Claims
- 1. A method of detecting a target nucleic acid in a sample, the method comprising:
a) contacting the sample with a nucleic acid polymerase; a first oligonucleotide primer comprising a sequence complementary to a first portion of the target nucleic acid; a second oligonucleotide primer comprising a first region and a second region, the first region comprising a sequence complementary to a second portion of the target nucleic acid and the second region comprising a non-natural base; b) amplifying the target nucleic acid, if present in the sample, by PCR using the first and second oligonucleotide primers to generate an amplification product having (i) a double-stranded region and (ii) a single-stranded region that comprises the non-natural base; c) contacting the sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; d) annealing at least a portion of the reporter to the single-stranded region of the amplification product; e) cleaving, after annealing, at least a portion of the reporter to release at least one reporter fragment; and f) correlating the release of the at least one reporter fragment with the presence of the target nucleic acid in the sample.
- 2. The method of claim 1, wherein the nucleic acid polymerase possesses 5′ to 3′ nuclease activity and the step of cleaving comprises cleaving with the nucleic acid polymerase at least a portion of the reporter to release at least one reporter fragment.
- 3. The method of claim 1, wherein the nucleic acid polymerase is a thermostable polymerase.
- 4. The method of claim 1, wherein amplifying the target nucleic acid by PCR comprises amplifying the target nucleic acid, if present in the sample, by Fast-shot™ amplification.
- 5. The method of claim 4, wherein amplifying the target nucleic acid, if present in the sample, by Fast-shot™ amplification comprises cycling the target nucleic acid and first and second oligonucleotide primers between about 90° C. to 100° C. and about 50° C. to 65° C. with about a one-second hold at each temperature.
- 6. The method of claim 1, wherein the step of contacting the sample with the first and second oligonucleotide primers comprises annealing the first and second oligonucleotide primers to the target nucleic acid, wherein the first and second oligonucleotide primers have a gap of zero to five bases between 3′ ends of the first and second oligonucleotide primers when annealed to the target nucleic acid.
- 7. The method of claim 1, wherein the non-natural base of the second region of the second oligonucleotide primer is positioned adjacent the first region of the second oligonucleotide primer.
- 8. The method of claim 7, wherein the reporter comprises (i) a first region that is complementary to at least a portion of the single-stranded region and comprises the non-natural base and (ii) a second region adjacent the non-natural base, wherein the second region of the reporter is not complementary to the first region of the second nucleotide primer.
- 9. The method of claim 1, wherein the non-natural base of the second oligonucleotide primer is selected from the group consisting of iso-cytosine and iso-guanine.
- 10. The method of claim 1, wherein the label of the reporter comprises a signal generating element and a signal quenching element and the step of cleaving comprises cleaving at least a portion of the reporter to release at least one reporter fragment, the at least one reporter fragment comprising one of, but not both, the signal generating element and the signal quenching element.
- 11. The method of claim 10, wherein the label is located at a 5′ terminus of the reporter.
- 12. The method of claim 10, wherein the signal generating element comprises a fluorophore and the signal quenching element comprises a fluorescence quencher.
- 13. The method of claim 1, wherein the label of the reporter comprises a signal generating element and a signal receiving element and the step of cleaving comprises cleaving at least a portion of the reporter to release at least one reporter fragment, the at least one reporter fragment comprising one of, but not both, the signal generating element and the signal receiving element.
- 14. The method of claim 1, wherein the reporter comprises an oligonucleotide and the step of annealing comprises annealing at least a portion of the oligonucleotide of the reporter to the single stranded region of the amplification product.
- 15. The method of claim 1, wherein the second region of the second oligonucleotide primer comprises at least two non-natural bases.
- 16. The method of claim 1, wherein the amplifying step comprises amplifying the target nucleic acid, if present in the sample, by PCR using the first and second oligonucleotide primers to generate the amplification product wherein the non-natural base of the second oligonucleotide substantially prevents extension of the first oligonucleotide primer beyond the non-natural base, resulting in the single-stranded region.
- 17. The method of claim 1, wherein the second region of the second oligonucleotide primer comprises at least two non-natural bases positioned adjacent the first region of the second oligonucleotide primer, and
the amplifying step comprises amplifying the target nucleic acid, if present in the sample, by PCR using the first and second oligonucleotide primers to generate the amplification product wherein the nucleic acid polymerase misincorporates a nucleotide opposite the non-natural base of second region of the second oligonucleotide primer that is nearest the first region, but does not incorporate a nucleotide opposite the non-natural base that is next nearest the first region of the second oligonucleotide primer.
- 18. The method of claim 1, wherein the annealing step comprises reducing the temperature to anneal at least a portion of the reporter to the singled stranded region of the amplification product.
- 19. The method of claim 1, wherein the correlating step comprises detecting the at least one reporter fragment.
- 20. The method of claim 1, wherein the correlating step comprises detecting the amplification product after the release of the at least one reporter fragment.
- 21. The method of claim 1, wherein steps a) and c) are performed simultaneously prior to amplifying the target nucleic acid.
- 22. A method of detecting a target nucleic acid in a sample, the method comprising:
a) contacting the sample with a nucleic acid polymerase; a first oligonucleotide primer comprising a sequence complementary to a first portion of the target nucleic acid; a second oligonucleotide primer comprising a first region and a second region, the first region comprising a sequence complementary to a second portion of the target nucleic acid and the second region comprising a non-natural base; b) amplifying the target nucleic acid, if present in the sample, by PCR using the first and second oligonucleotide primers to generate an amplification product having (i) a double-stranded region and (ii) a single-stranded region that comprises the non-natural base; c) contacting the sample with a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region; d) incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region; and e) correlating the incorporating of the reporter with the presence of the target nucleic acid in the sample.
- 23. The method of claim 22, wherein the step of contacting the sample with a reporter comprises contacting the sample with a reporter comprising a label and a nucleotide triphosphate of a non-natural base that is complementary to the non-natural base of the single-stranded region.
- 24. The method of claim 22, wherein the step of contacting the sample with a reporter comprises contacting the sample with a reporter consisting essentially of a label and a nucleotide triphosphate of a non-natural base that is complementary to the non-natural base of the single-stranded region.
- 25. The method of claim 22, wherein the incorporating step comprises incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region using the nucleic acid polymerase.
- 26. The method of claim 22, wherein the incorporating step comprises incorporating the reporter into the amplification product opposite the non-natural base of the single-stranded region using a ligase.
- 27. The method of claim 22, wherein the label comprises a fluorophore.
- 28. The method of claim 22, wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a signal generating/signal quenching pair.
- 29. The method of claim 22, wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a pair of fluorophores where the emission of one of the fluorophores stimulates the emission of the other fluorophore.
- 30. The method of claim 22, wherein the second region of the second oligonucleotide primer comprises at least one additional base.
- 31. A kit comprising:
a) a nucleic acid polymerase; b) a first oligonucleotide primer comprising a sequence complementary to a first portion of the target nucleic acid; c) a second oligonucleotide primer comprising a first region and a second region, the first region comprising a sequence complementary to a second portion of the target nucleic acid and the second region comprising a non-natural base; and d) a reporter comprising a label and a non-natural base that is complementary to the non-natural base of the single-stranded region.
- 32. The kit of claim 31, wherein the reporter comprises an oligonucleotide comprising the non-natural base.
- 33. The kit of claim 31, wherein the reporter does not include any base other than the non-natural base.
- 34. The kit of claim 31, wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a pair of fluorophores where the emission of one of the fluorophores stimulates the emission of the other fluorophore.
- 35. The kit of claim 31, wherein the second region of the second oligonucleotide primer further comprises a label and the labels of the reporter and the second region of the second oligonucleotide primer comprise a signal generating element and a signal quenching element.
CROSS-REFERENCE TO RELATED APPLICATION
[0001] This application claims the benefit of Provisional Applications Ser. Nos. 60/205,712, 60/240,398, and 60/282,831, all of which are hereby expressly incorporated by reference.
Provisional Applications (3)
|
Number |
Date |
Country |
|
60205712 |
May 2000 |
US |
|
60240398 |
Oct 2000 |
US |
|
60282831 |
Apr 2001 |
US |