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 polypeptide comprising a nuclease domain joined to a sequence non-specific double-stranded nucleic acid binding domain, wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 75% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
2. The polypeptide of claim 1 , wherein the nuclease domain is an exonuclease.
3. The polypeptide of claim 2 , wherein the exonuclease is a 5′ to 3′ exonuclease.
4. The polypeptide of claim 1 , wherein the nuclease domain is an endonuclease.
5. The polypeptide of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 75% identity to SEQ ID NO:2.
6. The polypeptide of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 75% identity to amino acids 7-71 of SEQ ID NO:10.
7. A recombinant fusion polypeptide comprising at least two heterologous domains wherein a first domain that is a sequence-non-specific double-stranded nucleic-acid-binding domain that comprises an amino sequence that has at least 75% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10, is joined to a second domain that is a catalytic nucleic-acid-modifying domain, wherein the catalytic nucleic acid-modifying domain is a polymerase and where the presence of the sequence non-specific double-stranded nucleic acid binding domain stabilizes the double-stranded conformation of the nucleic acid.
8. 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 amplification primers and a recombinant fusion polypeptide of claim 7 , wherein at least one of the amplification primers is a short primer; and
incubating the solution under conditions that amplify the subsequence.
9. The method of claim 8 , wherein the polymerase is ΔTaq.
10. The method of claim 8 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 75% identity to SEQ ID NO:2.
11. The method of claim 8 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 75% identity to amino acids 7-71 of SEQ ID NO:10.
12. The polypeptide of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 80% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
13. The polypeptide of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 90% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
14. The polypeptide of claim 1 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence of SEQ ID NO:2 or amino acids 7-71 of SEQ ID NO:10.
15. The recombinant fusion polypeptide of claim 7 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 80% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
16. The recombinant fusion polypeptide of claim 7 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 90% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
17. The recombinant fusion polypeptide of claim 7 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence of SEQ ID NO:2 or amino acids 7-71 of SEQ ID NO:10.
18. The method of claim 8 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 80% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
19. The method of claim 8 , wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 90% identity to SEQ ID NO:2 or to amino acids 7-71 of SEQ ID NO:10.
20. The method of claim 8 , wherein the sequence non-specific double-stranded nucleic acid binding domain comprises the amino acid sequence of SEQ ID NO:2 or amino acids 7-71 of SEQ ID NO:10.