Cloned DNA polymerases from thermotoga maritima and mutants thereof
First Claim
1. A mutant Thermotoga maritima DNA polymerase, which is modified to comprise a mutation in the 3'"'"'→
- 5'"'"' exonuclease domain of said polymerase to substantially reduce the 3'"'"'→
5'"'"' exonuclease activity of said polymerase, and is further modified in at least one way selected from the group consisting of;
(a) a first mutation in the 5'"'"'→
3'"'"' exonuclease domain of said polymerase to substantially reduce the 5'"'"'→
3'"'"' exonuclease activity of said polymerase; and
(b) a second mutation in the O helix of said polymerase to substantially reduce discriminatory activity against a dideoxynucleotide.
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Accused Products
Abstract
The invention relates to a substantially pure thermostable DNA polymerase from Thermotoga (Tne and Tma) and mutants thereof. The Tne DNA polymerase has a molecular weight of about 100 kilodaltons and is more thermostable than Taq DNA polymerase. The mutant DNA polymerase has at least one mutation selected from the group consisting of (1) a first mutation that substantially reduces or eliminates 3'"'"'→5'"'"' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'"'"'.increment.3'"'"' exonuclease activity of said DNA polymerase; (3) a third mutation in the O helix of said DNA polymerase resulting in said DNA polymerase becoming non-discriminating against dideoxynucleotides. The present invention also relates to the cloning and expression of the wild type or mutant DNA polymerases in E. coli, to DNA molecules containing the cloned gene, and to host cells which express said genes. The DNA polymerases of the invention may be used in well-known DNA sequencing and amplification reactions.
110 Citations
185 Claims
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1. A mutant Thermotoga maritima DNA polymerase, which is modified to comprise a mutation in the 3'"'"'→
- 5'"'"' exonuclease domain of said polymerase to substantially reduce the 3'"'"'→
5'"'"' exonuclease activity of said polymerase, and is further modified in at least one way selected from the group consisting of;(a) a first mutation in the 5'"'"'→
3'"'"' exonuclease domain of said polymerase to substantially reduce the 5'"'"'→
3'"'"' exonuclease activity of said polymerase; and(b) a second mutation in the O helix of said polymerase to substantially reduce discriminatory activity against a dideoxynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185)
- 5'"'"' exonuclease domain of said polymerase to substantially reduce the 3'"'"'→
Specification