Mutant RB69 DNA polymerase
Provided herein are mutant DNA-dependent polymerases which are derived from, or otherwise related to, wild type RB69 DNA polymerase. These mutant polymerases are capable of selectively binding labeled nucleotides. These mutant polymerases are also capable of incorporating a variety of naturally occurring and modified nucleotides, including, for example, terminator nucleotides.
1. An isolated DNA polymerase comprising the amino acid sequence of any one of SEQ ID NOS: 5-8.
2. The isolated DNA polymerase of claim 1 further comprising a reporter moiety.
3. The isolated DNA polymerase of claim 2 , wherein the reporter moiety is an energy transfer donor moiety.
4. The isolated DNA polymerase of claim 3 , wherein the energy transfer donor moiety is a fluorescent dye or a nanoparticle.
5. A system comprising a DNA polymerase of claim 1 bound to a DNA template and a primer, and a nucleotide transiently-bound to the DNA polymerase.
6. The system of claim 5 , further comprising a metal cation that inhibits incorporation of the transiently-bound nucleotide.
7. The system of claim 6 , wherein the metal cation is selected from a group consisting of calcium, scandium, titanium, vanadium, chromium, iron, cobalt, nickel, copper, zinc, gallium, germanium, arsenic, and selenium.
8. The system of claim 5 , wherein the transiently-bound nucleotide is linked to a reporter moiety.
9. The system of claim 5 , wherein the DNA polymerase further comprises a reporter moiety.
10. The system of claim 9 , wherein the reporter moiety comprises an energy transfer donor moiety.
11. The system of claim 9 , wherein the reporter moiety comprises a fluorescent dye or a nanoparticle.
12. The system of claim 5 , wherein the transiently-bound nucleotide comprises a reversible terminator nucleotide.
13. The system of claim 12 , wherein the reversible terminator nucleotide comprises an azidomethyl blocking group on the 3′ sugar position.
14. The isolated DNA polymerase of claim 1 , wherein the polymerase comprises SEQ ID NO: 7.
15. An isolated DNA polymerase comprising the amino acid sequence of SEQ ID NO:3.
16. The isolated DNA polymerase of claim 15 further comprising a reporter moiety.
17. The isolated DNA polymerase of claim 16 , wherein the reporter moiety is an energy transfer donor moiety.
18. A system comprising the DNA polymerase of claim 15 bound to a DNA template and a primer, and a nucleotide transiently-bound to the DNA polymerase.
19. The system of claim 18 , further comprising a metal cation that inhibits incorporation of the transiently-bound nucleotide, wherein the transiently-bound nucleotide is linked to a reporter moiety.
20. The system of claim 18 , wherein the transiently-bound nucleotide comprises a reversible terminator nucleotide.