SSB-polymerase fusion proteins

• US 8,828,700 B2
• Filed: 12/01/2008
• Issued: 09/09/2014
• Est. Priority Date: 09/09/2005
• Status: Active Grant

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Inventors: Lee, Jun, Potter, Robert, Mandelman, David
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US12/326,048
• Publication Number:
  US 20100075382A1
• Time in Patent Office: 2,108 Days
• Field of Search: None
• US Class Current: 435/194
• CPC Class Codes: C12N 15/62 DNA sequences coding for fusion proteins
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/1276 RNA-directed DNA polymerase (2.7.7.49), i.e. reverse transcriptase or telomerase
• CPC Class Codes: C12Q 1/6806 Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay C12Q1/6804 takes precedence
• CPC Class Codes: C12Q 1/6848 characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
• CPC Class Codes: C12Q 2522/101 Single or double stranded nucleic acid binding proteins

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).

Claims

1. An isolated or purified single stranded DNA binding protein (SSB)-nucleic acid polymerase fusion protein wherein said SSB comprises amino acids 1 to 150 of SEQ ID NO:

2, 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, and wherein the presence of said SSB enhances fidelity of said fusion protein compared to an identical nucleic acid polymerase that is not joined to said SSB.

2. The fusion protein of claim 1 wherein said nucleic acid polymerase is a DNA polymerase.

3. The fusion protein of claim 1, wherein said nucleic acid polymerase is a reverse transcriptase.

4. The fusion protein of claim 1, wherein said nucleic acid polymerase is a thermostable DNA polymerase.

5. The fusion protein of claim 4, wherein said thermostable DNA polymerase is Thermococcus zilligi (Tzi) DNA polymerase.

6. The fusion protein of claim 1, wherein said nucleic acid polymerase comprises amino acids 157 to 941 of SEQ ID NO:

2.

9. The fusion protein of claim 1, wherein said isolated or purified single stranded DNA binding protein (SSB)-nucleic acid polymerase fusion protein comprises no detectable endonuclease activity.

10. The fusion protein of claim 1, wherein said fidelity is enhanced by about 1.2 to 10,000 fold compared to the fidelity of an identical nucleic acid polymerase that is not joined to said SSB.

11. The fusion protein of claim 1, wherein said fusion protein exhibits a reduced misincorporation rate that is less than 90% of the relative misincoporation rate of an identical nucleic acid polymerase that is not joined to said SSB.

12. The fusion protein of claim 4, wherein said thermostable DNA polymerase retains at least 50% of its nucleic acid synthesis activity after bein heated in a nucleic acid synthesis mixture at 90°

C. for 30 seconds.

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

14. The fusion protein of claim 1, wherein said SSB is upstream (5′

) from said nucleic acid polymerase.

15. The fusion protein of claim 1, wherein said SSB and said nucleic acid polymerase are immediately adjacent to each other.

16. The fusion protein of claim 1, wherein said SSB and said nucleic acid polymerase are separated by an amino acid linker.

17. The fusion protein of claim 16, wherein said amino acid linker is between 1 and 100 amino acids in length.

7. An isolated or purified Sulfolobus sofataricus (Sso) SSB-Thermococcus zilligi (Tzi) DNA polymerase fusion protein wherein said Sso SSB comprises amino acids 1 to 150 of SEQ ID NO:

2 and has 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.

8. A fusion polymerase protein that comprises an amino acid sequence that has at least 75% sequence identity to SEQ ID NO:

2 and has at least two joined heterologous domains, wherein said heterologous domains comprise;

a. a single-stranded DNA binding protein (SSB) domain comprising amino acids 1 to 150 of SEQ ID NO;

2; and

b. a polymerase domain, and

wherein the relative fidelity of said polymerase fusion protein is enhanced compared to the fidelity of the corresponding full length wild type polymerase protein lacking said SSB domain.