Use of predetermined nucleotides having altered base pairing characteristics in the amplification of nucleic acid molecules
View Patent ↗The present invention provides improved methods for amplifying a nucleic acid molecule. More specifically, the invention provides methods for nucleic acid amplification which use primers having equivalent priming efficiency.
1. A kit for amplifying a nucleic acid, said kit comprising: a set of first and second amplification primers which exhibit a decreased difference between their Tms with respect to each other when hybridized to a template, a DNA polymerase, and a reverse transcriptase.
2. The kit of claim 1 , wherein said primers are contained separately from each other.
3. The kit of claim 2 , wherein said polymerase is contained separately from one or both of said primers.
4. The kit of claim 1 , wherein said primers have equalized Tms.
5. The kit of claim 1 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
6. The kit of claim 1 , wherein said reverse transcriptase lacks RNase H activity.
7. The kit of claim 1 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
8. A method for amplifying RNA, said method comprising: synthesizing cDNA complementary to said RNA using a reverse transcriptase, and amplifying said cDNA using a set of first and second amplification primers which exhibit a decreased difference between their Tms with respect to each other when hybridized to a template.
9. The method of claim 8 , wherein said primers have equalized Tms.
10. The method of claim 8 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
11. The method of claim 8 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
12. The method of claim 8 , wherein said reverse transcriptase lacks RNase H activity.
13. The method of claim 8 , wherein said RNA is polyadenylated at its 3 ′ end, and wherein one of said primers contains poly-T.
14. The method of claim 8 , wherein said RNA, copy thereof, or complementary copy thereof has been extended to contain a 3′ sequence to which one of said primers can hybridize.
15. An amplification mixture, said mixture comprising: a set of first and second amplification primers which exhibit a decreased difference between their Tms with respect to each other when hybridized to a template, a DNA polymerase, and a reverse transcriptase.
16. The mixture of claim 15 , wherein said reverse transcriptase lacks RNase H activity.
17. The mixture of claim 15 , wherein said primers have equalized Tms.
18. The mixture of claim 15 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
19. The mixture of claim 15 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
20. The mixture of claim 15 , further comprising an RNA template.
21. The mixture of claim 20 , wherein said RNA template is polyadenylated at its 3′ end, and wherein one of said primers contains poly-T.
22. The mixture of claim 20 , wherein said RNA template, copy thereof, or complementary copy thereof has been extended to contain a 3′ sequence to which one of said primers can hybridize.
23. The mixture of claim 20 , further comprising a nucleic acid complementary to said RNA template.