Nucleic acid libraries

• US 6,573,098 B1
• Filed: 01/29/1999
• Issued: 06/03/2003
• 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:
  US09/240,307
• Time in Patent Office: 1,586 Days
• Field of Search: 435/6, 435/440, 536/23.1, 536/24.3, 935/76, 935/77, 935/78
• US Class Current: 506/16
• 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 composition comprising a library of nucleic acids, the library comprising a mixture of a plurality of overlapping nucleic acids, wherein the plurality of overlapping nucleic acids are segments of the same gene from different species, and wherein the plurality of overlapping nucleic acids are capable of hybridizing to a portion of a selected target nucleic acid, or set of related sequence target nucleic acids, wherein a plurality of members of the library comprise one or more region of non-complementarity with the selected target nucleic acid, wherein a plurality of members of the library are capable of priming nucleotide extension upon hybridization to the selected target nucleic acid, and wherein the selected target nucleic acid is one of the genes used to provide the plurality of overlapping nucliec acids.

2. The composition of claim 1, wherein the library further comprises a plurality of members complementary to the selected target nucleic acid.

3. The composition of claim 1, wherein the overlapping nucleic acid segments comprise DNA fragments.

4. The composition of claim 1, wherein the overlapping nucleic acid segments comprise synthetic oligonucleotides.

5. The composition of claim 1, wherein the overlapping nucleic acid segments comprise synthetic DNA oligonucleotides.

6. The composition of claim 1, the composition further comprising a DNA polymerase.

7. The composition of claim 1, the composition further comprising a thermostable DNA polymerase.

8. The composition of claim 1, wherein the target nucleic acid encodes a protein.

9. The composition of claim 3, wherein the DNA fragments are random fragments.

10. The composition of claim 3, wherein the DNA fragments are produced by cleavage with a DNAse.

20. The method of claim 1 wherein the gene from different species encodes an enzyme.

21. The method of claim 1 wherein the gene from different species encodes a receptor binding protein.

22. The method of claim 1 wherein the gene from different species encodes a viral protein and wherein at least one of the genes used to provide the overlapping nucleic acids is a viral gene.

23. The method of claim 22 wherein the viral protein is selected from the group consisting of capsid glycoproteins, spike glycoproteins, polymerases, and proteases.

24. The method of claim 1 wherein the gene from different species encodes a bacterial protein and wherein at least one of the genes used to provide the overlapping nucleic acids is a bacterial gene.

25. The method of claim 24 wherein the bacterial protein is an enzyme.

26. The method of claim 1 wherein the same gene from different species encodes a human protein and wherein at least one of the genes used to provide the plurality of overlapping nucleic acids is a human gene.

27. The method of claim 26 wherein the human protein is a receptor binding protein.

28. The method of claim 26 wherein the human protein is a therapeutic protein.

29. The method of claim 28 wherein the therapeutic protein is selected from the group consisting of interleukin I, an antibody, tPA, and human growth hormone.

11. A composition comprising a library of nucleic acids, the library comprising a mixture of a plurality of overlapping DNA fragments, which overlapping DNA fragments are derived by fragmentation of one or more parental nucleic acids, wherein the plurality of overlapping DNA fragments are capable of hybridizing to a portion of a selected target nucleic acid, or set of related sequence target nucleic acids, wherein a plurality of members of the library comprise one or more region of non-complementarity with the selected target nucleic acid, wherein a plurality of members of the library are capable of priming nucleotide extension upon hybridization to the selected target nucleic acid.

12. The composition of claim 11, wherein the library comprises a plurality of DNA fragments, which DNA fragments comprise different alleles of the same gene.

13. The composition of claim 11, wherein the library comprises a plurality of DNA fragments, which DNA fragments comprise the same gene from different related species.

14. The composition of claim 11, wherein the library further comprises a plurality of members complementary to the selected target nucleic acid.

15. The composition of claim 11, wherein the library further comprises a plurality of synthetic oligonucleotides, which oligonucleotides are overlapping.

16. The composition of claim 11, the composition further comprising a DNA polymerase.

17. The composition of claim 11, the composition further comprising a thermostable DNA polymerase.

18. The composition of claim 11, wherein the target nucleic acid encodes a protein.

19. The composition of claim 11, wherein the DNA fragments are produced by cleavage with a DNAse.