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 T m s with respect to each other when hybridized to a template, wherein one or both of said primers is delectably labeled, and wherein said primers are contained in close compartmentalization.
2. The kit of claim 1 , wherein said primers are contained separately from each other.
3. The kit of claim 1 , further comprising a nucleic acid polymerase.
4. The kit of claim 3 , wherein said polymerase is contained separately from one or both of said primers.
5. The kit of claim 4 , wherein said polymerase is a DNA polymerase.
6. The kit of claim 4 , wherein said polymerase is a reverse transcriptase.
7. The kit of claim 1 , wherein said primers have equalized T m s.
8. The kit of claim 1 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
9. The kit of claim 1 , wherein said detectable label is an enzymatic label, radioisotopic label, fluorescent label, chemical label, or modified base.
10. The kit of claim 9 , 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. A method of making amplification primers, said method comprising:
synthesizing a set of first and second amplification primers which exhibit a decreased difference between their T m s with respect to each other when hybridized to a template, wherein one or both of said primers is detectable labeled.
13. The method of claim 12 , wherein said primers have equalized T m s.
14. The method of claim 12 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
15. The method of claim 12 , wherein said detectable label is an enzymatic label, radioisotopic label, fluorescent label, chemical label, or modified base.
16. The method of claim 15 , wherein said detectable label is a fluorescent label.
17. The method of claim 12 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
18. A method for amplifying a nucleic acid, said method comprising:
amplifying the nucleic acid using a set of first and second amplification primers which exhibit a decreased difference between their T m s with respect to each other when hybridized to a template, wherein one or both of said primers is detectable labeled.
19. The method of claim 18 , wherein said primers have equalized T m s.
20. The method of claim 18 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
21. The method of claim 18 , wherein said detectable label is an enzymatic label, radioisotopic label, fluorescent label, chemical label, or modified base.
22. The method of claim 21 , wherein said detectable label is a fluorescent label.
23. The method of claim 18 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
24. The method of claim 18 , wherein said nucleic acid is DNA.
25. The method of claim 18 , wherein said nucleic acid is RNA.
26. The method of claim 18 , wherein said nucleic acid is polyadenylated at its 3′ end, and wherein one of said primers contains poly-T.
27. The method of claim 18 , 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.
28. An amplification mixture, said mixture comprising:
a set of first and second amplification primers which exhibit a decreased difference between their T m s with respect to each other when hybridized to a template, wherein one or both of said primers is detectable labeled.
29. The mixture of claim 28 , further comprising a nucleic acid polymerase.
30. The mixture of claim 29 , wherein said polymerase is a DNA polymerase.
31. The mixture of claim 29 , wherein said polymerase is a reverse transcriptase.
32. The mixture of claim 28 , wherein said primers have equalized T m s.
33. The mixture of claim 28 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
34. The mixture of claim 28 , wherein said detectable label is an enzymatic label, radioisotopic label, fluorescent label, chemical label, or modified base.
35. The mixture of claim 34 , wherein said detectable label is a fluorescent label.
36. The mixture of claim 28 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.
37. The mixture of claim 28 , further comprising a nucleic acid template.
38. The mixture of claim 37 , wherein said template is DNA.
39. The mixture of claim 37 , wherein said template is RNA.
40. The mixture of claim 37 , wherein said template is polyadenylated at its 3′ end, and wherein one of said primers contains poly-T.
41. The mixture of claim 37 , 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.
42. A method for selecting amplification primers, said method comprising:
selecting a set of first and second amplification primers which exhibit a decreased difference between their T m s with respect to each other when hybridized to a template, wherein one or both of said primers is detectable labeled.
43. The method of claim 42 , wherein said primers have equalized T m s.
44. The method of claim 42 , wherein one or both of said primers comprises a nucleotide having an altered base pairing characteristic.
45. The method of claim 42 , wherein said detectable label is an enzymatic label, radioisotopic label, fluorescent label, chemical label, or modified base.
46. The method of claim 45 , wherein said detectable label is a fluorescent label.
47. The method of claim 42 , wherein one or both of said primers comprises a poly-G, poly-A, poly-T, or poly-C sequence.