Oligonucleotide mediated nucleic acid recombination

• US 6,319,714 B1
• Filed: 07/27/2000
• Issued: 11/20/2001
• Est. Priority Date: 01/19/1999
• Status: Expired due to Term

• 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:
  US09/626,929
• Time in Patent Office: 481 Days
• Field of Search: 435/440, 435/6, 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 two or more nucleic acids, the method comprising:

(i.) aligning two or more nucleic acid sequence strings representing the two or more nucleic acids to identify regions of identity and regions of diversity;

(ii.) providing a non-equimolar set of selected oligonucleotides, which set of selected oligonucleotides comprises a plurality of oligonucleotides which correspond in sequence to at least two of the two or more nucleic acids at at least one region of diversity, the oligonucleotides being present in non-equimolar amounts; and

,

(iii.) extending the oligonucleotides with a polymerase, thereby producing a plurality of recombinant nucleic acids.

2. The method of claim 1, wherein the two or more nucleic acids are homologous.

3. The method of claim 1, wherein the two or more nucleic acids are non-homologous.

4. The method of claim 1, further comprising:

(iv.) selecting the plurality of recombinant nucleic acids for a desired trait or property.

5. The method of claim 4, further comprising repeating any of steps (i.)-(iv.).

6. The method of claim 1, further comprising recombining the recombinant nucleic acid with an additional nucleic acid.

7. The method of claim 6, further comprising selecting the resulting further recombined nucleic acid for a desired trait or property.