Amplification of trace amounts of nucleic acids
Methods of reducing background during amplification of small amounts of nucleic acids employ careful analysis of sources of low level contamination. Ultraviolet light can be used to reduce nucleic acid contaminants in reagents and equipment. “Primer-dimer” background can be reduced by judicious design of primers. We have shown clean signal-to-noise with as little as starting material as one single human cell (˜6 picogram), E. coli cell (˜5 femtogram) or Prochlorococcus cell (˜3 femtogram).
1. A method of nucleic acid amplification, comprising:
exposing a sample of polymerase to ultraviolet light for a sufficient time to reduce nucleic acid contamination of said polymerase to produce an exposed sample of polymerase; and
amplifying a reaction mixture comprising a nucleic acid template, the exposed sample of polymerase, and one or more restricted-randomized oligonucleotide primers,
wherein the amplifying is real time amplification performed a plurality of times with a plurality of known amounts of a nucleic acid template and wherein data is obtained from the real time amplification and logistical regression analysis is performed on the data to calculate an amount of background nucleic acid contamination.
2. A method of nucleic acid amplification, comprising:
amplifying a nucleic acid template in a reaction mixture comprising:
one or more restricted-randomized oligonucleotide primers, wherein the one or more restricted-randomized primers do not comprise random hexamers (N 6 , wherein N=A, C, G, or T),
a template-dependent polymerase, and
mononucleotide triphosphates,
wherein the amplifying is real time amplification performed under conditions permitting hybridization of the primers to the template and wherein data is obtained from the real time amplification and logistical regression analysis is performed on the data.
3. The method of claim 1 wherein the concentration of nucleic acid template in the reaction mixture is at least 100-fold greater than the concentration of nucleic acid contamination.
4. The method of claim 2 wherein the one or more restricted-randomized oligonucleotide primers are selected from the group consisting of:
B 6 (wherein B=not A=C+G+T),
D 6 (wherein D=not C=A+G+T),
H 6 (wherein H=not G=A+C+T),
V 6 (wherein V=not T=A+G+C),
M 6 (wherein M=A+C),
W 6 (wherein W=A+T),
Y 6 (wherein Y=T+C),
R 6 (wherein R=A+G),
I 3 D 4 (wherein I=inosine, and wherein D=not C),
I 2 M 7 , (wherein I=inosine and M=A+C),
M 7 (wherein M=A+C), and combinations thereof.
5. The method of claim 2 wherein the restricted-randomized primers comprise K 6 (wherein K=G+T/U).
6. The method of claim 2 wherein the restricted-randomized primers are hexamers, heptamers, or combinations thereof.
7. The method of claim 2 wherein the restricted-randomized primers are hexamers.
8. The method of claim 2 wherein the restricted-randomized primers are heptamers.
9. The method of claim 1 wherein the step of exposing comprises applying between 100 and 500 mJ of UV to 5-10 ul of polymerase.
10. The method of claim 1 wherein the step of amplifying employs real-time isothermal amplification.
11. The method of claim 2 wherein the restricted-randomized oligonucleotide primer is R 6 .
12. The method of claim 2 wherein the restricted-randomized oligonucleotide primer is M 6 .
13. The method of claim 1 wherein the step of amplifying employs a restricted-randomized oligonucleotide primer selected from the group consisting of R 6 or M 6 .
14. The method of claim 1 wherein the nucleic acid contamination is reduced 100-fold by said step of exposing.
15. The method of claim 1 wherein the nucleic acid contamination is reduced 1000-fold by said step of exposing.
16. The method of claim 1 wherein the nucleic acid contamination is reduced 1,500-fold by said step of exposing.
17. The method of claim 1 wherein the nucleic acid contamination is reduced by said step of exposing to less than 3×10 −14 g in the exposed sample of polymerase.
18. The method of claim 1 wherein the nucleic acid contamination is reduced by said step of exposing to less than 3×10 −17 g in the exposed sample of polymerase.
19. The method of claim 1 wherein the nucleic acid contamination is reduced by said step of exposing to less than 3×10 −19 g in the exposed sample of polymerase.
20. The method of claim 1 wherein the nucleic acid contamination is reduced by said step of exposing to less than 3×10 −21 g in the exposed sample of polymerase.
21. The method of claim 1 wherein the nucleic acid contamination is reduced by said step of exposing to less than 3×10 −23 g in the exposed sample of polymerase.
22. The method of claim 2 wherein said step of amplifying is performed a plurality of times with a plurality of known amounts of a nucleic acid template.
23. The method of claim 2 wherein the logistical regression analysis is used to calculate an amount of background nucleic acid contamination.
24. The method of claim 23 wherein the amount of background nucleic acid contamination calculated is used to determine a sufficient amount of time to expose a sample of polymerase to ultraviolet light.
25. The method of claim 1 wherein the nucleic acid template is present at a concentration of less than 1 ng in the reaction mixture.
26. The method of claim 1 wherein the nucleic acid template is present at a concentration of less than 1 pg in the reaction mixture.
27. The method of claim 1 wherein the nucleic acid template is present at a concentration of less than 1 fg in the reaction mixture.
28. The method of claim 1 wherein the template is a single chromosome obtained by flow-sorting.
29. The method of claim 1 wherein the template is a single chromosome obtained by dilution.
30. The method of claim 1 wherein the template is a chromosome of a single cell.
31. The method of claim 1 wherein the one or more restricted-randomized oligonucleotide primers are selected from the group consisting of:
B 6 (wherein B=not A=C+G+T),
D 6 (wherein D=not C=A+G+T),
H 6 (wherein H=not G=A+C+T),
V 6 (wherein V=not T=A+G+C),
M 6 (wherein M=A+C),
W 6 (wherein W=A+T),
Y 6 (wherein Y=T+C),
R 6 (wherein R=A+G),
I 3 D 4 (wherein I=inosine, and wherein D=not C),
I 2 M 7 , (wherein I=inosine and M=A+C),
M 7 (wherein M=A+C), and combinations thereof.
32. The method of claim 1 wherein the restricted-randomized primers comprise K 6 (wherein K=G+T/U).
33. The method of claim 1 wherein the restricted-randomized primers are hexamers, heptamers, or combinations thereof.
34. The method of claim 1 wherein the restricted-randomized primers are hexamers.
35. The method of claim 1 wherein the restricted-randomized primers are heptamers.
36. The method of claim 1 wherein the restricted-randomized oligonucleotide primer is R 6 .
37. The method of claim 1 wherein the restricted-randomized oligonucleotide primer is M 6 .
38. The method of claim 2 wherein the step of amplifying employs a restricted-randomized oligonucleotide primer selected from the group consisting of R 6 or M 6 .
39. The method of claim 1 wherein the amount of background nucleic acid contamination calculated is used to determine a sufficient amount of time to expose a sample of polymerase to ultraviolet light.
40. The method of claim 2 wherein the template is a single chromosome obtained by flow-sorting.
41. The method of claim 2 wherein the template is a single chromosome obtained by dilution.
42. The method of claim 2 wherein the template is a chromosome of a single cell.