Fusion polymerase and method for using the same

Patent metadata

Field	Value
Publication number	US-9963687-B2
Application number	US-201615046166-A
Country	US
Kind code	B2
Filing date	Feb 17, 2016
Priority date	Aug 27, 2014
Publication date	May 8, 2018
Grant date	May 8, 2018

Abstract

This disclosure provides, among other things, a composition comprising: comprising a fusion protein comprising: (a) a DNA polymerase; and (b) a heterologous sequence-specific DNA binding domain. A method for copying a DNA template, as well as a kit for performing the same, are also described.

First claim

What is claimed is:

1. A composition comprising a fusion protein comprising:
(a) a DNA polymerase; and
(b) a sequence-specific DNA binding domain.

2. The composition of claim 1 , wherein the fusion protein exhibits increased processivity relative to the polymerase of (a) in the absence of the DNA binding domain of (b).

3. The composition of claim 1 , wherein the sequence-specific DNA binding domain is N-terminal of the polymerase.

4. The composition of claim 1 , wherein the sequence-specific DNA binding domain is C-terminal of the polymerase.

5. The composition of claim 1 , wherein the DNA polymerase is a type A polymerase.

6. The composition of claim 1 , wherein the DNA polymerase is a type B polymerase.

7. The composition of claim 1 , wherein the sequence-specific DNA binding domain has a helix-loop-helix, ribbon-helix-helix, helix-turn-helix, winged helix, or homeodomain structure.

8. The composition of claim 1 , wherein the sequence-specific DNA binding domain is from a transcriptional activator.

9. The composition of claim 1 , wherein the polymerase has proofreading activity.

10. The composition of claim 1 , wherein the wild type polymerase is bacterial or archaebacterial.

11. The composition of claim 1 , wherein the amino acid sequence of the sequence-specific DNA binding domain is at least 90% identical to a DNA binding domain of any of SEQ ID NOS 56-97.

12. The composition of claim 1 , wherein the amino acid sequence of the polymerase is at least 90% identical to the amino acid sequence of any of SEQ ID NOS. 33-55.

13. The composition of claim 12 , wherein the amino acid sequence of the polymerase is at least 90% identical to either the amino acid sequence of a Pyrococcal polymerase of any of SEQ ID NOS. 41, 47, or 48 or the amino acid sequence of a Thermococcal polymerase of any of SEQ ID NOS. 42-46, 51 or 52.

14. The composition of claim 1 , wherein the polymerase is thermostable.

15. The composition of claim 1 , wherein the sequence-specific DNA binding domain is thermostable.

16. A kit comprising:
(a) the composition of claim 1 ; and
(b) a reaction buffer.

17. The kit of claim 16 , wherein the composition further comprises glycerol.

18. The kit of claim 16 , wherein the buffer is in concentrated form.

19. A method comprising:
combining a DNA template, nucleotides, and the composition of claim 1 to produce a reaction mix; and
copying the DNA template.

20. The method of claim 19 , wherein the DNA template is a plurality of overlapping primers.

21. The method of claim 19 , wherein the DNA template is genomic DNA.

22. The method of claim 19 , wherein the copying step is done using isothermal conditions.

23. The method of claim 19 , wherein the copying step is done by thermocycling.

Assignees

• New England Biolabs Inc

Inventors

• Hsieh Pei-Chung
• Sun Luo
• Evans Jr Thomas C
• Davis Theodore B
• Gardner Andrew

Classifications

• C12P19/34

  Polynucleotides, e.g. nucleic acids, oligoribonucleotides · CPC title

• C12N15/66

  General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C07K2319/00

  Fusion polypeptide · CPC title

• C12Y207/07007

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title