Oligonucloetide mediated nucleic acid recombination

• US 6,521,453 B1
• Filed: 10/23/2000
• Issued: 02/18/2003
• Est. Priority Date: 01/19/1999
• Status: Expired due to Term

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: Patten, Phillip A., Gustafsson, Claes, Bass, Steven H., Welch, Mark, Ness, Jon E., Minshull, Jeremy, Stemmer, Willem P. C., Crameri, Andreas
• Primary Examiner(s): Whisenant, Ethan C.
• Application Number:
  US09/694,863
• Time in Patent Office: 848 Days
• Field of Search: 435/440, 435/6, 435/91.2, 536/23.1, 536/24.3, 536/25.3, 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 two or more nucleic acids, the method comprising:

chemically synthesizing a plurality of overlapping oligonucleotides and at least one cross-over oligonucleotide,

wherein sequences of the plurality of overlapping oligonucleotides correspond to subsequences from a plurality of different parental nucleic acids, wherein each of the different parental nucleic acids comprises or encodes at least one modular subsequence,

wherein a sequence of the cross-over oligonucleotide comprises the at least one modular subsequence from a first parental nucleic acid and a second subsequence from a second parental nucleic acid corresponding to a region outside of the at least one modular subsequence, and

wherein the plurality of overlapping oligonucleotides comprises chemically synthesized oligonucleotides that correspond to subsequences of one or more of the plurality of different parental nucleic acids other than the at least one modular subsequence;

hybridizing the plurality of overlapping oligonucleotides and the at least one cross-over oligonucleotide; and

,

elongating the plurality of overlapping oligonucleotides and the at least one cross-over oligonucleotide, thereby providing a population of recombined nucleic acids.

2. The method of claim 1, wherein the elongating step is performed with a polymerase.

3. The method of claim 1, wherein the elongating step is performed with a ligase.

4. The method of claim 1, wherein the at least one modular subsequence comprises at least one of the following:

an exon sequence, an intron sequence, a promoter sequence, or an enhancer sequence.

5. The method of claim 1, wherein the plurality of overlapping oligonucleotides comprise at least one multimer which comprises two or more modular subsequences from a parental nucleic acid.

6. The method of claim 5, wherein the two or more modular subsequences are different.

7. The method of claim 1, wherein the plurality of overlapping oligonucleotides are present in non-equimolar amounts.

8. The method of claim 1, further comprising PCR amplifying the population of recombined nucleic acids.

22. The method of claim 1, wherein the chemically synthesizing step comprises synthesizing the plurality of overlapping oligonucleotides or the at least one cross-over oligonucleotide via trinucleotide synthesis.

9. A method of recombining two or more nucleic acids, the method comprising:

chemically synthesizing a plurality of overlapping oligonucleotides which comprise a plurality of subsequences which correspond to a plurality of different parental nucleic acids, wherein each of the plurality of different parental nucleic acids comprises or encodes at least three modular subsequences, and wherein the plurality of overlapping oligonucleotides comprise at least one oligonucleotide which has one subsequence that corresponds to a first modular subsequence from a first parental nucleic acid and a second subsequence that corresponds to a second modular subsequence from the first or a second parental nucleic acid, and at least one cross-over oligonucleotide which has at least one subsequence corresponding to at least one modular subsequence from one of the plurality of different parental nucleic acids and at least one subsequence which corresponds to a region of a different second parental nucleic acid in a region outside of the least one modular subsequence, the plurality of overlapping oligonucleotides further comprising a plurality of additional chemically synthesized oligonucleotides which correspond to subsequences of one or more of the plurality of different parental nucleic acids other than the at least three modular subsequences;

hybridizing the plurality of overlapping oligonucleotides and the at least one cross- over oligonucleotide; and

,

elongating the plurality of overlapping oligonucleotides and the at least one cross- over oligonucleotide, thereby providing a population of recombined nucleic acids.

11. The method of claim 9, wherein the elongating step is performed with a polymerase.

12. The method of claim 9, wherein the elongating step is performed with a ligase.

13. The method of claim 9, wherein the at least three modular subsequences comprise at least one of the following:

an exon sequence, an intron sequence, a promoter sequence, or an enhancer sequence.

14. The method of claim 13, wherein the first and second modular subsequences are different.

15. The method of claim 9, wherein the plurality of overlapping oligonucleotides are present in non-equimolar amounts.

16. The method of claim 9, further comprising PCR amplifying the population of recombined nucleic acids.

23. The method of claim 9, wherein the chemically synthesizing step comprises synthesizing the plurality of overlapping oligonucleotides or the at least one cross-over oligonucleotide via trinucleotide synthesis.

10. A method of recombining two or more nucleic acids, comprising:

chemically synthesizing a plurality of overlapping oligonucleotides which comprise a plurality of subsequences which correspond to a plurality of different parental nucleic acids, wherein each of the different parental nucleic acids comprises or encodes a plurality of modular subsequences, and wherein the plurality of overlapping oligonucleotides comprise a plurality of different first cross-over oligonucleotides, each first cross-over oligonucleotide comprising one subsequence that corresponds to a first modular subsequence from a parental nucleic acid and a second modular subsequence that corresponds to a different modular subsequence from the parental nucleic acid, wherein the first modular subsequence and the different modular subsequence vary in order with respect to the parental nucleic acid, and at least one second cross-over oligonucleotide which has at least one subsequence corresponding to at least one modular subsequence from a first of the parental nucleic acids and at least one subsequence which corresponds to a region of a different second of the parental nucleic acids in a region outside of the least one modular subsequence, the plurality of overlapping oligonucleotides further comprising a plurality of additional chemically synthesized oligonucleotides which correspond to subsequences of one or more of the plurality of different parental nucleic acids other than the plurality of modular subsequences;

hybridizing the plurality of overlapping oligonucleotides and the at least one second cross-over oligonucleotide; and

,

elongating the plurality of overlapping oligonucleotides and the at least one second cross-over oligonucleotide, thereby providing a population of recombined nucleic acids.

17. The method of claim 10, wherein the elongating step is performed with a polymerase.

18. The method of claim 10, wherein the elongating step is performed with a ligase.

19. The method of claim 10, wherein the plurality of modular subsequences comprise at least one of the following:

an exon sequence, an intron sequence, a promoter sequence, or an enhancer sequence.

20. The method of claim 10, wherein the plurality of overlapping oligonucleotides are present in non-equimolar amounts.

21. The method of claim 10, further comprising PCR amplifying the population of recombined nucleic acids.

24. The method of claim 10, wherein the chemically synthesizing step compises synthesizing the plurality of overlapping oligonucleotides or the first or second cross-over oligonucleotide via trinucleotide synthesis.