Variant DNA polymerases

• US 5,928,919 A
• Filed: 06/06/1995
• Issued: 07/27/1999
• Est. Priority Date: 08/02/1993
• Status: Expired due to Fees

• Current Assignee: University of Alberta, University of Southern California
• Original Assignee: University of Alberta, University of Southern California
• Inventors: Goodman, Myron F., Reha-Krantz, Linda J.
• Primary Examiner(s): Houtteman, Scott W.
• Application Number:
  US08/465,994
• Time in Patent Office: 1,512 Days
• Field of Search: 530/350, 530/358, 935/77, 935/82, 935/11, 435/183
• US Class Current: 435/183
• CPC Class Codes: C07H 19/04 Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6869 Methods for sequencing

Abstract

Variant family B DNA polymerases, examples of which have reduced or no 3'"'"'→5'"'"' exonuclease activity. These variant polymerases have utility as DNA sequencing polymerases.

Claims

1. A variant family B DNA polymerase selected from the group consisting of:

a variant E coli DNA polymerase II with the aspartic acid present at its codon position 156 replaced with alanine; and

a variant E. coli DNA polymerase II with the glutamic acid present at its codon position 158 is replaced with alanine.

3. The variant E. coli DNA polymerase II of claim 1, characterized in that the aspartic acid at its codon position 156 is replaced with alanine and the glutamic acid present at its codon position 158 is replaced with alanine.

2. A variant family B DNA polymerase, said variant polymerase selected from the group consisting of T2, T4, and T6 DNA polymerases, characterized in that the aspartic acid present at its codon position 112 is replaced with alanine and the glutamic acid present at its codon position 114 is replaced with alanine.

4. A variant family B DNA polymerase for use in DNA sequencing, said variant polymerase selected from the group consisting of T2, T4, and T6 DNA polymerases, said variant polymerase comprising at least one mutation, wherein the mutation reduces 3'"'"'→

5'"'"' exonuclease activity and is selected from the group consisting of;

aspartic acid replaced with alanine at codon position 112;

glutamic acid replaced with alanine at codon position 114;

aspartic acid replaced with alanine at codon position 219; and

aspartic replaced with alanine at codon position 324.

5. A variant family B DNA polymerase for use in DNA sequencing, said variant polymerase selected from the group consisting of T2, T4, and T6 DNA polymerases, said variant polymerase comprising at least two mutations, wherein:

1) a first mutation reduces 3'"'"'→

5'"'"' exonuclease activity and is selected from the group consisting of;

a) aspartic acid replaced with alanine at codon position 112,

b) glutamic acid replaced with alanine at codon position 114,

c) aspartic acid replaced with alanine at codon position 219,

d) glycine replaced with serine at codon position 255, and

e) aspartic acid replaced with alanine at codon position 324; and

2) a second mutation is selected from the group consisting of;

a) isoleucine replaced with leucine at codon position 50,

b) glycine replaced with aspartic acid at codon position 82, and

c) glutamic acid replaced with lysine at codon position 743.

6. A variant family B DNA polymerase for use in DNA sequencing, said variant polymerase selected from the group consisting of T2, T4, and T6 DNA polymerases, the variant polymerase comprising at least one mutation, the mutation selected from the group consisting of:

isoleucine replaced with leucine at codon position 50; and

glutamic acid replaced with lysine at codon position 743.