Methods for analysis of molecular events

• US 7,776,524 B2
• Filed: 02/18/2003
• Issued: 08/17/2010
• Est. Priority Date: 02/15/2002
• Status: Expired due to Fees

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Genzyme Corporation (Sanofi )
• Inventors: Kann, Lisa, Shuber, Anthony P., Whitney, Duncan
• Primary Examiner(s): Benzion; Gary
• Assistant Examiner(s): Wilder; Cynthia B
• Application Number:
  US10/369,123
• Publication Number:
  US 20030203382A1
• Time in Patent Office: 2,737 Days
• Field of Search: 435/6
• US Class Current: 435/6.12
• 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 minimizing nucleotide misincorporation in a primer extension assay, the method comprising the steps of:

a) providing a heterogeneous nucleic acid sample comprising a target nucleic acid containing a predetermined position suspected of containing a first variant base and a second variant base;

b) contacting the heterogeneous nucleic acid sample with a primer complementary to a region adjacent to the predetermined position, a first nucleotide and a second nucleotide, wherein the first nucleotide is complementary to the first variant base and the second nucleotide is complementary to the second variant base and is a chain terminator;

c) providing a first polymerase that preferentially incorporates said first nucleotide and a second polymerase that preferentially incorporates said second nucleotide;

d) extending said primer in a primer extension reaction, wherein the first polymerase preferentially incorporates said first nucleotide into the extended primer on a template containing the first variant base at the predetermined position and the second polymerase preferentially incorporates said second nucleotide into the extended primer on a template containing the second variant base at the predetermined position, thereby reducing misincorporation of the first nucleotide into the extended primer on the template containing the second variant base.

2. The method of claim 1, wherein the second nucleotide is a dideoxynucleotide.

11. The method of claim 1, wherein the first nucleotide is labeled with a detectable signal.

12. The method of claim 1, wherein the first polymerase has between about 5% and a 100% preference for the first nucleotide relative to the second nucleotide.

13. The method of claim 1, wherein the second polymerase has between about 5% and a 100% preference for the second nucleotide relative to the first nucleotide.

3. A method for detecting the presence of a variation at a predetermined position in a heterogeneous nucleic acid sample, the method comprising the steps of:

a) contacting a heterogeneous nucleic acid sample with a primer substantially complementary to a region adjacent to the predetermined position suspected of containing a variant base, a first nucleotide complementary to the variant base, and a second nucleotide complementary to the wild-type base, wherein the first nucleotide is labeled with a detectable signal and the second nucleotide is a chain terminator,

b) providing a first polymerase that preferentially incorporates said first nucleotide, and a second polymerase that preferentially incorporates said second nucleotide,

c) performing a primer extension reaction to generate extended primers, and

d) detecting the presence of the detectable signal in the extended primers, wherein the presence of the signal indicates the presence of the variation at the predetermined position.

4. The method of claim 3, wherein the detectable signal is a fluorescent signal.

5. The method of claim 3, further comprising adding to the primer extension reaction a terminator nucleotide complementary to a nucleotide downstream from said predetermined position.

6. The method of claim 3, wherein the variant base is present in less than 10% of nucleic acid molecules in the heterogeneous nucleic acid sample.

7. The method of claim 3, wherein the variant base is present in less than 1% of nucleic acid molecules in the heterogeneous nucleic acid sample.

8. The method of claim 3, wherein the heterogeneous nucleic acid sample comprises nucleic acids prepared from cells, tissue, or body fluid.

9. The method of claim 8, wherein the cells are selected from blood cells, colon cells, buccal cells, cervicovaginal cells, epithelial cells, fetal cells, and/or cells present in tissue obtained by biopsy.

10. The method of claim 8, 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.