Mutant Pfu DNA polymerase

• US 8,435,775 B2
• Filed: 08/24/2007
• Issued: 05/07/2013
• Est. Priority Date: 09/06/2006
• Status: Active Grant

• Current Assignee: UK Research and Innovation
• Original Assignee: Medical Research Council (Government of United Kingdom)
• Inventors: Holliger, Phillip, Ramsay, Nicola, Jemth, Ann-Sofie
• Primary Examiner(s): KIM, ALEXANDER D
• Application Number:
  US12/440,374
• Publication Number:
  US 20100035767A1
• Time in Patent Office: 2,083 Days
• Field of Search: None
• US Class Current: 435/183
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence

Abstract

The present invention relates to an engineered polymerase with an expanded substrate range characterized in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-labeled nucleotide analogue into nucleic acid synthesized by that engineered polymerase as compared with the wild type polymerase from which it is derived.

Claims

1. A purified engineered DNA polymerase comprising an amino acid sequence, wherein the engineered polymerase has an expanded substrate range and incorporates an enhanced occurrence of a dye-labelled nucleotide analogue into nucleic acid synthesized by the engineered polymerase as compared with the wild type polymerase from which it is derived, wherein the engineered DNA polymerase has at least 90% identity to SEQ ID NO:

1, 4, 8, 12, 14, 16, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48, and wherein the engineered DNA polymerase comprises the following substitution mutations compared with the Pyrococcus furiosus (Pfu) wild-type DNA polymerase amino acid sequence set forth in SEQ ID NO;

134;

N400G/N400D and R407I.

2. The purified engineered DNA polymerase according to claim 1 wherein the dye-labelled nucleotide analogue is a fluorescent labelled nucleotide analogue.

3. The purified engineered DNA polymerase according to claim 2 wherein the fluorescent label is Alexa Fluor™

dye, diethylaminocoumarin, tetramethylrhodamine, N-methylanthraniloyl, trinitrophenyl, etheno derivatives, fluorescein, or carbocyanine.

4. The purified engineered DNA polymerase according to claim 3 wherein the dye-labelled nucleotide analogue is a carbocyanine labelled nucleotide analogue.

5. The purified engineered DNA polymerase according to claim 4, wherein the dye-labelled nucleotide analogue is Cy5-dNTP or Cy3-dNTP.

6. The purified engineered DNA polymerase according to claim 5, wherein the dye-labelled nucleotide analogue is Cy5-dCTP or Cy3-dCTP.

7. The purified engineered DNA polymerase according to claim 1, wherein the engineered polymerase further has one or more of the mutations V337I, E399D and Y546H compared with the Pfu wild-type amino acid sequence.

8. The purified engineered DNA polymerase according to claim 1 comprising the amino acid sequence set out in SEQ ID NO:

46 or an amino acid sequence at least identical to SEQ ID NO;

46.

9. The purified, engineered DNA polymerase of claim 1 that has substantially 0% of a 3′

-5′

exonuclease activity of the Pfu wild-type DNA polymerase.

10. A method for the incorporation of dye-labeled nucleotide analogues into newly synthesized nucleic acid comprising:

incubating a nucleic acid template, a primer, a polymerase, a buffer, and nucleotide synthesis reagents, including a dye-labeled nucleotide analogue, under conditions in which the polymerase synthesizes nucleic acid, wherein the polymerase is the purified engineered DNA polymerase according to claim 1.

11. The method of claim 10 wherein the dye-labeled nucleotide analogue is a fluorescent dye-labelled nucleotide analogue.

12. The method of claim 11 wherein the fluorescent dye-labelled nucleotide analogue is Cy3-CTP or Cy5-CTP.

13. The method of claim 11 wherein the method comprises a polymerase chain reaction;

a microarray analysis;

fluorescent in-situ hybridisation (FISH);

fibre FISH;

a comparative genome hybridisation;

DNA sequencing;

nucleic acid sequencing;

or single molecule detection.

14. The method of claim 11 wherein the engineered DNA polymerase has the amino acid sequence set out in SEQ ID NO:

46 or an amino acid sequence at least identical to SEQ ID NO;

46.

15. A kit for incorporating an enhanced occurrence of dye-labelled nucleotide analogue into nucleic acid, the kit comprising the purified engineered DNA polymerase according to claim 1.

16. The kit of claim 15 wherein the purified engineered DNA polymerase has the amino acid sequence set out in SEQ ID NO:

46 or an amino acid sequence at least 90% identical to SEQ ID NO;

46.

17. The kit according to claim 15, wherein the kit further includes a dye-labeled nucleotide analogue.