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, and a DNA polymerase.
2. A method for amplifying a nucleic acid, said method comprising amplifying said nucleic acid 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.
3. 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, and a DNA polymerase.
4. The kit of claim 1 , wherein said primers are contained separately from each other.
5. The kit of claim 1 , wherein said DNA polymerase is contained separately from one or both of said primers.
6. The kit of claim 1 , wherein said primers have equalized Tms.
7. The kit of claim 1 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
8. The kit of claim 1 , wherein said one or both of said primers comprises a detectable label.
9. The kit of claim 8 , wherein said detectable label is selected from the group consisting of an enzymatic label, radioisotopic label, fluorescent label, chemical label, and a modified base.
10. The kit of claim 8 , wherein said detectable label is a fluorescent label.
11. The kit of claim 1 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
12. The method of claim 2 , wherein said primers have equalized Tms.
13. The method of claim 2 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
14. The method of claim 2 , wherein one or both of said primers comprise a detectable label.
15. The method of claim 14 , wherein said detectable label is selected from the group consisting of an enzymatic label, radioisotopic label, fluorescent label, chemical label, and modified base.
16. The method of claim 14 , wherein said detectable label is a fluorescent label.
17. The method of claim 2 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
18. The method of claim 2 , wherein said nucleic acid is DNA.
19. The method of claim 2 , wherein said nucleic acid is RNA.
20. The method of claim 2 , wherein said nucleic acid is polyadenylated at its 3′ end, and wherein one of said primers contains poly-T.
21. The method of claim 2 , wherein said nucleic acid, copy thereof, or complementary copy thereof has been extended to contain a 3′ sequence to which one of said primers can hybridize.
22. The mixture of claim 3 , wherein said primers have equalized Tms.
23. The mixture of claim 3 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
24. The mixture of claim 3 , wherein one or both of said primers comprises a detectable label.
25. The mixture of claim 24 , wherein said detectable label is selected from the group consisting of an enzymatic label, radioisotopic label, fluorescent label, chemical label, and modified base.
26. The mixture of claim 24 , wherein said detectable label is a fluorescent label.
27. The mixture of claim 3 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
28. The mixture of claim 3 , further comprising a nucleic acid template.
29. The mixture of claim 28 , wherein said template is DNA.
30. The mixture of claim 28 , wherein said template is RNA.
31. The mixture of claim 28 , wherein said template is polyadenylated at its 3′ end, and wherein one of said primers contains poly-T.
32. The mixture of claim 28 , wherein said template, copy thereof, or complementary copy thereof has been extended to contain a 3′ sequence to which one of said primers can hybridize.