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 method for repairing DNA that is damaged or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of a blend for amplifying DNA, and deoxynucleoside 5′ triphosphates, 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; and
b) incubating the mixture at 0° C.-99° C. from about 0 sec. to about 3 hrs.
2 . The method of claim 1 , wherein the DNA has a size of at least about 200 base pairs.
3 . The method of claim 2 , wherein the DNA has a size of at least about 500 base pairs.
4 . The method of claim 1 , wherein the DNA has a size of less than about 1,000 base pairs.
5 . The method of claim 1 , wherein the DNA has a size of about 50 base pairs to about 500 base pairs.
6 . A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of a blend for amplifying DNA, deoxynucleoside 5′ triphosphates, and a pair of oligonucleotide primers, wherein the pair of primers is substantially complementary to segments of the DNA, and wherein said blend comprises a thermostable DNA polymerase and an AP endonuclease for repairing apurinic/apyrimidinic (AP) damage in DNA;
b) preincubating the mixture at 0° C.-99° C. from about 0 sec. to about 3 hrs.;
c) denaturing the DNA; and
d) amplifying the DNA.
7 . The method of claim 6 , wherein the mixture is incubated at 0° C.-50° C. from about 0 sec. to about 1 hr. in step (b).
8 . The method of claim 6 , wherein step (d) is a polymerase chain reaction that comprises the steps of denaturation, annealing, and extension.
9 . The method of claim 6 , wherein step (d) is a rolling circle amplification.
10 . The method of claim 6 , wherein the AP endonuclease DNA repair enzyme in the Enzyme Blend is AP endonuclease VI, REF1, APEX, Endonuclease IV, APNI, APE1 (human endonuclease 1), or FEN-1.
11 . The method of claim 10 , wherein the AP endonuclease DNA repair enzyme is AP endonuclease VI.
12 . The method of claim 11 , wherein the blend comprises:
a) 0.1-25 units/μl DNA polymerase; and
b) 5-50 units/μl AP endonuclease VI.
13 . The method of claim 12 , wherein the blend further comprises:
a) 3-15 mM DTT; and
b) 16-50% v/v glycerol.
14 . The method of claim 6 , wherein the DNA has a size of at least about 200 base pairs.
15 . The method of claim 14 , wherein the DNA has a size of at least about 500 base pairs.
16 . The method of claim 6 , wherein the DNA has a size of less than about 1,000 base pairs.
17 . The method of claim 6 , wherein the DNA has a size of about 50 base pairs to about 500 base pairs.
18 . A method for amplification of DNA that is damaged, undamaged, or suspected of being damaged, comprising:
a) forming a mixture comprising the DNA, an effective amount of an enzyme blend, deoxynucleoside 5′ triphosphates, and a pair of oligonucleotide primers, wherein the pair of primers is substantially complementary to segments of the DNA, and wherein said blend comprises:
i) 2.5 units/μl DNA polymerase;
ii) 5-50 units/μl AP endonuclease VI;
iii) 10 mM Tris-HCl pH 8.0;
iv) 150 mM KCl;
v) 100 μg/ml BSA;
vi) 0.075 mM EDTA;
vii) 7.5 mM DTT;
viii) 0.25% v/v Tween 20;
ix) 0.25% v/v IGEPAL CA-630; and
x) 50% v/v glycerol;
b) preincubating the mixture at 0° C.-99° C. from about 0 sec. to about 3 hrs.;
c) denaturing the DNA; and
d) amplifying the DNA.
19 . The method of claim 18 , wherein the mixture is incubated at 0° C.-50° C. from about 0 sec. to about 1 hr. in step (b).
20 . An improved method for amplification of DNA comprising:
a) forming a mixture comprising the DNA, an effective amount of a DNA polymerase, deoxynucleoside 5′ triphosphates, and a pair of oligonucleotide primers having thiophosphate linkages, wherein the pair of primers is substantially complementary to segments of the DNA;
b) denaturing the DNA; and
c) amplifying the DNA.
21 . The method of claim 20 , wherein step (c) is a polymerase chain reaction that comprises the steps of denaturation, annealing, and extension.
22 . The method of claim 20 , wherein step (c) is a rolling circle amplification.
23 . The method of claim 20 , wherein the thiophosphate linkages are located on the last two nucleotides at the 3′ end of each oligonucleotide primer.