Methods and Compositions for Amplification of DNA
The invention provides an Enzyme Blend comprising a DNA polymerase and a DNA repair enzyme. Methods and kits for amplification of DNA that is damaged, undamaged, or suspected of being damaged are also provided.
1 . A blend for amplifying DNA, said blend comprising a thermostable DNA polymerase and an AP endonuclease for repairing apurinic/apyrimidinic (AP) damage in DNA, wherein said blend does not contain primers and template.
2 . The blend of claim 1 , wherein the blend further comprises a second thermostable DNA polymerase, said second polymerase having a 3′→5′ exonuclease activity.
3 . The blend of claim 1 , wherein the blend further comprises a non-thermostable polymerase.
4 . The blend of claim 1 , wherein the AP endonuclease is AP endonuclease VI, REF1, APEX, Endonuclease IV, APNI, APE1 (human endonuclease 1), or FEN-1.
5 . The blend of claim 4 , wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.
6 . The blend of claim 1 , further comprising one or more of a stabilizing agent, a ligase, a DNA glycosylase, or a photolyase.
7 . The blend of claim 6 , wherein the stabilizing agent is 1,4-dithioerythritol, DL-dithiothreitol, 2-mercaptoethanol, 2-mercaptoethanolamine, fericyanide, hydrazine, borane, or phosphine.
8 . (canceled)
9 . The blend of claim 6 , wherein the ligase is T4 DNA ligase.
10 . (canceled)
11 . The blend of claim 6 , wherein the DNA glycosylase is uracil N-glycosylase.
12 . (canceled)
13 . (canceled)
14 . (canceled)
15 . The blend of claim 6 , wherein the photolyase is Thermus thermophilus photolyase.
16 . The blend of claim 5 comprising:
a) 0.1-25 units/ul DNA polymerase; and
b) 5-50 units/ul AP endonuclease VI.
17 . The blend of claim 16 , further comprising:
a) 1-15 mM DTT; and
b) 10-50% v/v glycerol.
18 . A blend for use in amplifying DNA, said blend comprising:
a) 2.5 units/ul DNA polymerase;
b) 5-50 units/ul AP endonuclease VI;
c) 10 mM Tris-HCl pH 8.0;
d) 150 mM KCl;
e) 100 ug/ml BSA;
f) 0.075 mM EDTA;
g) 7.5 mM DTT;
h) 0.25% v/v Tween 20;
i) 0.25% v/v IGEPAL CA-630; and
j) 50% v/v glycerol.
19 . A kit comprising the blend of claim 1 .
20 . The kit of claim 19 , wherein the blend further comprises a second thermostable DNA polymerase, said second polymerase having a 3′→5′ exonuclease activity.
21 . The kit of claim 19 , wherein the AP endonuclease in the blend is AP endonuclease VI, REF1, APEX, Endonuclease IV, APNI, APE1 (human endonuclease 1), or FEN-1.
22 . The kit of claim 21 , wherein the AP endonuclease is AP endonuclease VI.
23 . The kit of claim 22 , wherein the blend comprises:
a) 0.1-25 units/ul DNA polymerase; and
b) 5-50 units/ul AP endonuclease VI.
24 . The kit of claim 23 , wherein the blend further comprises:
a) 1-15 mM DTT; and
b) 10-50% v/v glycerol.
25 . A kit comprising the blend of claim 18 .
26 - 96 . (canceled)