Thermococcus barossii DNA polymerase mutants

• US 5,882,904 A
• Filed: 08/04/1997
• Issued: 03/16/1999
• Est. Priority Date: 08/04/1997
• Status: Expired due to Term

• Current Assignee: GE Healthcare Bio-Sciences Corporation (General Electric Company)
• Original Assignee: Amersham Pharmacia Biotech Inc. (General Electric Company)
• Inventors: Ting, Eve Y., Fly, Susan J., Riedl, William A., Kaboord, Barbara F., Nye, Steven H.
• Primary Examiner(s): Hendricks, Keith D.
• Application Number:
  US08/906,925
• Time in Patent Office: 589 Days
• Field of Search: 435/194, 435/91.2
• US Class Current: 435/91.2
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6837 using probe arrays or probe chips C12Q1/6874 takes precedence
• CPC Class Codes: C12Q 2521/101 DNA polymerase
• CPC Class Codes: C12Q 2533/101 Primer extension

Abstract

A Thermococcus barosii DNA polymerase with reduced 3'"'"'-5'"'"' exonuclease activity is disclosed. Additionally disclosed is a Thermococcus barosii DNA polymerase with increased ability to incorporate ribonucleotides and dideoxynucleotides.

Claims

1. A mutant of a native Thermococcus barossii DNA polymerase wherein the mutant polymerase has reduced 3'"'"'-5'"'"' exonuclease activity relative to the native polymerase, wherein the mutant polymerase comprises SEQ ID NO:

2, wherein the mutant polymerase further comprises a different amino acid residue 489 than the native polymerase, and wherein the mutant polymerase also has increased ability to incorporate dideoxy nucleotides or ribonucleotides relative to the native polymerase.

2. The polymerase of claim 1 wherein amino acid residue 489 is selected from the group consisting of tyrosine and phenylalanine.

3. The polymerase of claim 2 wherein residue 489 is a tyrosine.

4. The polymerase of claim 1 wherein the increase is at least 2-fold.

5. The polymerase of claim 4 wherein the increase is at least 3-fold.

6. The polymerase of claim 1 wherein the increase is at least 5-fold.

7. A method of DNA polymerization, comprising reaching the polymerase of claim 8 with a DNA template, primer, dNTPs, and other reagents necessary for polymerization.

8. A method of arrayed primer extension, comprising the steps of

a. exposing the polymerase of claim 1 to bound oligonucleotide primers that are arrayed on a solid support, chain terminators, and to other reagents necessary for primer extension, and

b. incorporating chain terminators, whereby primer extension ceases.