Dna polymerases

Abstract

The present invention provides a DNA polymerase including the sequence of SEQ ID NO. 1 or a sequence which is at least 70% identical thereto, but wherein the aspartic acid residue at position 18 of SEQ ID NO. 1, or the equivalent aspartic acid residue in other sequences, has been replaced by a non-negatively charged amino acid residue. It further provides DNA polymerases comprising the amino acid sequences of SEQ ID NO. 2, 11 and 12 and variants thereof. The present invention also provides nucleic acids encoding the DNA polymerases, a method of producing said DNA polymerases, and compositions, expression vectors and host cells or viruses comprising said DNA polymerases. The present invention also provides uses of said DNA polymerases in nucleotide polymerisation, amplification, and sequencing reactions.

1 . A DNA polymerase comprising the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine. 
     
     
         2 . The DNA polymerase according to  claim 1 , wherein said DNA polymerase comprises an amino acid sequence which is at least 80% identical to SEQ ID NO:11, wherein the aspartic acid residue at position 422 of SEQ ID NO:11, or the equivalent aspartic acid residue in the variant sequence, is replaced by alanine. 
     
     
         3 . The DNA polymerase according to  claim 1 , wherein said DNA polymerase comprises the amino acid sequence of SEQ ID NO: 11, or an amino acid sequence which is at least 90% identical to SEQ ID NO: 11, wherein the aspartic acid residue at position 422 of SEQ ID NO: 11, or the equivalent aspartic acid residue at the equivalent position in the variant sequence, is replaced by alanine. 
     
     
         4 . The DNA polymerase according to  claim 1 , wherein said DNA polymerase has at least 30% greater strand displacement activity as compared to a DNA polymerase with SEQ ID NO:11 but with aspartic acid at position 422, relative to SEQ ID NO:11, or at the equivalent position in the variant sequence. 
     
     
         5 . The DNA polymerase according to  claim 1 , wherein across a concentration range from 20 mM to 200 mM of NaCl, KCl, or a mixture thereof, said DNA polymerase exhibits at least 40% of its maximum polymerase activity. 
     
     
         6 . A composition, comprising:
 the DNA polymerase according to  claim 1 , and   a buffer.   
     
     
         7 . A nucleic acid molecule, comprising:
 a nucleotide sequence encoding the DNA polymerase according to  claim 1 .   
     
     
         8 . The nucleic acid molecule of  claim 7 , wherein the nucleotide sequence has at least 70% sequence identity to SEQ ID NO: 14. 
     
     
         9 . An expression vector comprising the nucleic acid molecule of  claim 8 , and one or more regulatory sequences enabling transcription and translation of a protein encoded by said nucleic acid molecule. 
     
     
         10 . A host cell or virus, comprising one or more expression vectors according to  claim 9 . 
     
     
         11 . A host cell or virus, comprising one or more nucleic acid molecules according to  claim 8 . 
     
     
         12 . A method of producing a DNA polymerase, which comprises:
 (i) culturing a host cell in a growth medium, wherein the host cell comprises one or more recombinant expression vectors or one or more nucleic acid molecules encoding the DNA polymerase, under conditions suitable for expression of the encoded DNA polymerase; and optionally   (ii) isolating the expressed DNA polymerase from the host cell or from the growth medium or supernatant of the growth medium,   wherein the DNA polymerase comprises the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, and   wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine.   
     
     
         13 . A method of nucleotide polymerization, said method comprising:
 (i) providing a reaction mixture comprising
 a DNA polymerase, 
 a template nucleic acid molecule, 
 an oligonucleotide primer which is capable of annealing to a portion of the template nucleic acid molecule and 
 one or more species of nucleotide; and 
   (ii) incubating said reaction mixture under conditions whereby the oligonucleotide primer anneals to the template nucleic acid molecule and said DNA polymerase extends said oligonucleotide primer by polymerizing one or more nucleotides,   wherein the DNA polymerase comprises the amino acid sequence of SEQ ID NO:11 or a variant sequence which is at least 70% identical to SEQ ID NO:11, and   wherein an aspartic acid residue at position 422 of SEQ ID NO:11, or an equivalent aspartic acid residue at an equivalent position in the variant sequence, is replaced by alanine.   
     
     
         14 . The method of  claim 13 , wherein said method is performed at a constant temperature. 
     
     
         15 . The method of  claim 14 , wherein said constant temperature is from 0° C. to 42° C. 
     
     
         16 . The method of  claim 14 , wherein said constant temperature is from 10° C. to 25° C.