One component and two component DNA Pol III replicases and uses thereof

• US 8,192,960 B2
• Filed: 04/07/2005
• Issued: 06/05/2012
• Est. Priority Date: 04/07/2004
• Status: Expired due to Fees

• Current Assignee: QIAGEN North American Holdings Inc. (Qiagen NV)
• Original Assignee: QIAGEN North American Holdings Inc. (Qiagen NV)
• Inventors: Peters, Lars-Erik, Ettinger, Anna
• Primary Examiner(s): HUTSON, RICHARD G
• Application Number:
  US11/101,977
• Publication Number:
  US 20060211003A1
• Time in Patent Office: 2,616 Days
• Field of Search: None
• US Class Current: 435/91.1
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12Q 1/6867 Replicase-based amplification, e.g. using Q-beta replicase
• CPC Class Codes: C12Q 2521/101 DNA polymerase

Abstract

The invention provides one-component and two-component DNA polymerases, as well as kits comprising the same, and methods of using the same for nucleic amplification and nucleic acid sequencing.

Claims

1. A method for replicating a DNA molecule, comprising subjecting a DNA molecule to a replication reaction in a replication reaction mixture, which replication reaction mixture comprises a minimal functional Pol III replicase, wherein said minimal functional Pol III replicase comprises a first component, which first component comprises an α

subunit from a gram negative bacteria, wherein said α

subunit comprises amino acid consensus sequence motifs A, B, and C arranged in the order C-A-B from amino terminus to carboxyl terminus, wherein said minimal functional Pol III replicase and said replication reaction mixture lack each subunit (τ

/γ

, δ and

δ

′

) of the clamp loader, and wherein said DNA molecule is replicated in said replication reaction in said replication reaction mixture by said minimal functional Pol III at a rate of at least 100 nucleotides per second.

2. The method according to claim 1, wherein said minimal functional Pol III replicase is a thermostable minimal Pol III.

3. The method according to claim 1, wherein said gram negative bacteria is selected from the group consisting of the genera Thermus, Aquifex, and Thermatoga.

4. The method according to claim 1, wherein said DNA is double stranded DNA.

5. The method according to claim 1, wherein said first component further comprises an ε

subunit

6. The method according to claim 1, wherein said minimal functional Pol III replicase is a single component Pol III replicase.

7. The method according to claim 1, wherein said minimal functional Pol III replicase is a two component Pol III replicase further comprising a second component, which second component comprises a processivity clamp.

8. The method according to claim 1, wherein said method is an amplification method, wherein said replication reaction is an amplification reaction, and said replication reaction mixture is an amplification reaction mixture.

9. The method according to claim 8, wherein said amplification method is a PCR method, wherein said amplification reaction is a polymerase chain reaction, and wherein said amplification reaction mixture is a PCR mixture.

10. The method according to claim 1, wherein said method is a sequencing method, wherein said replication reaction is a sequencing reaction, wherein said replication reaction mixture is a sequencing reaction mixture.

11. The method according to claim 1, wherein said replication reaction mixture lacks CaCl2.

12. The method according to claim 1, wherein said replication reaction mixture is a zwitterionic buffer comprising a weak organic acid and a weak organic base.

13. The method according to claim 1, wherein said replication reaction mixture has a pH of between about pH7.5-8.9.

14. The method according to claim 1, wherein said a subunit is present in said replication reaction mixture at a concentration of not less than 6 ng/μ

L.

15. The method according to claim 1, wherein said a subunit is a DnaE a subunit from a gram negative bacteria and the spacing between said motif C and said motif A ranges from about 153 to about 155 amino acids, and wherein the spacing between said motif A and said motif B ranges from about 195 to about 201 amino acids.

16. The method according to claim 15, wherein said motif A comprises the sequence set forth in SEQ ID NO:

15.

17. The method according to claim 15, wherein said motif B comprises the sequence set forth in SEQ ID NO:

16.

18. The method according to claim 15, wherein said motif C comprises the sequence set forth in SEQ ID NO:

17.

19. The method according to claim 15, wherein said motif A comprises the sequence set forth in SEQ ID NO:

21.

20. The method according to claim 15, wherein said motif B comprises the sequence set forth in SEQ ID NO:

22.

21. The method according to claim 15, wherein said motif C comprises the sequence set forth in SEQ ID NO:

23.