Methods for analysis of molecular events

• US 8,409,829 B2
• Filed: 06/09/2010
• Issued: 04/02/2013
• Est. Priority Date: 02/15/2002
• Status: Expired due to Fees

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Inventors: Shuber, Anthony P., Kann, Lisa, Whitney, Duncan
• Primary Examiner(s): Benzion, Gary
• Assistant Examiner(s): WILDER, CYNTHIA B
• Application Number:
  US12/797,397
• Publication Number:
  US 20100248252A1
• Time in Patent Office: 1,028 Days
• Field of Search: 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12Q 1/6858 Allele-specific amplification
• CPC Class Codes: C12Q 2525/186 incorporating a non-extendable or blocking moiety
• CPC Class Codes: C12Q 2527/125 Specific component of sample, medium or buffer
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

Methods and compositions are provided for detecting the presence of nucleic acid sequence variants in a subpopulation of nucleic acid molecules in a biological sample. These methods are particularly useful for identifying individuals with mutations indicative of cancer.

Claims

1. A method for detecting the presence of one or more variations at a predetermined region in a heterogeneous nucleic acid sample, the method comprising the steps of:

a) providing a heterogeneous nucleic acid sample comprising a target nucleic acid suspected of containing one or more variant bases at a predetermined region;

b) providing a primer substantially complementary to a region adjacent to the predetermined region, one or more nucleotides complementary to the to the corresponding one or more suspected variant bases, one or more chain terminators complementary to corresponding wild type bases in the predetermined region, wherein the one or more nucleotides complementary to the corresponding one or more suspected variant bases are labeled with distinctively detectable signals;

c) providing two or more different polymerases wherein the two or more different polymerases comprise a first polymerase that preferentially incorporates the one or more nucleotides complementary to the corresponding wild type base in the predetermined region and at least one additional polymerase that preferentially incorporates the one or more nucleotides complementary to the corresponding one or more suspected variant bases;

d) extending said primer in a primer extension reaction, under conditions such that the primer binds to the target nucleic acid and the first polymerase can incorporate the one or more chain terminators complementary to the corresponding wild type base in the predetermined region into an extended primer and the at least one additional polymerase can incorporate the one or more nucleotides complementary to the corresponding one or more suspected variant bases labeled with distinctively detectable signals into an extended primer; and

e) detecting the presence of the distinctively detectable signals in the extended primers, thereby determining the presence of the one or more variant bases in the predetermined region.

2. The method of claim 1, wherein the one or more variant bases are present in less than about 10% of nucleic acid molecules in the heterogeneous nucleic acid sample.

3. The method of claim 1, wherein the one or more variant bases are present in less than about 1% of nucleic acid molecules in the heterogeneous nucleic acid sample.

4. The method of claim 1, wherein the heterogeneous nucleic acid sample comprises nucleic acids prepared from cells, tissue, or body fluid.

5. The method of claim 4, wherein the cells are selected from blood cells, colon cells, buccal cells, cerivcovaginal cells, epithelial cells, fetal cells, and/or cells present in tissue obtained by biopsy.

6. The method of claim 4, wherein the body fluid is selected from blood, sputum, pancreatic fluid, bile, lymph, plasma, urine, cerebrospinal fluid, seminal fluid, saliva, breast nipple aspirate, pus, amniotic fluid and/or stool.