SSB-polymerase fusion proteins

Patent metadata

Field	Value
Publication number	US-9580743-B2
Application number	US-201414450925-A
Country	US
Kind code	B2
Filing date	Aug 4, 2014
Priority date	Sep 9, 2005
Publication date	Feb 28, 2017
Grant date	Feb 28, 2017

Abstract

Fusion proteins comprising a single strand DNA binding protein and a nucleic acid polymerase (e.g. DNA polymerase or reverse transcriptase). These high fidelity proteins are suitable for use in nucleic acid amplification methods, including the polymerase chain reaction (PCR).

First claim

What is claimed is:

1. A method of increasing the yield of a polymerase reaction on a target nucleic acid comprising:
a. contacting said target nucleic acid with a primer which specifically hybridizes thereto, and an isolated or purified fusion protein comprising a single-stranded DNA binding protein (SSB) fused to a nucleic acid polymerase, wherein said SSB comprises amino acids 1 to 150 of SEQ ID NO:2, and wherein the fidelity of said fusion protein is enhanced relative to the fidelity of said nucleic acid polymerase which does not have said SSB fused thereto; and
b. incubating the resulting mixture under conditions whereby said primer is extended by said nucleic acid polymerase.

2. The method of claim 1 , wherein said nucleic acid polymerase comprises amino acids 157 to 929 of SEQ ID NO:2.

3. The method of claim 1 , wherein said fusion protein comprises at least 50% polymerase activity, 3′-5′ exonuclease activity and/or 5′-3′ exonuclease activity of the fusion protein shown in SEQ ID NO:2.

4. The method of claim 1 , wherein said fusion protein comprises the amino acid sequence of SEQ ID NO:2.

5. The method of claim 1 , wherein said nucleic acid polymerase is a DNA polymerase.

6. The method of claim 5 , wherein said DNA polymerase is a thermostable DNA polymerase.

7. The method of claim 6 , wherein said thermostable DNA polymerase is Thermococcus zilligii (Tzi) DNA polymerase.

8. The method of claim 1 , wherein said nucleic acid polymerase is a reverse transcriptase.

9. The method of claim 1 , wherein said fusion protein comprises no detectable endonuclease activity.

10. The method of claim 1 , wherein said fidelity of said fusion protein is enhanced by about 1.2 to 10,000 fold relative to the fidelity of said DNA polymerase which does not have said SSB fused thereto.

11. The method of claim 1 , wherein said SSB is at the N-terminal end and said nucleic acid polymerase is at the C-terminal end of said fusion protein.

12. The method of claim 1 , wherein said SSB and said nucleic acid polymerase are separated by an amino acid linker.

13. The method of claim 12 , wherein said amino acid linker comprises between 1 and 100 amino acids.

14. The method of claim 12 , wherein said amino acid linker comprises the amino acid sequence GSGGVD (Position 151-156 of SEQ ID NO:2).

15. The method of claim 1 , wherein said SSB and said nucleic acid polymerase are immediately adjacent to one another.

16. The method of claim 6 , wherein said thermostable DNA polymerase retains at least 50% of its DNA polymerase activity after being heated at 90° C. for 30 seconds.

17. The method of claim 1 , wherein said fusion protein exhibits a reduced misincorporation rate that is less than 90% of the relative misincorporation rate of said DNA polymerase which does not have said SSB fused thereto.

Assignees

• Life Technologies Corp

Inventors

• Lee Jun
• Potter Robert
• Mandelman David

Classifications

• C12Q1/6848

  characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction · CPC title

• C12N15/62

  DNA sequences coding for fusion proteins · CPC title

• C12N9/1252 Primary

  DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase · CPC title

• C12Q1/6806 Primary

  Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence) · CPC title

• C12N9/1276

  RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase · CPC title