Methods of using improved polymerases

• US 9,708,598 B2
• Filed: 08/20/2015
• Issued: 07/18/2017
• Est. Priority Date: 11/28/2001
• Status: Expired due to Term

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: Bio-Rad Laboratories Incorporated
• Inventors: Wang, Yan, Vander Horn, Peter, Xi, Lei
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US14/831,042
• Publication Number:
  US 20160060616A1
• Time in Patent Office: 698 Days
• Field of Search: None
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2319/00 Fusion polypeptide
• CPC Class Codes: C07K 2319/80 containing a DNA binding domain, e.g. Lacl or Tet-repressor
• CPC Class Codes: C12N 15/10 Processes for the isolation, preparation or purification of DNA or RNA chemical preparation of DNA or RNA C07H21/00; preparation of non-structural polynucleotides from microorganisms or with enzymes C12P19/34
• 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: C12P 19/34 Polynucleotides, e.g. nucleic acids, oligoribonucleotides
• CPC Class Codes: C12Q 1/686 Polymerase chain reaction [PCR]
• CPC Class Codes: C12Y 207/07007 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase

Abstract

This invention provides for methods of sequencing and performing polymerase reactions using an improved generation of nucleic acid polymerases. The improvement is the fusion of a sequence-non-specific nucleic-acid-binding domain to the enzyme in a manner that enhances the processivity of the polymerase.

Claims

1. A method of increasing the yield from a polymerase reaction on a target nucleic acid present in a solution that comprises a fluorescent dye that detects double-stranded DNA, the method comprising:

(a) contacting the target nucleic acid with a DNA polymerase, wherein the DNA polymerase comprises a polymerase domain fused to a sequence-non-specific double-stranded nucleic-acid-binding domain that comprises at least 75% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO;

2 and enhances the processivity of the DNA polymerase compared to an identical DNA polymerase not having the sequence non-specific double-stranded nucleic-acid binding domain fused to it;

wherein the solution comprises the fluorescent dye 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 polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;

(b) incubating the solution under conditions in which the primer is extended by the polymerase.

2. The method of claim 1, wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 90% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO:

2.

3. 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.

4. A method of claim 1, wherein the fluorescent dye is SYBR Green I.

5. A method of claim 1, wherein the polymerase domain has thermally stable polymerase activity.

6. A method of claim 5, wherein the thermally stable polymerase domain is a Δ

Taq polymerase domain.

7. A method of claim 5, wherein the polymerase domain is from Pyrococcus.

8. A method of increasing the yield from a polymerase reaction on a target nucleic acid present in a crude cellular extract, the method comprising:

(a) contacting the target nucleic acid with a DNA polymerase, wherein the DNA polymerase comprises a polymerase domain fused to a sequence-non-specific double-stranded nucleic-acid-binding domain that comprises at least 75% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO;

2 and enhances the processivity of the DNA polymerase compared to an identical DNA polymerase not having the sequence non-specific double-stranded nucleic-acid binding domain fused to it;

wherein the solution comprises impurities from the crude cellular extract 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 polymerase domain to extend a primer that is hybridized to the target nucleic acid sequence;

(b) incubating the solution under conditions in which the primer is extended by the polymerase.

9. The method of claim 8, wherein the sequence non-specific double-stranded nucleic acid binding domain has at least 90% amino acid sequence identity to the Sso7d amino acid sequence set forth in SEQ ID NO:

2.

10. 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.

11. A method of claim 8, wherein the polymerase domain has thermally stable polymerase activity.

12. A method of claim 11, wherein the thermally stable polymerase domain is a Δ

Taq polymerase domain.

13. A method of claim 11, wherein the polymerase domain is from Pyrococcus.