Oligonucleotide mediated nucleic acid recombination

• US 6,376,246 B1
• Filed: 09/28/1999
• Issued: 04/23/2002
• Est. Priority Date: 02/05/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Ness, Jon E., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C., Bass, Steven H., Crameri, Andreas, Welch, Mark
• Primary Examiner(s): WHISENANT, ETHAN C
• Application Number:
  US09/408,392
• Time in Patent Office: 938 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: 435/440
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• CPC Class Codes: B01J 2219/00689 using computers
• CPC Class Codes: C07K 14/005 from viruses
• CPC Class Codes: C07K 14/505 Erythropoietin [EPO]
• CPC Class Codes: C07K 14/535 Granulocyte CSF; Granulocyte-macrophage CSF
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• CPC Class Codes: C12N 15/1034 Isolating an individual clone by screening libraries
• CPC Class Codes: C12N 15/11 DNA or RNA fragments; Modified forms thereof DNA or RNA not used in recombinant technology, C07H21/00; Non-coding nucleic acids having a biological activity
• CPC Class Codes: C12N 2740/16122 New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
• CPC Class Codes: C12N 9/16 acting on ester bonds (3.1)

Abstract

Methods of recombining nucleic acids, including homologous nucleic acids, are provided. Families of gene shuffling oligonucleotides and their use in recombination procedures, as well as polymerase and ligase mediated recombination methods are also provided.

Claims

1. A method of recombining a plurality of parental nucleic acids, the method comprising:

ligating a set of a plurality of chemically synthesized oligonucleotides, the set comprising a plurality of nucleic acid sequences from a plurality of the parental nucleic acids to produce a recombinant nucleic acid encoding a full length protein.

2. The method of claim 1, the set comprising at least a first oligonucleotide which is complementary to at least a first of the parental nucleic acids at a first region of sequence diversity and at least a second oligonucleotide which is complementary to at least a second of the parental nucleic acids at a second region of diversity.

3. The method of claim 1, wherein the nucleic acids are ligated with a ligase.

4. The method of claim 1, wherein the oligonucleotides are hybridized to a first parental nucleic acid and ligated with a ligase.

5. The method of claim 1, wherein the parental nucleic acids are homologous.

6. The method of claim 1, wherein the set of oligonucleotides comprises a set of family gene shuffling oligonucleotides.

7. The method of claim 1, the method further comprising hybridizing the set of oligonucleotides to one or more of the parental nucleic acids, elongating the oligonucleotides with a polymerase and ligating the resulting elongated oligonucleotides to produce a nucleic acid encoding a substantially full-length protein.

29. The method of claim 1, wherein a plurality of the plurality of oligonucleotide members are hybridized to at least one parental nucleic acid prior to ligation.

30. The method of claim 29, wherein the resulting hybridized oligonucleotide members are treated with a polymerase or nuclease prior to ligation.

8. The method of 1, wherein the plurality of oligonucleotides comprises at least one bridging oligonucleotide comprising a first subsequence derived from a first parental nucleic acid and a second subsequence derived from a second parental nucleic acid.

9. The method of 1, the method comprising hybridizing the set of a plurality of oligonucleotides, the set comprising a set of overlapping family gene shuffling oligonucleotides;

and extending the set of overlapping family gene shuffling oligonucleotides with a polymerase or a ligase, thereby providing a population of recombined nucleic acids.

10. The method of claim 9, the method further comprising:

denaturing the population of recombined nucleic acids, thereby providing denatured recombined

nucleic acids;

reannealing the denatured recombined nucleic acids;

extending the resulting reannealed recombined nucleic acids; and

, optionally;

selecting one or more of the resulting recombined nucleic acids for a desired property.

11. The method of claim 10, wherein, the reannealed recombined nucleic acids are recombined prior to said selecting.

12. The method of claim 11, further comprising recombining the resulting selected recombined nucleic acids.

13. The method of claim 12, further comprising selecting the resulting multiply selected multiply recombined nucleic acids for a desired trait or property.

14. The method of claim 9, the method further comprising the steps of:

denaturing the population of recombined nucleic acids, thereby providing denatured recombined

nucleic acids;

reannealing the denatured nucleic acids;

extending the resulting reannealed recombined nucleic acids; and

,

repeating the denaturing, reannealing and extending at least once.

15. The method of claim 9, wherein a plurality of members of the recombined nucleic acids are screened for a desired property and are determined to have the desired property, thereby providing first round screened nucleic acids, the method further comprising:

hybridizing a second set of overlapping family gene shuffling oligonucleotides, which second set of overlapping family gene shuffling oligonucleotides are derived from the first round screened nucleic acids; and

,

elongating the second set of overlapping family gene shuffling oligonucleotides, thereby providing a population of further recombined nucleic acids.

16. The method of claim 15, further comprising sequencing the first round screened nucleic acids, wherein the second set of overlapping family gene shuffling oligonucleotides is derived from the first round screened nucleic acids by aligning sequences of the first round screened nucleic acids to identify regions of identity and regions of diversity in the first round screened nucleic acids, and synthesizing the second set of overlapping family gene shuffling oligonucleotides to comprise a plurality of oligonucleotides, each of which comprise subsequences corresponding to at least one region of diversity.

17. The method of claim 16, wherein the first round screened nucleic acids encode polypeptides of about 50 amino acids or less.

18. The method of claim 16, wherein the second set of overlapping family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of the first round screened nucleic acids.

19. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of homologous target nucleic acids.

20. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprise at least one module shuffling oligonucleotide(s).

21. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprise a plurality of module shuffling oligonucleotides, each comprising at least a first subsequence from a first sequence module and a second subsequence from a second sequence module.

22. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprise a plurality of module shuffling oligonucleotides, wherein one or more of the plurality of oligonucleotides each comprising at least a first subsequence from a first sequence module and a second subsequence from a second sequence module.

23. The method of claim 9, wherein the set of overlapping family shuffling oligonucleotides comprise a plurality of codon-varied oligonucleotides.

24. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprises at least 3 member types.

25. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprises at least 5 member types.

26. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprises at least 10 member types.

27. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprises a plurality of homolgous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in approximately equimolar amounts.

28. The method of claim 9, wherein the set of overlapping family shuffling gene oligonucleotides comprises a plurality of homolgous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in non-equimolar amounts.