Fusion polymerase and method for using the same
View Patent ↗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 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.