Thermococcus zilligii DNA polymerases and variants thereof
Native and variant Thermococcus zilligii DNA polymerases are disclosed, as are methods for using the same for nucleic acid synthesis, DNA sequencing, nucleic acid amplification and cDNA synthesis.
1 . An isolated native or variant Thermococcus zilligii (Tzi) DNA polymerase having an amino acid sequence at least 80% identical to SEQ ID NO: 2.
2 . The isolated Tzi DNA polymerase of claim 1 , having a molecular weight of about 90 kDa, and being stable at 95° C. for 60 minutes.
3 . An expression vector encoding the Tzi DNA polymerase of claim 1 .
4 . A host cell comprising vector of claim 3 .
5 . An isolated monoclonal antibody that binds to the Tzi DNA polymerase of claim 1 .
6 . 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- and/or didexoyribonucleoside triphosphates and the Tzi DNA polymerase of claim 1 under conditions sufficient to synthesize a second DNA molecule complementary to all or a portion of said first DNA molecule.
7 . 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 DNA polymerase of claim 1 , 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.
8 . 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 DNA polymerase of claim 1 and dATP, dCTP, dGTP and dTTP, whereby a cDNA-RNA hybrid is obtained.
9 . 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 DNA polymerase of claim 1 , dATP, dCTP, dGTP and dTTP, and an oligonucleotide or primer which is complementary to the first strand cDNA; whereby dsDNA is obtained.