Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

• US 6,995,017 B1
• Filed: 01/28/1999
• Issued: 02/07/2006
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Stemmer, Willem P. C.
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/239,395
• Time in Patent Office: 2,567 Days
• Field of Search: 435/440, 435/6, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 435/440
• CPC Class Codes: C12N 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries

Abstract

A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.

Claims

1. A method of identifying a recombined nucleic acid with a desired functional property, the method comprising:

providing a set of nucleic acids comprising a plurality of overlapping nucleic acids, the plurality of overlapping nucleic acids comprising one or more region of complementarity with a target nucleic acid, or with set of related sequence target nucleic acids, wherein;

the plurality of overlapping nucleic acids comprise nucleic acids that are capable of priming polynucleotide elongation upon hybridization to the target nucleic acid, or to one or more member of the set of related sequence target nucleic acids; and

the plurality of overlapping nucleic acids comprises one or more partially non-complementary nucleic acid, each partially non-complementary nucleic acid comprising at least one region of non-complementarity with the target nucleic acid or with the set of related sequence target nucleic acids and at least one region of complementarity with the target nucleic acid or set of related sequence target nucleic acids;

the method further comprising;

elongating at least one of the one or more partially non-complementary nucleic acids which has hybridized to another member of the set of nucleic acids, thereby forming one or more double-stranded sequence recombined nucleic acids;

denaturing the double-stranded sequence recombined nucleic acids and conducting at least one additional cycle of hybridization and elongation to provide further sequence recombined nucleic acids; and

screening or selecting the sequence recombined polynucleotides for the desired functional property.

2. The method of claim 1, wherein the plurality of overlapping nucleic acids comprise more than two overlapping nucleic acid.

3. The method of claim 1, comprising incompletely elongating the at least one partially non-coplementary nucleic acid.

4. The method of claim 1, wherein the plurality of overlapping nucleic acids comprise a plurality of overlapping nucleic acids with different 3′

and 5′

termini.

5. The method of claim 1, wherein the sequence recombined nucleic acid comprises a plurality of subsequences which correspond to a plurality of members of a set of polynucleotides and wherein the set of polynucleotides comprise related sequence polynucleotides which are related in sequence to the target nucleic acid.

6. The method of claim 1, wherein the plurality of overlapping nucleic acids comprise a plurality of regions of identity to the target nucleic acid.

7. The method of claim 1, wherein elongating the at least one or more partially non-complementary nucleic acid is performed by primer extension of the one or more partially non-complementary nucleic acid with a polymerase.

8. The method of claim 1, wherein extension of the at least one partially non-complementary nucleic acid is performed with a thermostable polymerase.

9. The method of claim 1, wherein extension of the at least one partially non-complementary nucleic acid is performed with a DNA polymerase.

10. The method of claim 1, wherein the plurality of overlapping nucleic acids are oligonucleotides.

11. The method of claim 1, wherein the plurality of overlapping nucleic acids comprise a library of nucleic acids.

12. The method of claim 1, wherein the plurality of partially non-complementary nucleic acids are between about 70 percent and at least about 90 percent identical to the target nucleic acid over a region corresponding to the full length of the partially non-complementary nucleic acid.

13. The method of claim 1, comprising repeating the steps of the method using the further sequence recombined nucleic acid as a target nucleic acid.

14. The method of claim 1, comprising recursively repeating the steps of the method for at least two cycles using the further sequence recombined nucleic acids as a target nucleic acid in the first cycle and the resulting second further sequence recombined nucleic acids as a target nucleic acid in the second cycle.

15. The method of claim 1, comprising repeating the steps of the method using fragments of the further sequence recombined nucleic acids in the set of nucleic acids.

16. The method of claim 1, wherein the set of nucleic acids comprises one or more artificially synthesized nucleic acid.

17. The method of claim 1 wherein the target nucleic acids encode a protein.

18. The method of claim 17, wherein the protein is an enzyme.

19. The method of claim 18, wherein the protein is an antibody.

20. The method of claim 17, wherein the protein is a therapeutic protein.

21. The method of claim 1, wherein the overlapping nucleic acids are of varying length.