Oligonucleotide mediated nucleic acid recombination

• US 6,423,542 B1
• Filed: 07/27/2000
• Issued: 07/23/2002
• Est. Priority Date: 01/19/1999
• 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:
  US09/626,930
• Time in Patent Office: 726 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 producing a recombinant nucleic acid, the method comprising:

(i) transducing a population of cells with a set of chemically synthesized overlapping family gene shuffling oligonucleotides; and

,

(ii) permitting recombination to occur between the set of overlapping family gene shuffling oligonucleotides and one or more nucleic acid contained within a plurality of cells of the population of cells, thereby providing a population of recombined nucleic acids within the resulting population of recombinant cells.

2. The method of claim 1, further comprising selecting the population of recombinant cells for a desired trait or property.

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

4. The method of claim 3, further comprising transducing the PCR amplified nucleic acids into a cell, vector, or virus.

5. The method of claim 1, wherein the set of overlapping family gene shuffling oligonucleotides comprise one or more chimeraplasts.

6. The method of claim 5, wherein the chimeraplasts comprise one or more codon-varied oligonucleotides.

7. The method of claim 1, wherein the set of overlapping family gene shuffling oligonucleotides comprises a plurality of codon-varied oligonucleotides.