Fusion polymerase and method for using the same
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.
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.