POLYMERASE

• US 20100035767A1
• Filed: 08/24/2007
• Published: 02/11/2010
• 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: Ramsay, Nicola, Jemth, Ann-Sofie, Holliger, Phillip
• Granted Patent: US 8,435,775 B2
• Time in Patent Office: Days
• US Class Current: 506/12
• 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. An engineered polymerase with an expanded substrate range characterised in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase as compared with the wild type polymerase from which it is derived.

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

3. The engineered polymerase according to claim 2 wherein the agent-labelled nucleotide analogue is a fluorescent labelled nucleotide analogue.

4. The engineered polymerase according to claim 3 wherein the fluorescent label is selected from the group consisting of Alexa Flour™

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

5. The engineered polymerase according to claim 4 wherein the agent-labelled nucleotide analogue is a carbocyanine labelled nucleotide analogue.

6. The engineered polymerase according to claim 1 which is a DNA polymerase.

7. The engineered polymerase according to claim 2, wherein the dye labelled nucleotide analogue is selected from the group consisting of Cy5-dNTP and Cy3-dNTP.

8. The engineered polymerase according to claim 2, wherein the dye labelled nucleotide analogue is selected from the group consisting of Cy5-dCTP and Cy3-dCTP.

9. The engineered polymerase according to claim 6 wherein the polymerase has one or more of the following mutations:

V337I, E399D, N400D, R407I and/or Y546H.

10. The engineered polymerase according to claim 1 comprising the amino acid sequence designated SEQ ID NO:

1.

11. The engineered polymerase according to claim 1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs:

4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38 and 40.

13. The engineered DNA polymerase according to claim 6 wherein the engineered DNA polymerase is derived from wild type DNA polymerase by substitution, deletion or insertion of one or more amino acids.

14. The engineered DNA polymerase according to claim 6, wherein the engineered DNA polymerase is derived from a DNA polymerase by substitution, deletion or insertion of one or more amino acids.

15. The engineered polymerase according to claim 14, wherein the DNA polymerase is selected from the group consisting of polA polymerases, polB polymerases and pfu polymerases.

16. The engineered polymerase according to claim 1, wherein the amino acid sequence of the polymerase is at least 70% identical to that designated herein as ElO and set forth in SEQ ID NO:

1.

22. A method for the incorporation of detection agent-labelled nucleotide analogues into newly synthesised nucleic acid which method comprises the use of an engineered polymerase according to claim 1.

24. The method according to claim 22 wherein the detection agent-labelled nucleotide is a dye-labelled nucleotide analogue.

25. The method according to claim 24 wherein the dye-labelled nucleotide analogue is a fluorescent dye-labelled nucleotide analogue.

26. The method according to claim 25 wherein the fluorescent dye-labelled nucleotide analogue is selected from the group consisting of the following:

(Cy3-CTP, Cy5-CTP.

28. The use of an engineered polymerase with an expanded substrate range according to claim 1 in one or more biotechnological techniques in the group consisting of the following:

polymerase chain reaction (PCR);

microarray analysis (such as gene expression microarray analyses, tissue microarrays, array Comparative Genome Hybridisations);

fluorescent in-situ hybridisation (FISH);

fibre FISH;

comparative genome hybridisations;

DNA sequencing, nucleic acid sequencing, (eg. single-stranded nucleic acid sequencing) and single molecule detection.

29. The use according to claim 28 wherein the engineered polymerase is a Pfu mutant polymerase having, a nucleotide sequence which is at least 70%, identical to the Pfu mutant expressed by one of the clone selected from the group consisting of the following:

23, AH12, 55, 15, 33, 34, 35 and ElO.

30. The use according to claim 29 wherein the engineered DNA polymerase is E10.

31. A kit for incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid comprising the isolated, engineered DNA polymerase according to claim 1.

17. An isolated nucleic acid molecule which encodes an engineered polymerase polypeptide wherein the nucleotide sequence is at least 80% identical to any of the nucleotide sequences selected from the group consisting of SEQ ID NOS:

2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39, and wherein said polypeptide is capable of incorporating an enhanced occurrence of detection agent-labelled nucleotide analogue into nucleic acid synthesised by that engineered polymerase.

12. The isolated nucleic acid molecule according to claim 17 comprising a nucleotide sequence encoding an engineered polymerase and selected from the group consisting of SEQ ID NOS:

2, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37 and 39.

18. A method for the generation of an engineered polymerase with an expanded substrate range characterised in that the polymerase is capable of incorporating an enhanced occurrence of detection agent-label led nucleotide analogue into nucleic acid synthesised by that engineered polymerase as compared with the wild type polymerase from which it is derived, which method comprises the steps of:

(a) generating a polymerase repertoire/library;

(b) performing compartmentalised self replication (CSR) using one of the libraries of step (a), wherein the CSR is performed in emulsion, utilising primers which anneal 3′ and

5′

of the region diversified in the library according to step (a) and wherein the emulsion comprises detection agent-labelled dNTP in one embodiment dCTP, in place of the corresponding dNTP;

(c) expressing those engineered polymerase repertoire members selected according to step (b) to obtain the protein product (mutant enzyme);

(d) selecting those expressed mutant enzymes which are capable of incorporating one or more dye-labelled nucleotide analogues; and

(e) optionally, isolating and purifying the selected mutant polymerase.

19. The method according to claim 18 wherein the mutant polymerase is selected from the group consisting of the following;

a Pfu polymerase;

a PoIA polymerase and PoIB polymerase and combinations thereof.

20. The method according to claim 18 wherein the engineered polymerase is a Pfu polymerase generated from one or more repertoires of Pfu genes in which diversity is targeted to discrete regions.

21. The method according to claim 18 wherein the engineered polymerase is a Pfu polymerase generated from repertoires which are themselves generated by recombining related Pfu genes.

23. (canceled)

27. (canceled)

32-38. -38. (canceled)