Methods for detecting mutations using primer extension for detecting disease

{
  "publication_number": "US6475738B2",
  "country": "US",
  "kind": "B2",
  "title": "Methods for detecting mutations using primer extension for detecting disease",
  "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.",
  "claims": [
    "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 polynucleotide 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 selected in step (a), 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.",
    "2. The method of claim 1, further comprising the step of terminating the primer extension product by incorporating a terminator nucleotide in said product that is complementary to a nucleotide downstream from the target region in a wild type nucleic acid, wherein said terminator nucleotide is not complementary to any of the nucleotides of the target region selected in step (a), said step of terminating the primer extension product being performed simultaneously with or immediately after step (d).",
    "3. The method of claim 2, wherein the labeled nucleotide and the terminator necleotide are the same.",
    "4. The method of claim 1, wherein the labeling reaction of step (d) is performed in the presence of labeled nucleotide and unlabeled nucleotide of the same type.",
    "5. The method of claim 4, wherein the ratio of labeled nucleotide base to unlabeled nucleotide base is 1:1.6 (unlabeled:labeled).",
    "6. The method of claim 4, wherein more than one nucleotide from step (d) is incorporated into the labeled extension product.",
    "7. The method of claim 1, wherein said sample contains a heterofeneous mixture of mutant nucleic acid having a deletion in the target region and wild type nucleic acid with no deletion in the target region.",
    "8. The method of claim 1, wherein said sample is selected from the group consisting of stool, homogenized stool, urine, semen, blood, saliva, sputum, cerebrospinal fluid, pancreatic juice, pus, and a spirate.",
    "9. The method of claim 1, wherein a deletion in the target region is indicative of the presence of cancerous or precancerous tissue in the biological sample.",
    "10. The method of claim 1, wherein said sample includes a buffer comprising at least 100 nM DTA.",
    "11. The method of claim 1, wherein said target region is the poly-A tract at the BAT-26 locus.",
    "12. The method of claim 1, wherein said target region is a microstatellite region.",
    "13. The method of claim 1, wherein the presence of a deletion in said target region is associated with the presence of a mutation at a separate genetic locus selected from the group consisting of APC, DCC, P53, and RAS."
  ],
  "description_excerpt": "The invention relates generally to methods of detecting cancer, precancer, or other diseases or disorders using nucleic acid markers.\n\nNumerous diseases are associated with disruptions in genomic stability. For example, sickle cell anemia, phenylketonuria, hemophilia, cystic fibrosis, and various cancers have been associated with one or more genetic mutation(s). Cancer is thought to arise from a multi-step process that typically involves multiple genetic mutations leading to uncontrolled cell growth. Many cancers are curable if detected early in their development. For example, colorectal cancers typically originate in the colonic epithelium, and are not extensively vascularized (and therefore not invasive) during early stages of development. The transition to a highly-vascularized, invasive and ultimately metastatic cancer commonly takes ten years or longer. If the presence of cancer is detected prior to extensive vascularization, surgical removal typically is an effective cure. However, colorectal cancer is often detected only upon manifestation of clinical symptoms, such as pain and bloody stool. Generally, such symptoms are present only when the disease is well established, and often after metastasis has occurred. Similarly, with the exception of the Pap smear for detection of pre-malignant cervical lesions, diagnostic screening methods for other types of cancer are best at detecting established disease. Increased knowledge of the molecular basis for disease has lead to a proliferation of screening assays capable of detecting disease-associated nucleic acid mutations.",
  "cpc": [
    "C12Q 1/6827",
    "C12Q 1/6886",
    "C12Q 1/703",
    "C12Q 2600/156",
    "Y02A 90/10"
  ],
  "ipc": [
    "C12Q 1/68",
    "C12Q 1/70"
  ],
  "assignees": [
    "Exact Sciences Corp"
  ],
  "inventors": [
    "Anthony P. Shuber",
    "William Pierceall"
  ],
  "filing_date": "2001-06-18",
  "publication_date": "2002-11-05",
  "grant_date": "2002-11-05",
  "priority_date": "1999-01-10",
  "application_number": "US-88354801-A",
  "family_id": "25049848",
  "cited_by_count": 91,
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