Methods of using improved polymerases

• US 20040002076A1
• Filed: 11/27/2002
• Published: 01/01/2004
• Est. Priority Date: 11/28/2001
• Status: Active Grant

• Current Assignee: Bio-Rad Laboratories Incorporated
• Original Assignee: MJ Bioworks Incorporated (Bio-Rad Laboratories Incorporated)
• Inventors: Vander Horn, Peter, Wang, Yan, Xi, Lei
• Granted Patent: US 8,232,078 B2
• Time in Patent Office: Days
• US Class Current: 435/6
• 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 polymerase inhibitor, the method comprising:

(a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that;

(i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and

wherein the solution is of a composition that permits the 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. A method of claim 1, wherein the polymerase inhibitor is a fluorescent dye.

3. A method of claim 2, wherein the dye is SYBR Green I.

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

5. A method of claim 4, wherein the thermally stable polymerase domain is a Δ

Taq polymerase domain.

6. A method of claim 4, wherein the polymerase domain comprises a Pyrococcus polymerase domain.

7. A method of claim 1, wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.

8. A method of claim 1, wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7d.

9. A method of claim 1, wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.

10. A method of claim 1, wherein the target nucleic acid is a crude sample.

11. A method of sequencing a nucleic acid in an aqueous solution using an improved polymerase, the method comprising:

(a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that;

(i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and

wherein the solution is of a composition that permits the 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.

12. A method of claim 13, wherein the thermally stable polymerase domain is a Δ

Taq polymerase domain.

13. A method of claim 11, wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.

14. A method of claim 11, wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7d.

15. A method of claim 11, wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.

16. A method of performing a quantitative polymerase reaction on a target nucleic acid present in a solution that comprises a DNA-binding fluorescent dye, the method comprising:

(a) contacting the target nucleic acid with a polymerase, wherein the polymerase is joined to a sequence-non-specific nucleic-acid-binding domain that;

(i) binds to double-stranded nucleic acid, and (ii) enhances the processivity of the polymerase compared to an identical polymerase not having the sequence non-specific nucleic-acid-binding domain fused to it, and

wherein the solution is of a composition that permits the 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, and

(c) exposing the solution to a suitable excitation light and measuring fluorescence emission from the fluorescent dye.

17. A method of claim 16, wherein the polymerase domain has thermally stable polymerase activity.

18. A method of claim 17, wherein the thermally stable polymerase domain is a Δ

Taq polymerase domain.

19. A method of claim 17, wherein the polymerase domain comprises a Pyrococcus polymerase domain.

20. A method of claim 16, wherein the sequence-non-specific nucleic-acid-binding domain specifically binds to polyclonal antibodies generated against Sso7d.

21. A method of claim 16, wherein the sequence-non-specific nucleic-acid-binding domain contains a 50 amino acid subsequence containing 50% amino acid similarity to Sso7d.

22. A method of claim 16, wherein the sequence-non-specific nucleic-acid-binding domain is Sso7d.