Multi-site mutagenesis

• US 8,685,676 B2
• Filed: 10/11/2011
• Issued: 04/01/2014
• Est. Priority Date: 07/26/2001
• Status: Expired due to Term

• Current Assignee: Agilent Technologies, Inc.
• Original Assignee: Agilent Technologies, Inc.
• Inventors: Hogrefe, Holly H., Cline, Janice M.
• Primary Examiner(s): Lu, Frank
• Application Number:
  US13/270,698
• Publication Number:
  US 20130122550A1
• Time in Patent Office: 903 Days
• Field of Search: 435/6.1, 435/6.11, 435/6.12, 435/91.1, 435/91.2, 435/183, 436/94, 436/501, 536/23.1, 536/24.3, 536/24.33, 536/25.3
• US Class Current: 435/91.2
• CPC Class Codes: C12N 15/102 Mutagenizing nucleic acids
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 9/1252 DNA-directed DNA polymerase (2.7.7.7), i.e. DNA replicase
• CPC Class Codes: C12N 9/22 Ribonucleases RNAses, DNAses catalytic nucleic acids C12N15/113

Abstract

The present invention provides compositions and improved methods for multi-site directed mutagenesis and DNA shuffling. The present compositions and methods provide increased mutation frequency and increased number of transformants which allow one to sequence only a few clones in order to identify the correct mutants and to obtain the desired mutant by screening large number of transformants in a short time. Moreover, the inclusion of FEN-1, PEF and optimized buffer and cycling conditions provided in the present invention should also facilitate random mutagenized library construction and the mutagenesis of large or difficult templates.

Claims

1. A method for producing a DNA product comprising two or more mutations by introducing said two or more mutations to a double-stranded target DNA molecule, said method comprising:

a) annealing one or more primers to the same strand of said target DNA molecule in a solution, wherein said one or more primers comprise two or more mutation sites with respect to said target DNA molecule;

b) using said target DNA molecule as a template, synthesizing a mutagenized single strand DNA comprising said primers and a flap structure in said solution by an amplification reaction in the presence of a DNA polymerase, a DNA ligase, and a flap endonuclease; and

c) adding a selection enzyme to the solution after step (b) to perform a digestion reaction, wherein said selection enzyme digests said target DNA molecule but not significantly digests said mutagenized single strand, thereby producing said DNA product.

2. The method of claim 1, further comprising transforming a host cell with said DNA product in step c).

3. The method of claim 1, wherein said DNA polymerase is a thermostable DNA polymerase.

4. The method of claim 3, wherein said thermostable DNA polymerase is selected from the group consisting of:

Taq DNA polymerase, Pfu DNA polymerase, Tma DNA polymerase, Tli DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase and Pyrolobus furmarius DNA polymerase.

5. The method of claim 1, wherein said DNA ligase is a thermostable DNA ligase.

6. The method of claim 5, wherein said thermostable DNA ligase is selected from the group consisting of:

Pfu DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Rhodothermus marinus DNA ligase, Thermus scotoductus DNA ligase and Bacillus stearothermophilus DNA ligase.

7. The method of claim 1, wherein said selection enzyme is a restriction endonuclease.

8. The method of claim 7, wherein said restriction endonuclease is selected from the group consisting of:

DpnI, Nan II, NmuD I, and NmuE I.

9. The method of claim 1, wherein each primer of said one or more primers comprises a different mutation site with respect to said target DNA molecule.

10. The method of claim 1, further comprising annealing one or more primers to a second strand of said double-stranded target DNA molecule.

11. A method for producing a host cell comprising a DNA product having two or more mutations, said method comprising:

a) annealing one or more primers to the same strand of a double-stranded target DNA molecule in a solution, wherein said one or more primers comprise two or more mutation sites with respect to said target DNA molecule;

b) using said target DNA molecule as a template, synthesizing a mutagenized single strand DNA comprising said primers and a flap structure in said solution by an amplification reaction in the presence of a DNA polymerase, a DNA ligase, and a flap endonuclease; and

c) adding a selection enzyme to the solution after step (b) to perform a digestion reaction, wherein said selection enzyme digests said target DNA molecule but not significantly digests said mutagenized single strand,

d) generating a double-stranded mutagenized DNA intermediate comprising said two or more mutations from said mutagenized single strand DNA; and

e) transforming a host cell with said double-stranded mutagenized DNA intermediate, thereby producing a host cell comprising said DNA product having said two or more mutations.

12. The method of claim 11, wherein said DNA polymerase is a thermostable DNA polymerase.

13. The method of claim 12, wherein said thermostable DNA polymerase is selected from the group consisting of:

Taq DNA polymerase, Pfu DNA polymerase, Tma DNA polymerase, Tli DNA polymerase, KOD DNA polymerase, JDF-3 DNA polymerase, PGB-D DNA polymerase, Tgo DNA polymerase and Pyrolobus furmarius DNA polymerase.

14. The method of claim 11, wherein said DNA ligase is a thermostable DNA ligase.

15. The method of claim 14, wherein said thermostable DNA ligase is selected from the group consisting of:

Pfu DNA ligase, Tth DNA ligase, Taq DNA ligase, Thermus filiformis DNA ligase, Rhodothermus marinus DNA ligase, Thermus scotoductus DNA ligase and Bacillus stearothermophilus DNA ligase.

16. The method of claim 11, wherein said selection enzyme is a restriction endonuclease.

17. The method of claim 16, wherein said restriction endonuclease is selected from the group consisting of:

DpnI, NanII, NmuDI, and NmuEI.

18. The method of claim 11, wherein each primer of said one or more primers comprises a different mutation site with respect to said target DNA molecule.

19. The method of claim 11, further comprising annealing one or more primers to a second strand of said double-stranded target DNA molecule.