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

• US 6,372,497 B1
• Filed: 06/08/2000
• Issued: 04/16/2002
• 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:
  US09/590,774
• Time in Patent Office: 677 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/1
• CPC Class Codes: C07K 14/43595 from coelenteratae, e.g. medusae
• CPC Class Codes: C07K 14/545 IL-1
• CPC Class Codes: C07K 16/00 Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies antibodies with enzymatic activity, e.g. abzymes C12N9/0002
• CPC Class Codes: C07K 2317/565 Complementarity determining region [CDR]
• CPC Class Codes: C07K 2317/622 Single chain antibody (scFv)
• 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
• CPC Class Codes: C12N 15/1037 Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
• CPC Class Codes: C12N 15/1058 Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms
• CPC Class Codes: C12N 15/52 Genes encoding for enzymes or proenzymes
• CPC Class Codes: C12N 15/64 General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
• CPC Class Codes: C12N 9/2471 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12N 9/86 acting on amide bonds in cyclic amides, e.g. penicillinase (3.5.2)
• CPC Class Codes: C12Q 1/68 involving nucleic acids
• CPC Class Codes: C12Q 1/6811 Selection methods for production or design of target specific oligonucleotides or binding molecules
• CPC Class Codes: C12Q 2525/179 incorporating arbitrary or random nucleotide sequences
• CPC Class Codes: C12Y 302/01023 Beta-galactosidase (3.2.1.23), i.e. exo-(1-->4)-beta-D-galactanase
• CPC Class Codes: C12Y 305/02006 Beta-lactamase (3.5.2.6)
• CPC Class Codes: C40B 40/02 Libraries contained in or displayed by microorganisms, e.g. bacteria or animal cells; Libraries contained in or displayed by vectors, e.g. plasmids; Libraries containing only microorganisms or vectors
• CPC Class Codes: H10N 60/128 having three or more electrodes, e.g. transistor-like structures

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 producing a recombined polynucleotide having a desired characteristic, comprising:

(a) providing a plurality of related-sequence double-stranded template polynucleotides, comprising polynucleotides with non-identical sequences;

(b) providing a plurality of single-stranded nucleic acid fragments capable of hybridizing to the template polynucleotides;

(c) hybridizing single-stranded nucleic acid fragments to the template polynucleotides and extending the hybridized fragments on the template polynucleotides with a polymerase, thereby forming a plurality of sequence-recombined polynucleotides;

(d) subjecting the sequence recombined polynucleotides of step (c) to at least one additional cycle of recombination to produce further sequence-recombined poly-nucleotides; and

,

(e) selecting or screening the further sequence-recombined polynucleotides for the desired characteristic.

2. The method of claim 1, wherein the plurality of single-stranded nucleic acid fragments comprise fragments produced by digestion with a nuclease.

3. The method of claim 2, wherein the plurality of single-stranded nucleic acid fragments are DNAs and the nuclease is a DNAse.

4. The method of claim 1, wherein the plurality of single-stranded nucleic acid fragments comprise fragments produced by incomplete polymerase extension.

5. The method of claim 1, wherein the plurality of single-stranded nucleic acid fragments comprise synthetic oligonucleotides.

6. The method of claim 1, wherein said selecting or screening step comprises:

(a) introducing the plurality of further sequence-recombined polynucleotides into a population of cells;

(b) expressing the plurality of further sequence-recombined polynucleotides in said cells; and

,

(c) selecting or screening said population of cells for the desired characteristic.

7. The method of claim 1, wherein the population of nucleic acid fragments is a mixed population.

8. The method of claim 1, wherein the set of related-sequence polynucleotides comprises species variants.

9. The method of claim 1, wherein said set of related-sequence polynucleotides comprises allelic variants.

10. The method of claim 1, wherein the template polynucleotide comprises uracil and wherein the template polynucleotide is substantially non-replicable in one or more cells.

11. The method of claim 1, wherein said plurality of template polynucleotides comprises naturally occurring variants of a polynucleotide.

12. The method of claim 1, wherein the plurality of template polynucleotides comprises mutagenized polynucleotides.

13. The method of claim 12, wherein said mutagenized polynucleotides are produced by error-prone PCR.

14. The method of claim 12, wherein said mutagenized polynucleotides are produced by oligonucleotide-directed mutagenesis.

15. The method of claim 12, wherein said mutagenized polynucleotides are produced by chemical mutagenesis.

16. The method of claim 12, wherein said mutagenized polynucleotides are produced by random mutagenesis.

17. The method of claim 1, wherein the plurality of template polynucleotides comprises bacterial polynucleotides.

18. The method of claim 1, wherein the plurality of template polynucleotides comprises fungal polynucleotides.

19. The method of claim 1, wherein the plurality of template polynucleotides comprises viral polynucleotides.

20. The method of claim 1, wherein the set of plurality of template comprises plant polynucleotides.

21. The method of claim 1, wherein the plurality of template comprises animal polynucleotides.

22. The method of claim 1, wherein the plurality of template polynucleotides comprise DNA.

23. The method of claim 1, wherein the plurality of template polynucleotides comprise RNA.

24. The method of claim 1, wherein the plurality of single-stranded nucleic acid fragments comprises overlapping fragments.

25. The method of claim 1, wherein the template polynucleotides encode a protein.

26. The method of claim 25, wherein the template polynucleotides are in a vector.

27. The method of claim 25, wherein the template polynucleotides encode an enzyme.

28. The method of claim 27, wherein the enzyme is a polymerase.

29. The method of claim 25, wherein the template polynucleotides encode an antibody chain.

30. The method of claim 25, wherein the template polynucleotides encode a single chain antibody.

31. The method of claim 1, wherein the additional cycle of recombination is performed in vitro.

32. The method of claim 1, wherein the additional cycle of recombination is performed in vivo.

33. The method of claim 1, wherein the extension reaction of step (c) is performed under conditions of incomplete polymerase extension on the template and further comprising at least one additional cycle of denaturation, renaturation, and extension of the partially extended sequence-recombined polynucleotides with the template to produce the further sequence-recombined polynucleotides.

34. The method of claim 1, further comprising expressing and isolating a protein encoded by a further sequence-recombined polynucleotide.

35. The method of claim 1, wherein the desired characteristic is the capacity of a protein encoded by a further sequence-recombined nucleic acid to bind a receptor or a ligand.

36. The method of claim 1, wherein the desired characteristic is the capacity of a protein encoded by a further sequence-recombined nucleic acid to bind to an antigen.

37. The method of claim 1, wherein the desired characteristic is suitability of a protein encoded by a further sequence-recombined nucleic acid as an agent for DNA-based vaccination.