Methods of attaching adapters to sample nucleic acids
View Patent ↗Methods of preparing double-stranded nucleic acids with single-stranded overhangs for amplification and sequencing are disclosed. Contacting a blunt-ended double-stranded nucleic acid molecules with Taq results in non-templated directed addition of a single nucleotide to the 3′ ends of the nucleic acid with A added most frequently followed by G followed by C and T. G tailing is sufficiently frequent that the efficiency of ligation of nucleic acid molecules to adapters can be significantly increased by including adapters tailed with T and C. The ligation efficiency can be increased even further with blunted-ended adapters to ligate to blunt-ended nucleic acid molecules that failed to undergo tailing.
1. A method of preparing nucleic acids for analysis comprising:
(a) blunt-ending double-stranded nucleic acids with single-stranded overhangs in a sample by the action of one or more enzymes providing a 5′-3′ polymerase activity and 3′-5′ proof reading activity, and four standard nucleotide types, wherein single-stranded overhangs with 5′ ends serve as templates for extension of a complementary strand by the polymerase activity and single-stranded overhangs with 3′ ends are digested by the proof reading activity producing blunt-ended nucleic acids;
(b) without separating the blunt-ended nucleic acids from other components of the sample, end-tailing the blunt-ended nucleic acids by action of a polymerase without a 3′-5′ proof reading function, which performs a non-template directed addition of a nucleotide to the 3′ ends of blunt-ended nucleic acids, wherein A is added most frequently followed by G followed by C and T;
(c) annealing the nucleic acids from step (b) with at least partially double-stranded adapters with a single nucleotide T overhang at a 3′-end and with at least partially double-stranded adapters with a single nucleotide C overhang at a 3′-end; and
(d) ligating the nucleic acids to the adapters; wherein
the method further comprises contacting the sample with at least partially double-stranded blunt-ended adapters, which in step (d), ligate with blunt-ended double-stranded nucleic acids which have not undergone the non-template directed addition of a nucleotide to the 3′ ends.
2. The method of claim 1 , further comprising denaturing the one or more enzymes after step (a).
3. The method of claim 1 , further comprising contacting the sample with the one or more enzymes, the four standard nucleotide types and the polymerase without a 3′-5′ proof reading function.
4. The method of claim 1 , wherein the sample is contacted with the one or more enzymes, the four standard nucleotide types and the polymerase without a 3′-5′ proof reading function together.
5. The method of claim 1 , wherein step (b) is performed at a higher temperature than step (a).
6. The method of claim 1 , wherein step (a) is performed at ambient temperature and step (b) at a temperature over 60° C.
7. The method of claim 1 , wherein the one or more enzymes are a polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity.
8. The method of claim 7 , wherein the polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity is T4 polymerase or Klenow large fragment.
9. The method of claim 1 , wherein the polymerase without a 3′-5′ proof reading function is a thermostable polymerase and the method further comprises increasing temperature of the sample after step (a) to inactivate the polymerase with 5′-3′ polymerase activity and 3′-5′ proof reading activity.
10. The method of claim 1 further comprising (e) amplifying the nucleic acids ligated to the adapters; and (f) analyzing the nucleic acids.
11. The method of claim 10 , wherein steps (a)-(e) or (a) to (f) are performed in a single tube.
12. The method of claim 10 , wherein at least 70% of the double-stranded nucleic acids with single-stranded overhangs in the sample are analyzed.
13. The method of claim 10 , wherein step (f) comprises sequencing the nucleic acids ligated to the adapters.
14. The method of claim 13 , wherein the sequencing sequences a nucleotide that formed an overhang in step (c) or (d).
15. The method of claim 1 , wherein the polymerase without a 3′-5′ proof reading function is a Taq polymerase.
16. The method of claim 1 , wherein at least steps (a)-(d) are performed in a single tube.
17. The method of claim 1 , wherein for at least steps (a)-(d) no component is removed from the sample.
18. The method of claim 1 , wherein at least partially double-stranded adapters with a single nucleotide T to a single nucleotide C has a molar ratio of 4:1 to 2:1.
19. The method of claim 18 , wherein the molar ratio of blunt-ended adapters to tailed adapters is 1:5 to 1:500.
20. The method of claim 1 , wherein at least 70% of the double-stranded nucleic acids in the sample are joined to adaptors.