;
(f) repeating steps (b) through (e) at least once; and
(g) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target single nucleotide polymorphic variant is present at said genetic locus.
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Specification