Methods of using improved polymerases

• US 9,139,873 B2
• Filed: 06/21/2013
• Issued: 09/22/2015
• 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:
  US13/924,353
• Publication Number:
  US 20140220577A1
• Time in Patent Office: 823 Days
• Field of Search: None
• US Class Current: 1/1
• 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 sequencing a target nucleic acid in an aqueous solution using an improved DNA polymerase, the method comprising:

(a) contacting the target nucleic acid with a thermally stable DNA polymerase, wherein the DNA polymerase is joined 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, and

wherein the solution 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 DNA polymerase; and

(c) identifying the nucleotides that are incorporated into the synthesized nucleic acid strand upon the template-dependent extension of the primer by the DNA polymerase, thereby sequencing the nucleic acid.

2. The method of claim 1, wherein the thermally stable DNA polymerase has a Δ

Taq polymerase domain.

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

2.

4. The method of claim 1, wherein the sequence non-specific double-stranded nucleic acid binding domain comprises Sso7d (SEQ ID NO:

2) or Sac7d (amino acids 7-71 of SEQ ID NO;

10).

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

(a) contacting the target nucleic acid with a thermally stable DNA polymerase, wherein the DNA polymerase is joined to a sequence non-specific double-stranded nucleic acid binding domain that specifically binds to polyclonal antibodies generated against either Sso7d (SEQ ID NO;

2) or Sac7d (amino acids 7-71 of SEQ ID NO;

10), 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, and

wherein the solution 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 DNA polymerase; and

(c) identifying the nucleotides that are incorporated into the synthesized nucleic acid strand upon the template-dependent extension of the primer by the DNA polymerase, thereby sequencing the nucleic acid.

6. The method of claim 5, wherein the thermally stable DNA polymerase has a Δ

Taq polymerase domain.