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

• US 6,413,774 B1
• Filed: 01/29/1999
• Issued: 07/02/2002
• Est. Priority Date: 02/17/1994
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Cramieri, Andreas M., Stemmer, Willem P. C.
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/240,734
• Time in Patent Office: 1,250 Days
• Field of Search: 435/6, 435/172.3, 435/440, 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 recombinant nucleic acid with a desired property from at least two nucleic acids, the method comprising:

(a) providing single-stranded segments of the at least two nucleic acids;

(b) hybridizing the single-stranded segments to produce partially or fully annealed nucleic acid strands and elongating the partially or fully annealed nucleic acid strands to produce recombinant nucleic acids;

(c) denaturing the recombinant nucleic acids to form recombinant single-stranded nucleic acids;

(d) hybridizing the single-stranded recombinant nucleic acids of step (c) to produce additional partially or fully annealed nucleic acid strands;

(e) elongating the partially or fully-annealed nucleic acid strands of step (d) to produce transferrable recombinant nucleic acids;

(f) transferring the transferrable recombinant nucleic acids into one or more cells; and

,

(g) recombining the transferrable recombinant nucleic acids in the one or more cells to produce additional recombinant nucleic acids.

2. The method of claim 1, further comprising selecting or screening the additional recombinant nucleic acids for one or more trait or property.

8. The method of claims 2 or 3, wherein the selection or screening step comprises placing cells which comprise the additional recombinant nucleic acids under selective pressure.

9. The method of claim 1 or 3, wherein the transferrable nucleic acids are stably integrated into a genome of the cell.

10. The method of claim 1 or 3, wherein the transferrable nucleic acids are cloned into one or more episomally replicable vectors.

11. The method of claim 10, wherein the episomally replicable vector is capable of stable replication in the cell.

12. The method of claim 1 or 3, wherein the vector comprises a selectable marker.

13. The method of claim 1 or 3, wherein the transferrable nucleic acids are cloned into one or more episomally replicable vectors, wherein the resulting cloned nucleic acids comprise direct or indirect repeats.

14. The method of claim 13, wherein the cloned nucleic acids are recombined in the cell by intra-vector or inter-vector recombination.

15. The method of claim 1 or 3, wherein the transferrable nucleic acids recombine with each other, or with other nucleic acids, via homologous recombination, in the cell.

16. The method of claim 1 or 3, wherein the cell is treated with a chemical or radiological muatgen.

17. The method of claim 1 or 3, wherein the cell is treated with a chemical or radiological muatgen selected from MNU, ENU, MNNG, nitrosourea, BuDR, UV light, ionizing radiation, and a clastogenic agent.

18. The method of claim 1 or 3, wherein the transferrable nucleic acids are transferred into the cell using one or more transfer techniques selected from:

electroporation, natural competence, transduction, transfection, lipofection, biolistics and conjugation.

19. The method of claim 1 or 3, wherein the transferrable nucleic acids are single stranded.

20. The method of claim 1 or 3, wherein the transferrable nucleic acids comprise viral sequences.

21. The method of claim 1 or 3, wherein the transferrable nucleic acids are associated with a recombinase prior to or subsequent to transfer into the cell.

22. The method of claim 21, wherein the recombinase is RecA.

23. The method of claim 1 or 3, wherein the cell is a bacterial cell, a yeast cell, or a mammalian cell.

24. The method of claim 1 or 3, further comprising recombining the additional recombinant nucleic acid with itself or with one or more additional selected nucleic acid.

25. The method of claim 1 wherein the single-stranded segments are produced by cleaving the at least two nucleic acids to produce a population of double-stranded fragments, and denaturing the double-stranded fragments to produce the single-stranded segments.

26. The method of claim 1 wherein the overlapping single-stranded segments are produced on a DNA synthesizer.

27. The method of claim 1 wherein the overlapping single-stranded segments are produced by PCR amplification.

28. The method of claim 1 wherein the single-stranded segments are random segments of the polynucleotides.

29. The method of claim 1 wherein the single-stranded segments are non-random segments of the polynucleotides.

30. The method of claim 25 wherein the fragments are random fragments.

31. The method of claim 25 wherein the fragments are non-random fragments.

3. A method of recombining one or more nucleic acids, the method comprising:

(a.) producing a plurality of transferrable nucleic acids in vitro;

(b.) transferring the transferrable nucleic acids into one or more cells;

(c.) recombining the transferrable nucleic acids with each other or with one or more first additional nucleic acids in the cell to produce one or more recombinant nucleic acids;

(d.) recombining the one or more recombinant nucleic acids with each other or with the one or more first additional nucleic acids or with one or more second additional nucleic acids to produce one or more additional recombinant nucleic acids; and

,

(e.) selecting or screening the one or more recombinant nucleic acids or the one or more additional recombinant nucleic acids for one or more desirable trait or property.

4. The method of claim 3, wherein step (a) comprises producing fragments of at least two corresponding nucleic acids, hybridizing the resulting fragments;

and, elongating the resulting hybridized fragments to produce the transferrable nucleic acids.

5. The method of claim 3, wherein step (a) comprises producing fragments derived from more than one source from nature.

6. The method of claim 3, wherein step (a) comprises producing fragments derived from more than one species.

7. The method of claim 6, wherein said elongating comprises initially extending the hybridized fragments with a first polymerase, denaturing the resulting initially extended hybridized fragments, re-hybridizing the resulting single-stranded initially extended fragments and extending the resulting re-hybridized initially extended fragments with the first polymerase, or with a second polymerase, to produce the elongated nucleic acids.

32. The method of claim 3, wherein the plurality of transferable nucleic acids comprises mutagenized nucleic acids.

33. The method of claim 32, wherein the mutagenized nucleic acids are produced by error prone PCR.

34. The method of claim 32, wherein the mutagenized nucleic acids are produced by oligonucleotide directed mutagenesis.

35. The method of claim 32, wherein the mutagenized nucleic acids are produced by chemical mutagenesis.

36. The method of claim 3, wherein the plurality of transferable nucleic acids comprises allelic or species variants of a polynucleotide sequence.