Repair of nucleic acids for improved amplification
View Patent ↗Methods and compositions are provided for repairing a polynucleotide so that it can be copied with improved fidelity and/or 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. If used, these enzymes may be selected according to their ability to withstand high temperatures. For example, 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 in as much as the repair is effected rapidly and prolonged incubation is not generally adverse.
1. A composition, comprising:
a reaction mixture comprising an NAD + -dependent ligase, an NAD + cofactor, a polymerase having flap endonuclease activity and an AP endonuclease, the reaction mixture not including Endo VI.
2. A composition according to claim 1 , wherein the AP endonuclease is at a concentration of 1-100 units, the polymerase at a concentration of 0.05-5 units, and the ligase at a concentration of 5-500 units in a volume 10-1000 μl of the reaction mixture.
3. A composition according to claim 1 , further comprising a glycosylase/lyase, wherein the glycosylase/lyase is selected from the group consisting of E. coli Endo III, E. coli Endo VIII, E. coli fpg, human OGGI and T4 pyrimidine dimer glycosylase (T4pdg).
4. A composition according to claim 1 , wherein the AP endonuclease is a Type II AP Endonuclease.
5. A composition according to claim 1 , wherein the AP endonuclease is selected from the group consisting of E. coli Endo IV, Tth Endo IV, and human AP endonuclease.
6. A composition according to claim 1 , wherein the glycosylase/lyase is T4 pyrimidine dimer glycosylase (pdg).
7. A composition according to claim 6 , wherein the glycosylase/lyase is [FAPY]-DNA glycosylase (Fpg).
8. A composition according to claim 1 , further comprising at least one of UvrA, UvrB, UvrC, UvrD and Cho.
9. A composition according to claim 1 , further comprising a glycosylase/lyase, wherein the glycosylase/lyase is selected from the group consisting of at least one of Endo VIII, E. coli fpg, human OGGI, T4Pdg and Endo III.
10. A composition according to claim 1 , further comprising a glycosylase wherein the glycosylase is selected from at least one of UDG, E. coli AlkA and Aag.
11. A method for sequencing a polynucleotide, comprising:
(a) contacting the polynucleotide with the composition of claim 1 ; and
(b) sequencing the polynucleotide.
12. A composition according to claim 1 , wherein the polymerase is Bst polymerase, the AP endonuclease is Endo IV, the ligase is Taq ligase, the glycosylase is E. coli UDG, the glycosylase/lyase is E. coli Fpg, T4 pdg and E. coli Endo VIII.
13. A composition according to claim 1 , wherein the polymerase is an E. coli Y family polymerase.
14. A composition according to claim 1 , wherein the NAD + -dependent ligase, and the polymerase are purified enzymes.
15. A composition according to claim 1 , further comprising an effective amount of each of the NAD + -dependent ligase, an NAD + cofactor, the polymerase having flap endonuclease activity and the AP endonuclease, wherein the reaction mixture when added to a damaged polynucleotide prior to amplification enhances at least one of yield and fidelity of an amplified polynucleotide compared with the yield or fidelity of the amplified polynucleotide without prior addition of the reaction mixture.