Methods for detecting mutations using primer extension for detecting disease
First Claim
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1. A method for detecting a nucleic acid insertion or deletion, the method comprising the steps of:
- (a) selecting a nucleic acid having a known wild-type sequence and having a target region comprising a repeat sequence having at most three different types of nucleotide bases selected from the group consisting of dGTP, dATP, dTTP, and dCTP;
(b) contacting a sample with an oligonucleotide primer that is complementary to a portion of said nucleic acid immediately upstream of said target region;
(c) extending said primer in the presence of nucleotide bases that are complementary to the nucleotide bases of the target region, thereby to form a primer extension product;
(d) extending the primer extension product in the presence of a labeled nucleotide complementary to a nucleotide base downstream from the target region in said nucleic acid, wherein said labeled nucleotide is not complementary to any of the nucleotide bases of the target region, thereby to produce a labeled extension product comprising a sequence that is complementary to the entire target region;
(e) detecting the labeled extension product; and
(f) comparing the size of the labeled extension product detected in step e) to a standard, wherein a labeled extension product smaller than the standard is indicative of the presence of a deletion in the target region and a labeled extension product larger than the standard is indicative of the presence of an insertion in the target region.
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Abstract
Methods of the invention comprise assays for markers indicative of cancer, precancer, and other diseases or disorders. Assays of the invention are preformed on heterogeneous samples obtained from patients by non-invasive or minimally-invasive methods. Such assays may be employed alone or in combination with other disease screening techniques.
19 Citations
43 Claims
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1. A method for detecting a nucleic acid insertion or deletion, the method comprising the steps of:
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(a) selecting a nucleic acid having a known wild-type sequence and having a target region comprising a repeat sequence having at most three different types of nucleotide bases selected from the group consisting of dGTP, dATP, dTTP, and dCTP;
(b) contacting a sample with an oligonucleotide primer that is complementary to a portion of said nucleic acid immediately upstream of said target region;
(c) extending said primer in the presence of nucleotide bases that are complementary to the nucleotide bases of the target region, thereby to form a primer extension product;
(d) extending the primer extension product in the presence of a labeled nucleotide complementary to a nucleotide base downstream from the target region in said nucleic acid, wherein said labeled nucleotide is not complementary to any of the nucleotide bases of the target region, thereby to produce a labeled extension product comprising a sequence that is complementary to the entire target region;
(e) detecting the labeled extension product; and
(f) comparing the size of the labeled extension product detected in step e) to a standard, wherein a labeled extension product smaller than the standard is indicative of the presence of a deletion in the target region and a labeled extension product larger than the standard is indicative of the presence of an insertion in the target region. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 17, 18, 20, 21, 22, 23, 27, 28, 29, 30, 31, 32)
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14. A method for detecting a nucleic acid deletion in a sample, the method comprising the steps of:
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a) selecting a nucleic acid with a known wild-type sequence and having a target region suspected of containing a deletion, wherein said target region comprises a repeat sequence and contains at most three different types of nucleotides selected from the group consisting of dGTP, dATP, dTTP, and dCTP;
b) hybridizing an oligonucleotide primer to a region upstream of said target region, in a nucleic acid sample;
c) contacting said hybridized oligonucleotide primer with an extension reaction mixture comprising;
i) the nucleotides that are complementary to the nucleotides in the target region, ii) a labeled nucleotide that is complementary to a nucleotide found downstream from the target region, but is not complementary to any nucleotide found within the target region, and iii) a terminator nucleotide that is complementary to a nucleotide found downstream from the target region, but is not complementary to any nucleotide found in the target region;
d) extending the hybridized oligonucleotide primer to generate a labeled extension product comprising a sequence that is complementary to the entire target region;
e) detecting the labeled extension product; and
f) comparing the size of the labeled extension product detected in step e) to a standard, wherein a labeled extension product smaller than the standard is indicative of the presence of a deletion in the target region, and a labeled extension product larger than the standard is indicative of the presence of an insertion.
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19. A method for diagnosing colorectal cancer or precancer, the method comprising the steps of:
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performing an assay to detect, in a stool sample from a patient, a nucleic acid mutation indicative of a colorectal lesion;
performing a sigmoidoscopy on said patient; and
diagnosing colorectal cancer or precancer in said patient if at least one of said assay and said sigmoidoscopy is positive.
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24. A method for localizing a colorectal lesion in a patient, the method comprising the steps of:
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performing an assay to detect, in a stool sample from a patient, a nucleic acid mutation indicative of said colorectal lesion;
performing a sigmoidoscopy on said patient;
diagnosing a proximal colonic lesion if said assay is positive for the mutation and said sigmoidoscopy is negative; and
diagnosing a distal colonic lesion if said sigmoidoscopy is positive and said assay is negative for the mutation.
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25. A method for diagnosing hereditary non-polyposis colorectal cancer, the method comprising the steps of:
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performing an assay to detect, in a stool sample from a patient, a nucleic acid mutation indicative of said hereditary non-polyposis colorectal cancer;
performing a colonoscopy on said patient; and
diagnosing hereditary non-polyposis colorectal cancer if said assay is positive and said colonoscopy reveals an adenoma.
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26. A method for determining whether a target nucleotide is present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target nucleotide position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to the target nucleotide;
(c) further extending said primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, but is not complementary to the target nucleotide, thereby to generate an extension product; and
(d) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target nucleotide is present at said genetic locus.
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33. A method for determining whether a target point mutation is present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target point mutation position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to a target nucleotide suspected to be present at said target position;
(c) further extending said primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, but is not complementary to the target nucleotide, thereby to generate an extension product; and
(d) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target point mutation is present at said genetic locus. - View Dependent Claims (34, 35, 37, 38)
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36. A method for identifying a target single nucleotide polymorphic variant present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of said genetic locus immediately upstream of a target single nucleotide polymorphic variant position;
(b) extending said primer in the presence of at least a first and a second differentially labeled nucleotide, said first labeled nucleotide being complementary to a first nucleotide suspected to be present at said target position, said second labeled nucleotide being complementary to a second nucleotide alternatively suspected to be present at said target position;
(c) further extending the primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target position, wherein said terminator nucleotide is not complementary to said first or second nucleotides, thereby to produce an extension product; and
(d) determining the identity of the labeled nucleotide present in said extension product, thereby to determine the identity of the target single nucleotide polymorphic variant present at the genetic locus.
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39. A method for determining whether a target single nucleotide polymorphic variant is present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target single nucleotide polymorphic variant position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to a nucleotide suspected to be present at said target position;
(c) further extending the primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from said target nucleotide, wherein said terminator nucleotide is not complementary to said target nucleotide, thereby to produce an extension product; and
(d) 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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40. A method for determining whether a target nucleotide deletion is present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target nucleotide deletion position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to the target nucleotide;
(c) further extending said primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, but is not complementary to the target nucleotide, thereby to generate an extension product; and
(d) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target nucleotide deletion is present at said genetic locus.
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41. A method for determining whether a target nucleotide insertion is present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target nucleotide insertion position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to the target nucleotide;
(c) further extending said primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, but is not complementary to the target nucleotide, thereby to generate an extension product; and
(d) determining whether said labeled nucleotide is present in said extension product, thereby to determine whether said target nucleotide insertion is present at said genetic locus.
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42. A method for quantifying the number of a nucleic acid having a target nucleotide present at a genetic locus of interest, the method comprising the steps of:
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(a) contacting a nucleic acid in a biological sample with a primer that is complementary to a portion of a genetic locus immediately upstream of a target nucleotide position;
(b) extending said primer in the presence of a labeled nucleotide that is complementary to target nucleotide, (c) further extending the primer in the presence of a terminator nucleotide that is complementary to a nucleotide downstream from the target nucleotide, wherein said terminator nucleotide is not complementary to said target nucleotide, thereby to form an extension product; and
(d) enumerating the number of extension products that comprise the labeled nucleotide, thereby determining the number of nucleic acids having the target nucleotide at the genetic locus. - View Dependent Claims (43)
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Specification