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.
- 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.
- 27. The method of claim 16 or 19, wherein said first nucleotide is an alkynylamino acycloterminator or analog thereof.
- 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.
- 30. The method of claim 16 wherein said variant is present in about 10% of target nucleic acid molecules in a biological sample.
- 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.
- 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.
RELATED APPLICATIONS
[0001] This application claims priority to, and the benefit of U.S. Ser. No. 60/357,585, filed Feb. 15, 2002, the disclosure of which is incorporated by reference herein.
Provisional Applications (1)
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Number |
Date |
Country |
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60357585 |
Feb 2002 |
US |