Patent ID: 7148049

Claim:
An isolated thermostable or thermoactive DNA polymerase comprising a 3′-5′ exonuclease domain and exhibiting an attenuated 3′-5′ exonuclease activity of about 6.5 or less, but greater than 0, U/pmol, wherein one unit (U) of 3′-5′ exonuclease activity catalyzes the conversion of 50 pmol of a labeled single-stranded oligonucleotide of SEQ ID NO:80 to shorter length oligonucleotides in 15 minutes at 63° C., the enzymatic activity being measured in a reaction mixture containing 4 pmol (100 nM) of the oligonucleotide of SEQ ID NO: 80 in 50 mM Tricine, pH 8.3, 25 mM KOAc, 5% w/v DMSO, 0.5 mM Mn(OAc) 2 and the polymerase, and incubated at 63° C. for 15 minutes, wherein degradation of the oligonucleotide of SEQ ID NO: 80 is measured, wherein said 3′-5′ exonuclease domain comprises a sequence motif selected from the following sequence motifs: (a) A sequence motif having the formula DX 1 EX 2 X 3 SX 4 , wherein D is an aspartate residue, X 1 is any amino acid residue E is a glutamate residue, X 2 is a threonine, X 3 is any amino acid residue, S is a serine residue, and X 4 is a leucine or an alanine, or (b) A sequence motif having the formula X 5 X 6 X 7 X 8 X 9 X 10 X 11 X 12 X 13 X 14 (SEQ ID NO:1), wherein X 5 is a glutamine or an alanine, X 6 is an asparagine or an alanine, X 7 is a cysteine or a leucine residue, X 8 is a lysine, X 9 is a phenylalanine or a tyrosine residue, X 10 is an aspartate or a glutamate, X 11 is any amino acid residue, X 12 is any amino acid residue, X 13 is an isoleucine or valine residue, and X 14 is a leucine or phenylalanine residue.