Methods and Compositions for PCR
A modified thermostable Pol B DNA polymerase, produced by a reaction, under essentially aqueous conditions, of a thermostable Pol B DNA polymerase and a modifier reagent of Formula I wherein the reaction results in a thermally reversible inactivation of the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity, which polymerase is suitable for hot-start PCR. Also disclosed are the method for the modification, a polynucleic acid amplification method and PCR reaction mixture and kit comprising the modified thermostable Pol B DNA polymerase.
1 . A modified thermostable Pol B DNA polymerase having both a DNA polymerase activity and a 3′-5′ exonuclease activity, wherein the modified thermostable Pol B DNA polymerase is produced by reacting, under essentially aqueous conditions, a thermostable Pol B DNA polymerase with a modifier reagent of Formula I
wherein R 1 and R 2 are hydrogen or a C 1 -C 4 alkyl which may be linked,
wherein the reaction results in inactivation of the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity, and wherein the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity are restorable.
2 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the Pol B DNA polymerase is Pfu, KOD, Tli, or Pfx, or a DNA polymerase sold under the trade names Vent®, Deep Vent®, Phusion™, or iProof™, or a fragment or variant thereof having DNA polymerase activity and 3′-5′ exonuclease activity.
3 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the Pol B DNA polymerase is a fusion protein comprises Pfu, KOD, Tli, or Pfx, or a DNA polymerase sold under the trade names Vent®, Deep Vent®, Phusion™, or iProof™, or a fragment or variant thereof having DNA polymerase activity and 3′-5′ exonuclease activity.
4 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the Pol B DNA polymerase is a fusion protein which comprises Pfu.
5 . The modified thermostable Pol B DNA polymerase of claim 4 , wherein the Pol B DNA polymerase is 10His-Pfu-Pae3192.
6 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the modifier reagent is selected from the group consisting of maleic anhydride or a substituted maleic anhydride.
7 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the modifier reagent is citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride, exo-cis-3,6-endoxo-δ 4 -tetrahydropthalic anhydride; or 3,4,5,6-tetrahydrophthalic anhydride.
8 . The modified thermostable Pol B DNA polymerase according to claim 1 , wherein the modifier reagent is citraconic anhydride.
9 . The modified thermostable Pol B DNA polymerase of claim 1 , wherein the polymerase activity and the 3′-5′ exonuclease activity are restorable by incubating the thermostable Pol B DNA polymerase at an elevated temperature.
10 . The modified thermostable Pol B DNA polymerase of claim 9 , wherein the elevated temperature is >50° C.
11 . The modified thermostable Pol B DNA polymerase of claim 10 , wherein the elevated temperature is ≧95° C.
12 . The modified thermostable Pol B DNA polymerase of claim 9 , wherein the elevated temperature is ≧96.5° C.
13 . The modified thermostable Pol B DNA polymerase of claim 9 , wherein the elevated temperature is ≧98° C.
14 . The modified thermostable Pol B DNA polymerase of claim 1 , wherein the polymerase activity and the 3′-5′ exonuclease activity have been restored by incubating the thermostable Pol B DNA polymerase at an elevated temperature.
15 . The modified thermostable Pol B DNA polymerase of claim 14 , wherein the polymerase activity and the 3′-5′ exonuclease activity have been restored by incubating the thermostable Pol B DNA polymerase in a reaction buffer having a pH of between about 7.5 and about 8.5.
16 . The modified thermostable Pol B DNA polymerase of claim 14 , wherein the polymerase activity and the 3′-5′ exonuclease activity have been restored by incubating the thermostable Pol B DNA polymerase in a reaction buffer having a pH of about 8.0.
17 . The modified thermostable Pol B DNA polymerase of claim 4 , wherein a ratio between the polymerase activity and the 3′-5′ exonuclease activity is restored to a value that is essentially the same as before modification.
18 . A method for modifying a thermostable Pol B DNA polymerase, the method comprising reacting a thermostable Pol B DNA polymerase and a modifier reagent of Formula I
wherein R 1 and R 2 are hydrogen or a C 1 -C 4 alkyl which may be linked, and wherein the reaction results in inactivation of the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity, and wherein the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity are restorable.
19 . The method according to claim 18 , wherein the Pol B DNA polymerase is Pfu, KOD, Tli, or Pfx, or a DNA polymerase sold under the trade names Vent®, Deep Vent®, Phusion™, or iProof™, or a fragment or variant thereof having DNA polymerase activity and 3′-5′ exonuclease activity.
20 . The method according to claim 18 , wherein the Pol B DNA polymerase is a fusion protein comprises Pfu, KOD, Tli, or Pfx, or a DNA polymerase sold under the trade names Vent®, Deep Vent®, Phusion™, or iProof™, or Pfu Ultra Fusion™, or a fragment or variant thereof having DNA polymerase activity and 3′-5′ exonuclease activity.
21 . The method according to claim 18 , wherein the Pol B DNA polymerase is a fusion protein comprises Pfu.
22 . The method of claim 21 , wherein the Pol B DNA polymerase is 10His-Pfu-Pae3192.
23 . The method according to claim 1 , wherein the modifier reagent is selected from the group consisting of maleic anhydride or a substituted maleic anhydride.
24 . The method according to claim 18 , wherein the modifier reagent is citraconic anhydride, cis-aconitic anhydride, 2,3-dimethylmaleic anhydride, exo-cis-3,6-endoxo-δ 4 -tetrahydropthalic anhydride; or 3,4,5,6-tetrahydrophthalic anhydride.
25 . The method according to claim 18 , wherein the modifier reagent is citraconic anhydride.
26 . The method according to claim 1 , further comprising restoring the polymerase activity and the 3′-5′ exonuclease activity by incubating the thermostable Pol B DNA polymerase at an elevated temperature.
27 . The method of claim 26 , wherein the elevated temperature is >50° C.
28 . The method of claim 26 , wherein the elevated temperature is ≧98° C.
29 . The method of claim 18 , further comprising restoring the polymerase activity and the 3′-5′ exonuclease activity by incubating the thermostable Pol B DNA polymerase in a reaction buffer having a pH of between about 7.5 and about 8.5.
30 . The method of claim 29 , wherein the pH is about 8.0.
31 . The method of claim 28 , further comprising restoring the polymerase activity and the 3′-5′ exonuclease activity by incubating the thermostable Pol B DNA polymerase in a reaction buffer having a pH of about 8.0.
32 . The method of claim 21 , wherein a ratio between the polymerase activity and the 3′-5′ exonuclease activity is restored to a value that is essentially the same as before modification.
33 . A method for amplifying a target nucleic acid contained in a sample comprising the steps of:
a) contacting the sample with an amplification reaction mixture containing a primer complementary to the target nucleic acid and a modified thermostable Pol B DNA polymerase according to claim 1 ; and
b) incubating the resulting mixture of step (a) at a temperature which is greater than about 50° C. for a time sufficient to reactivate the pol B DNA polymerase and allow formation of primer extension products.
34 . The method of claim 33 , wherein said amplification is a polymerase chain reaction.
35 . A modified thermostable Pol B DNA polymerase having both a DNA polymerase activity and a 3′-5′ exonuclease activity, wherein the modified thermostable Pol B DNA polymerase has been reacted, under essentially aqueous conditions, with a modifier reagent of Formula I
wherein R 1 and R 2 are hydrogen or a C 1 -C 4 alkyl which may be linked,
whereby both the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity have been inactivated, and wherein both the thermostable Pol B DNA polymerase activity and the 3′-5′ exonuclease activity are restored by incubation at an elevated temperature, whereby a ratio between the polymerase activity and the 3′-5′ exonuclease activity is restored to a value that is essentially the same as before the thermostable Pol B DNA polymerase has been reacted with the modifier reagent.
36 . The modified thermostable Pol B DNA polymerase according to claim 35 , which is able to produce detectable amplification product in a polymerase chain reaction.
37 . A polymerase chain reaction amplification reaction mixture, comprising at least a primer and a modified thermostable Pol B DNA polymerse of claim 1 .
38 . A kit comprising a modified thermostable Pol B DNA polymerse of claim 1 , and a suitable container.