MODIFIED TYPE A DNA POLYMERASES
The present invention provides improved DNA polymerases, in particular, type A DNA polymerases, that may be better suited for applications in recombinant DNA technologies. Among other things, the present invention provides modified DNA polymerases derived from directed evolution experiments designed to select mutations that confer advantageous phenotypes under conditions used in industrial or research applications.
1 . A method of performing a DNA polymerization reaction, the method comprising:
a) providing a mixture wherein the mixture comprises:
i) a template nucleic acid;
ii) at least one primer;
iii) nucleotides; and
iv) a modified Taq DNA polymerase whose amino acid sequence includes a lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1; and
b) incubating the mixture under conditions that:
i) permit hybridization of the at least one primer to the template nucleic acid; and
ii) permit extension of the at least one primer by polymerization of the nucleotides, which polymerization is catalyzed by the modified Taq DNA polymerase.
2 . The method of claim 1 , wherein the template nucleic acid is DNA.
3 . The method of claim 1 , wherein the modified Taq DNA polymerase has an amino acid sequence that shows at least 95% overall identity with that of wild type Taq of SEQ ID NO:1.
4 . The method of claim 1 , wherein the conditions comprise presence of one or more of a nucleic acid intercalating agent, phenol, high salt concentration, an inhibitor of processivity and an inhibitor of enzyme activity.
5 . The method of claim 1 , wherein the DNA polymerization reaction produces a product of at least 2 kilobases long.
6 . The method of claim 1 , wherein the DNA polymerization reaction produces a product of at least 5 kilobases long.
7 . The method of claim 1 , wherein the DNA polymerization reaction produces a product of at least 8 kilobases long.
8 . The method of claim 1 , wherein the DNA polymerization reaction produces a product of at least 10 kilobases long.
9 . In a method of performing a polymerase chain reaction (PCR) using a modified Taq DNA polymerase, the improvement comprising utilizing a modified Taq DNA polymerase whose amino acid sequence includes a lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1.
10 . A method of providing an improvement in performance in a polymerase chain reaction (PCR) relative to that of wild type Taq DNA polymerase of SEQ ID NO:1, which improvement in performance is selected from the group consisting of increase of one or more of enzyme activity, processivity, resistance to nucleic acid intercalating dyes, salt-resistance, and combinations thereof, the method comprising:
using a modified Taq DNA polymerase whose sequence includes a lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1 to perform the PCR.
11 . A method of providing an improvement in performance in a heparin binding assay relative to that of wild type Taq DNA polymerase of SEQ ID NO:1, the method comprising:
using a modified Taq DNA polymerase whose sequence includes a lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1 to perform the heparin binding assay.
12 . A kit comprising:
a modified Taq DNA polymerase whose sequence includes a substitution of lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1; and
at least one buffer suitable for use in a PCR reaction.
13 . A nucleic acid whose nucleotide sequence encodes a modified Taq DNA polymerase whose amino acid sequence has at least 95% overall identity with that of wild type Taq of SEQ ID NO:1. but differs from that of wild type Taq DNA polymerase in that it a) includes a lysine at a position corresponding to E507 of wild type Taq of SEQ ID NO: 1; and b) is characterized by ability to catalyze a DNA-template-dependent DNA polymerization activity that is improved relative to that of the wild type Taq DNA polymerase.
14 . A vector comprising the nucleic acid of claim 13 .
15 . A cell comprising the nucleic acid of claim 13 .
16 . A cell comprising the vector of claim 14 .
17 . A modified Taq DNA polymerase having an amino acid sequence that is at least 95% identical to a sequence selected from the group consisting of SEQ ID NO:2 (A3E), SEQ ID NO:3 (G9S), SEQ ID NO:4 (D5S), SEQ ID NO:5 (D2), SEQ ID NO:6 (A5E), SEQ ID NO:7 (B6S), SEQ ID NO:8 (E2S), SEQ ID NO:9 (A3), SEQ ID NO:10 (H10), SEQ ID NO:11 (H1S), SEQ ID NO:12 (F9E), SEQ ID NO:13 (A5S), SEQ ID NO:14 (C10E), SEQ ID NO:15 (F5S), SEQ ID NO:16 (E7S), SEQ ID NO:17 (G6S), SEQ ID NO:18 (E1E), SEQ ID NO:19 (C7), SEQ ID NO:20 (E12), SEQ ID NO:21 (D9), SEQ ID NO:22 (F10), SEQ ID NO:23 (H7), SEQ ID NO:24 (A5) but differs from that of wild type Taq DNA polymerase as set forth in SEQ ID NO:1 and is characterized by ability to catalyze a DNA-template-dependent DNA polymerization activity that is improved relative to that of the wild type Taq DNA polymerase.
18 . The modified Taq DNA polymerase of claim 17 , having an amino acid sequence that includes a lysine residue at a position corresponding to E507 of the wild type Taq DNA polymerase.
19 . A kit comprising:
at least one modified Taq DNA polymerase as set out in claim 17 ; and
at least one buffer suitable for use in a PCR reaction.