Methods for analysis of molecular events

• US 20030203382A1
• Filed: 02/18/2003
• Published: 10/30/2003
• Est. Priority Date: 02/15/2002
• Status: Active Grant

• Current Assignee: Esoterix Genetic Laboratories LLC (Laboratory Corporation Of America Holdings)
• Original Assignee: Exact Sciences Corporation
• Inventors: Shuber, Anthony P., Kann, Lisa, Whitney, Duncan
• Granted Patent: US 7,776,524 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 a variant in a primer extension assay, the method comprising the steps of:

a) contacting a target nucleic acid with a nucleic acid primer complementary to a region of said target nucleic acid; and

b) extending said primer in the presence of;

i) a first nucleotide that is complementary to a first variant nucleotide suspected to be at a position downstream of said region; and

,

ii) a second nucleotide that is complementary to a second variant nucleotide at said position, thereby to reduce misincorporation of said first nucleotide on a template comprising said second variant nucleotide..

2. The method of claim 1 wherein said first nucleotide is a terminator nucleotide.

3. The method of claim 1 wherein said second nucleotide is a terminator nucleotide.

4. The method of claim 2 wherein said first nucleotide is labeled.

5. The method of claim 3 wherein said second nucleotide is labeled.

6. The method of claim 1 comprising the step of adding a plurality of terminator nucleotides, wherein each of said nucleotides is complementary to a variant suspected to be present in said target nucleic acid.

7. The method of claim 1 wherein said first nucleotide is an extension nucleotide.

8. The method of claim 1 wherein the sequence of said region in said target nucleic acid is a wild-type sequence.

9. The method of claim 1 wherein said target nucleic acid is a genetic locus selected from the group consisting of BAT26, APC, DCC, P53 and RAS.

10. The method of claim 1 wherein said target nucleic acid is a genetic locus associated with cancer or precancer.

11. The method of claim 1 wherein said cancer or precancer is a colorectal cancer or precancer.

12. The method of claim 1 wherein said target nucleic acid sequence is obtained from a heterogeneous biological sample.

13. The method of claim 12 wherein said biological sample is a stool sample.

14. The method of claim 12 wherein said biological sample is selected from the group consisting of urine, semen, blood, sputum, cerebrospinal fluid, pus, and aspirate.

15. The method of claim 13 wherein said stool sample is a homogenized stool sample.

16. A method for minimizing nucleotide misincorporation in a primer extension assay, the method comprising the steps of:

a) contacting a target nucleic acid with a nucleic acid primer complementary to a region of said target nucleic acid; and

b) extending said primer in the presence of;

i) a first nucleotide that is complementary to a first variant base suspected to be at a position downstream of said region, and a first polymerase that preferentially incorporates said first nucleotide; and

,

ii) a second nucleotide that is complementary to a second variant base at said position and a second polymerase that preferentially incorporates said second nucleotide, thereby to reduce misincorporation of said first nucleotide in a primer extension reaction at said second variant base.

17. The method of claim 16, further comprising the step of providing a terminator nucleotide to terminate a primer extension product that incorporates said first nucleotide.

18. The method of claim 16, wherein said first nucleotide is labeled.

27. The method of claim 16 or 19, wherein said first nucleotide is an alkynylamino acycloterminator or analog thereof.

30. The method of claim 16 wherein said variant is present in about 10% of target nucleic acid molecules in a biological sample.

32. The kit of claim 16 wherein said first nucleotide is an acycloterminator and said second nucleotide is a NTP.

33. The kit of claim 16 wherein said first polymerase preferentially incorporates acycloterminators and said second polymerase preferentially is a thermostable polymerase that preferentially incorporates dNTPs.

19. 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 target nucleic acid with a nucleic acid primer substantially complementary to said target nucleic acid to form a nucleic acid complex upstream from a predetermined position in said target nucleic acid; and

b) contacting said nucleic acid complex with a first nucleotide, a first polymerase, a second nucleotide, and a second polymerase, wherein said first nucleotide is complementary to a first variant base at said predetermined position, and said second nucleotide is complementary to a second variant base at said predetermined position, and wherein said first and second polymerases are different, thereby to reduce misincorporation of said first nucleotide in a primer extension reaction at a said second variant base.

20. The method of claim 19, wherein said first nucleotide is labeled.

21. The method of claim 20, wherein said label is a fluorescent label.

22. The method of claim 19, wherein said first nucleotide is a terminator nucleotide.

23. The method of claim 22, wherein said second nucleotide is a terminator nucleotide.

24. The method of claim 20, wherein said terminator nucleotide is labeled.

25. The method of claim 19, further comprising the step of adding a terminator nucleotide complementary to a nucleotide downstream from said predetermined position.

26. The method of claim 25, wherein said terminator is labeled.

28. The method of claim 19, wherein said first polymerase preferentially incorporates an alkynylamino acycloterminator or analog thereof.

29. The method of claim 28 wherein said second polymerase preferentially incorporates a dNTP.

31. A kit comprising:

a) a first nucleotide, a second nucleotide; and

b) a first polymerase adapted for said first nucleotide and a second polymerase adapted for said second nucleotide.