Cloned DNA polymerases from thermotoga and mutants thereof

Details Cloned DNA polymerases from thermotoga and mutants thereof Cloned DNA polymerases from thermotoga and mutants thereof

1996-09-06

2000-01-18

Hendricks, Keith D.

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 911, 435 912, 435194, 4353201, 435325, 435419, 4352523, 536 232, C12Q 168, C12P 1934, C12N 912

Patent

active

060156686

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'.fwdarw.5' exonuclease activity of said DNA polymerase; (2) a second mutation that substantially reduces or eliminates 5'.fwdarw.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.

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