DNA polymerase blends and mutant DNA polymerases
A thermostable DNA polymerase composition comprising at least two DNA polymerases, one of which is substantially reduced in 5′-exonuclease activity and one of which has 5′-exonuclease activity. This polymerase may be used in methods including, but not limited to, nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis,
1 . An isolated recombinant nucleic acid molecule encoding a Thermus filiformis (Tfi) DNA polymerase having a D144A point mutation, wherein said point mutation substantially reduces the 5′-exonuclease activity of said polymerase.
2 . The isolated nucleic acid molecule of claim 1 , further comprising an E437D point mutation.
3 . An isolated mutant Tfi DNA polymerase produced from the nucleic acid molecule of claim 1 .
4 . An isolated mutant Tfi DNA polymerase having a D144A point mutation.
5 . An isolated mutant Tfi DNA polymerase having D144A and E437D point mutations.
6 . A composition comprising at least two thermostable DNA polymerases wherein at least one of said polymerases is substantially reduced in 5′ exonuclease activity (exo−) and wherein at least one of said polymerases has 5′ exonuclease activity (exo+).
7 . The composition of claim 6 , wherein said polymerases are from the same species of thermophilic bacteria.
8 . The composition of claim 6 , wherein said 5′-exo− and said 5′-exo+ polymerases are combined in a ratio of between 9:1 and 1:9 (exo−:exo+).
9 . The composition of claim 8 , wherein the ratio is 7:3 (exo−:exo+)
10 . A vector comprising the isolated nucleic acid molecule of claim 1 .
11 . The vector of claim 10 wherein said nucleic acid molecule is operably linked to a promoter.
12 . A host cell comprising the vector of claim 11 .
13 . The composition of claim 6 , further comprising at least two components selected from the group consisting of a detergent, buffer salt, deoxynucleoside triphosphate (dNTP) and dideoxynucleoside triphosphate (dNTP).
14 . The composition of claim 13 , wherein said components are a detergent, buffer salt and dNTP.
15 . A method of synthesizing a double-stranded DNA molecule, comprising:
(a) hybridizing a primer to a first DNA molecule; and
(b) incubating said DNA molecule recited in (a) in the presence of one or more deoxy- or didexoyribonucleoside triphosphates and the composition of claim 6 under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.
16 . A method of amplifying a double stranded DNA molecule, comprising:
(a) providing a first and second primer, wherein said first primer is complementary to a sequence at or near the 3′-terminus of the first strand of said DNA molecule and said second primer is complementary to a sequence at or near the 3′-terminus of the second strand of said DNA molecule;
(b) hybridizing said first primer to said first strand and said second primer to said second strand in the presence of the composition of claim 6 , under conditions such that the third strand complementary to said first strand and a fourth strand complementary to said second strand are synthesized;
(c) denaturing said first and third strands and said second and fourth strands; and
(d) repeating steps (a) to (c) one or more times.
17 . (canceled)
18 . (canceled)
19 . A method of preparing cDNA from mRNA, comprising:
(a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and
(b) contacting said hybrid formed in (a) with the composition of claim 6 and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid is obtained.
20 . A method of preparing dsDNA from mRNA, comprising:
(a) contacting mRNA with an oligo(dT) primer or other complementary primer to form a hybrid; and
(b) contacting said hybrid formed in (a) with the composition of claim 6 , dATP, dCTP, dGTP and dTTP, and an oligonucleotide or primer which is complementary to the first strand cDNA; whereby dsDNA is obtained.
21 . An isolated mutant Tfi DNA polymerase produced from the nucleic acid molecule of claim 2.