Methods and Compositions for Amplification of DNA
Compositions for the amplification of damaged nucleic acids are disclosed. The compositions generally comprise a mesophilc polymerase, a thermostable polymerase, and an AP endonuclease. Methods of using the same for the amplification of damaged DNA are also disclosed.
1 . A composition for use in amplifying a nucleic acid, said composition comprising at least two different polymerases, wherein one of the polymerases is a mesophilic polymerase, and an AP endonuclease.
2 . The composition of claim 1 , wherein the composition is free of template and primers.
3 . The composition of claim 1 , wherein the at least two different polymerases are each DNA polymerases.
4 . The composition of claim 1 , wherein at least one of the polymerases is a thermostable polymerase.
5 . The composition of claim 1 , wherein the composition comprises two different DNA polymerases.
6 . The composition of claim 5 , wherein one of the DNA polymerases is a mesophilic DNA polymerase and the other DNA polymerase is a thermostable DNA polymerase.
7 . The composition of claim 6 , wherein the mesophilic DNA polymerase is selected from the group consisting of Pol I, Klenow, Klenow exo-, M-MuLV, phi 29, T4, and T7 and the AP endonuclease is selected from the group consisting of AP endonuclease VI, REF1, APEX, Endonuclease IV, APNI, APE1 (human endonuclease 1), and FEN-1.
8 . The composition of claim 6 , wherein each of the thermostable DNA polymerase, the mesophilic DNA polymerase, and the AP endonuclease is independently present in an amount of about 0.1 to about 25 units/μl.
9 . The composition of claim 8 , wherein the thermostable DNA polymerase is AccuTaq LA present in an amount of about 2.5 units/μl, the mesophilic DNA polymerase is Klenow exo- present in an amount of about 0.75 units/μl, and the AP endonuclease is Exonuclease III present in an amount of about 10 units/μl.
10 . The composition of claim 9 , wherein the composition further comprises 10 mM Tris-HCL, pH 8.0, 2.5 mM potassium phosphate, 150 mM KCL, 0.075 mM EDTA, 1 mM DTT, 100 μg/μl BSA, 0.25% TWEEN® 20, 0.25% IGEPAL® CA-630, and 50% glycerol.
11 . A composition for use in amplifying a nucleic acid, said composition comprising at least three different polymerases, wherein one of the polymerases exhibits 3′ to 5′ exonucleolytic activity, and an AP endonuclease.
12 . The composition of claim 11 , wherein the composition is free of template and primers.
13 . The composition of claim 11 , wherein the at least three different polymerases are each DNA polymerases.
14 . The composition of claim 13 , wherein at least two of the DNA polymerases are thermostable DNA polymerases.
15 . The composition of claim 14 , wherein at least one of the DNA polymerases is a mesophilic DNA polymerase.
16 . The composition of claim 15 , wherein the mesophilic DNA polymerase is selected from the group consisting of Pol I, Klenow, Klenow exo-, M-MuLV, phi 29, T4, and T7 and the AP endonuclease is selected from the group consisting of AP endonuclease VI, REF1, APEX, Endonuclease IV, APNI, APE1 (human endonuclease 1), and FEN-1.
17 . A method of repairing a nucleic acid, said method comprising forming a mixture, the mixture comprising the composition of claim 2 , a deoxyribonucleoside 5′ triphosphate, and the nucleic acid to be repaired, and incubating the mixture at about 0° C. to about 99° C. for less than about 5 hours.
18 . The method of claim 17 , wherein the mixture is incubated at a temperature of about 0° C. to about 50° C.
19 . The method of claim 17 , wherein the mixture is incubated for less than about 1 hour
20 . The method of claim 17 , wherein the mixture is incubated at about 0° C. to 50° C. for less than about an hour, at about 50° C. to about 80° C. for less than about an hour, and at about 80° C. to about 99° C. for less than about an hour.
21 . The method of claim 20 , wherein at least one of the DNA polymerases is a thermostable DNA polymerase.
22 . The method of claim 21 , wherein the amplification mixture further comprises a thermostable DNA polymerase having 3′ to 5′ exonucleolytic activity.
23 . The method of claim 21 , wherein the incubated nucleic acid is thereafter amplified, said method further comprising the step of subjecting the mixture to a nucleic acid amplification procedure.
24 . A kit for amplifying a nucleic acid, said kit comprising a composition for use in amplifying a nucleic acid, said composition comprising at least two different polymerases, wherein at least one of the polymerases is a mesophilic polymerase, and an AP endonuclease, and instructions for use of the kit in a method for amplifying a nucleic acid.
25 . The kit of claim 24 , wherein the composition is free of template and primers.
26 . The kit of claim 25 , wherein the at least two different polymerases are each DNA polymerases.
27 . The kit of claim 26 , wherein at least one of the polymerases is a thermostable polymerase.
28 . The kit of claim 27 , wherein the thermostable polymerase is AccuTaq LA and the mesophilic polymerase is Exonuclease III.