Fusion polymerase and method for using the same

Patent metadata

Field	Value
Publication number	US-10081799-B2
Application number	US-201715432031-A
Country	US
Kind code	B2
Filing date	Feb 14, 2017
Priority date	Aug 27, 2014
Publication date	Sep 25, 2018
Grant date	Sep 25, 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) a fusion protein comprising a DNA polymerase and a sequence-specific DNA binding domain;
(b) an exonuclease; and
(c) a ligase;
wherein the composition optionally comprises dNTPs.

2. The composition of claim 1 , wherein the fusion protein comprises:
(i) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:3,
(ii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:1; and
(iii) an amino acid sequence that has at least 90% sequence identity to SEQ ID NO:2.

3. The composition of claim 1 , wherein the fusion protein comprises:
(i) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:3,
(ii) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:1; and
(iii) an amino acid sequence that has at least 95% sequence identity to SEQ ID NO:2.

4. The composition of claim 1 , wherein the fusion protein comprises the amino acid sequence of SEQ ID NO:3.

5. The composition of claim 1 , wherein the exonuclease is a 5′-3′ exonuclease.

6. The composition of claim 5 , wherein the exonuclease is exonuclease T5.

7. The composition of claim 1 , wherein the ligase is thermostable.

8. The composition of claim 1 , wherein the ligase is NAD+ dependent and the composition further comprises NAD+.

9. The composition of claim 8 , wherein the ligase is Taq ligase.

10. The composition of claim 1 , wherein the ligase is ATP dependent and the composition further comprises ATP.

11. The composition of claim 1 , further comprising a single-stranded DNA binding protein.

12. The composition of claim 11 , wherein the single-stranded DNA binding protein is ET SSB (Extreme Thermostable Single-Stranded DNA Binding Protein), E. coli recA, T7 gene 2.5 product, phage lambda RedB or Rac prophage RecT.

13. The composition of claim 1 , wherein the composition does not comprise a crowding agent.

14. The composition of claim 1 , wherein the composition does not comprise a non strand-displacing polymerase.

15. The composition of claim 1 , wherein the composition comprises a potassium salt.

16. The composition of claim 15 , wherein the potassium salt is potassium chloride and the potassium chloride is at a concentration in the range of 7 mM to 150 mM.

17. The composition of claim 1 , wherein the composition comprises dNTPs.

18. The composition of claim 1 , wherein the composition comprises a non-naturally occurring buffering agent.

19. The composition of claim 1 , wherein the composition further comprises a set of overlapping polynucleotides.

20. The composition of claim 19 , wherein at least two of the oligonucleotides in the set have an overlap of at least 15 nucleotides.

21. The composition of claim 19 , wherein the set of overlapping polynucleotides has at least two members.

22. The composition of claim 19 , wherein the set of overlapping polynucleotides has at least five members.

23. The composition of claim 19 , wherein the set of overlapping polynucleotides comprises:
double-stranded polynucleotides;
at least one double strand polynucleotide and at least one single strand oligonucleotide;
single stranded oligonucleotides; and/or
a subpopulation of polynucleotides that are otherwise identical to one another except for a sequence that varies between the members of the sub-population.

24. 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 (a).

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

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

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

28. 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.

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

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

31. 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 SEQ ID NOS 56-97.

32. 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.

33. The composition of claim 1 , wherein the polymerase is bacterial or archaebacterial.

34. The composition of claim 1 , wherein the amino acid sequence of the polymerase is at least 90% identical to the amino acid sequence of a wild type Pyrococcal polymerase of any of SEQ ID NOS. 41, 47, or 48 or the amino acid sequence of a wild type Thermococcal polymerase of any of SEQ ID NOS. 42-46, 51 or 52.

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

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

Assignees

• New England Biolabs Inc

Inventors

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

Classifications

• C07K2319/00

  Fusion polypeptide · CPC title

• C12N15/1027

  by DNA shuffling, e.g. RSR, STEP, RPR · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N9/1252 Primary

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

• C12P19/34

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