Repair of nucleic acids for improved amplification
View Patent ↗Methods and compositions are provided for repairing a polynucleotide so that it can be synthesized efficiently with improved fidelity and yield in, for example, an amplification reaction. This involves the use of a reaction mixture that includes a ligase and a cofactor selected from NAD+ or ATP and incubating the polynucleotide with the reaction mixture in the absence of Endonuclease VI. The reaction mixture may further contain an AP endonuclease and a polymerase. These enzymes are optionally selected according to their ability to withstand high temperatures so they can be included in an amplification mixture. The reaction mixture may be used prior to a polynucleotide synthesis reaction in which case enzymes that are not thermophilic may be used. The repair reaction is not time sensitive with respect to seconds, minutes or hours of incubation in the enzyme mixture.
1. A method for enhancing at least one of fidelity and yield of an amplification product of a damaged polynucleotide, comprising the steps of:
(a) incubating the polynucleotide containing one or more damaged sites with a reaction mixture comprising an effective amount of ligase selected from Taq ligase or E. coil ligase in the presence of NAD + , and in the absence of Endonuclease (Endo) VI, for repairing the damaged sites in the polynucleotide; and
(b) adding amplification reagents to the reaction mixture so as to permit in vitro polymerase-dependent amplification of the polynucleotide in the reaction mixture.
2. A method according to claim 1 , wherein step (a) is performed at a single temperature.
3. A method according to claim 1 , wherein the damaged polynucleotide contains one or more damaged sites selected from apurinic/apyrimidinic (AP) bases, mutagenized nucleotides, modified nucleotides, nicks, gaps and DNA-DNA cross-links.
4. A method according to claim 1 , wherein the polynucleotide is obtained from a natural source.
5. A method according to claim 1 , wherein the polynucleotide is obtained from preserved biological material.
6. A method according to claim 1 , wherein the polynucleotide is obtained from forensic evidence.
7. A method according to claim 1 , wherein the polynucleotide is ancient.
8. A method according to claim 1 , wherein the polynucleotide is obtained from a tissue biopsy.
9. A method according to claim 1 , wherein the reaction mixture in step(a) further comprises a T7 Endo I or mutant thereof.
10. A method according to claim 1 , wherein the reaction mixture in step (a) further comprises a polymerase and an AP endonuclease.
11. A method according to claim 10 , wherein the AP endonuclease is a class II AP endonuclease.
12. A method according to claim 10 , wherein the AP endonuclease is selected from the group consisting of a T4 endonuclease, an E. coil endonuclease, Tth Endo IV, and human AP endonuclease.
13. A method according to claim 10 , wherein the AP endonuclease is E. coil Endo IV.
14. A method according to claim 10 , wherein the polymerase is selected from the group consisting of Taq DNA polymerase, Bst DNA polymerase, T4 DNA polymerase, T7 DNA polymerase, E. coil DNA polymerase I and an archaeal DNA polymerase or modifications thereof.
15. A method according to claim 10 , wherein the polymerase is an archaeal DNA polymerase and the archaeal polymerase is selected from Pfu, Vent®, Deep Vent®, 9° North, and GBD DNA polymerase.
16. A method according to claim 10 , wherein the polynucleotide is DNA and the reaction mixture in step (a) comprises 1-100 units of the AP endonuclease, 0.05-0.25 units of the polymerase and 5-500 units of the ligase.
17. A method according to claim 10 or 16 , wherein the reaction mixture in step (a) further comprises T4 pyrimidine dimer glycosylase (pdg).
18. A method according to claim 10 or 16 , wherein the reaction mixture in step (a) further comprises [fapy]-DNA glycosylase (Fpg).
19. A method according to claim 10 or 16 , wherein the reaction mixture in step (a) further comprises at least one of UvrA, UvrB, UvrC, UvrD and Cho.
20. A method according to claim 10 or 16 , wherein the reaction mixture in step (a) further comprises at least one glycosylase/lyase selected from the group consisting of Endo III, Endo VIII, E coil Fpg, OGGI, and T4 pdg.
21. A method according to claim 10 or 16 or 20 , wherein the reaction mixture in step (a) further comprises at least one glycosylase selected from the group consisting of UDG, AlkA and Aag.
22. A method according to claim 10 or 16 , wherein the polymerase is a Bst DNA polymerase, the AP endonuclease is Endo IV, the ligase is Taq ligase, the reaction mixture in step (a) further comprising one or more glycosylases and one or more glycosylase/lyases.
23. A method according to claim 1 , wherein the amplification is PCR amplification, helicase-dependent amplification, transcription-mediated amplification, strand-displacement amplification, rolling circle amplification or whole genome amplification.
24. A method according to claim 1 , wherein the polynucleotide is a single-stranded RNA and the amplification is RT-amplification.
25. A method according to claim 1 , wherein amplification of the polynucleotide is capable of producing an amplicon in a size range of 50 nucleotides to 100,000 nucleotides in a polymerase chain reaction.
26. A method according to claim 22 , wherein the one or more glycosylases is UDG and the one or more glycosylase/lyases are Fpg, T4 pdg and Endo VIII.
27. A method according to claim 10 , wherein the polymerase is an E. coli Y family DNA polymerase.