Nucleic acid modifying enzymes
This invention provides for an improved generation of novel nucleic acid modifying enzymes. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the ability of the enzyme to bind and catalytically modify the nucleic acid.
1. A method of amplifying a 5kb or longer subsequence of a target nucleic acid in an aqueous solution using a polymerase chain reaction, the method comprising:
(i) contacting the target nucleic acid with a DNA polymerase comprising a sequence-non-specific double-stranded nucleic-acid-binding domain that comprises an amino acid sequence that has at least 75% identity to the amino acid sequence of SEQ ID NO:2 joined to a DNA polymerase domain with error-correcting activity, where the sequence non-specific double-stranded nucleic-acid-binding domain enhances the processivity of the DNA polymerase domain compared to an identical DNA polymerase domain not having the sequence non-specific double-stranded nucleic acid binding domain; and
wherein the solution is of a composition that permits the sequence non-specific double-stranded nucleic acid binding domain to bind to the target nucleic acid and the DNA polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence to a length of 5 kb or longer;
(ii) incubating the solution using a polymerase chain reaction temperature profile that amplifies the 5 kb or longer subsequence.
2. The method of claim 1 , wherein the polymerase domain has thermally stable polymerase activity.
3. The method of claim 1 , wherein the polymerase domain comprises a Pyrococcus polymerase domain.
4. The method of claim 1 wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 85% identity to SEQ ID NO:2.
5. The method of claim 1 wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 90% identity to SEQ ID NO:2.
6. The method of claim 1 wherein the sequence-non-specific double-stranded nucleic-acid-binding domain comprises the amino acid sequence of SEQ ID NO:2.
7. A method of amplifying a subsequence of a target nucleic acid in an aqueous solution using a polymerase chain reaction, the method comprising:
contacting the target nucleic acid with a DNA polymerase comprising a sequence-non-specific double-stranded nucleic-acid-binding domain that comprises an amino acid sequence that has at least 75% identity to the amino acid sequence of SEQ ID NO:2 joined to a DNA polymerase domain with error-correcting activity, where the sequence non-specific nucleic-acid-binding domain enhances the processivity of the DNA polymerase domain compared to an identical DNA polymerase domain not having the sequence non-specific double-stranded nucleic acid binding domain; and
wherein the solution comprises 10 5 or fewer copies/ml of the target nucleic acid and is of a composition that permits the sequence non-specific double-stranded nucleic acid binding domain to bind to the target nucleic acid and the DNA polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;
(ii) incubating the solution using a polymerase chain reaction temperature profile that amplifies the subsequence.
8. The method of claim 7 wherein the polymerase domain has thermally stable polymerase activity.
9. The method of claim 7 wherein the polymerase domain comprises a Pyrococcus polymerase domain.
10. The method of claim 7 wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 85% identity to SEQ ID NO:2.
11. The method of claim 7 wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 90% identity to SEQ ID NO:2.
12. The method of claim 7 , wherein the sequence-non-specific double-stranded nucleic-acid-binding domain comprises the amino acid sequence of SEQ ID NO:2.
13. A method of amplifying a target nucleic acid in an aqueous solution, the method comprising:
(i) contacting the target nucleic acid with a DNA polymerase comprising a sequence non-specific double-stranded nucleic acid binding domain joined to a DNA polymerase domain, wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 75% identity to the amino acid sequence of SEQ ID NO:2, where the presence of the sequence non-specific double-stranded nucleic acid binding domain enhances the processivity of the DNA polymerase domain compared to an identical DNA polymerase domain not having the sequence non-specific double-stranded nucleic acid binding domain; and
wherein the solution is of a composition that permits the sequence non-specific double-stranded nucleic acid binding domain to bind to the target nucleic acid and the polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;
(ii) incubating the solution such that the primer is extended, thereby amplifying the target nucleic acid.
14. The method of claim 13 , wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 85% identity to SEQ ID NO:2.
15. The method of claim 13 , wherein the sequence-non-specific double-stranded nucleic-acid-binding domain has at least 90% identity to SEQ ID NO:2.
16. The method of claim 13 , wherein the sequence-non-specific double-stranded nucleic-acid-binding domain comprises the amino acid sequence of SEQ ID NO:2.
17. The method of claim 13 , wherein the polymerase domain comprises a family A polymerase domain.
18. The method of claim 13 , wherein the polymerase domain comprises a family B polymerase domain.
19. The method of claim 13 , wherein the polymerase domain is thermostable.
20. The method of claim 13 , wherein the polymerase domain is a Thermus polymerase domain or a Pyrococcus polymerase domain.
21. A method of claim 13 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 75% sequence identity to the Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10.
22. The method of claim 19 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 85% sequence identity to the Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10.
23. The method of claim 20 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises an amino acid sequence that has at least 90% sequence identity to the Sac7d sequence set forth in amino acids 7-71 of SEQ ID NO:10.
24. The method of claim 13 , wherein the sequence non-specific nucleic-acid-binding domain is Sac7d.