Identifying oligonucleotides for in vitro recombination

• US 7,421,347 B2
• Filed: 03/07/2005
• Issued: 09/02/2008
• Est. Priority Date: 01/19/1999
• Status: Expired due to Fees

• Current Assignee: Codexis, Inc.
• Original Assignee: Maxygen Inc.
• Inventors: del Cardayre, Stephen, Tobin, Matthew, Patten, Phillip A., Selifonov, Sergey A., Gustafsson, Claes, Minshull, Jeremy, Stemmer, Willem P. C.
• Primary Examiner(s): Zhou; Shubo (Joe)
• Application Number:
  US11/075,231
• Publication Number:
  US 20050191688A1
• Time in Patent Office: 1,275 Days
• Field of Search: None
• US Class Current: 702/19
• CPC Class Codes: A61K 39/00 Medicinal preparations containing antigens or antibodies materials for immunoassay G01N33/53
• 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 15/1027 by DNA shuffling, e.g. RSR, STEP, RPR
• CPC Class Codes: C12N 15/1031 mutagenesis by gene assembly, e.g. assembly by oligonucleotide extension PCR
• 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

“In silico” nucleic acid recombination methods, related integrated systems utilizing genetic operators and libraries made by in silico shuffling methods are provided.

Claims

1. A computer program product comprising a machine readable medium on which is provided program code for identifying a set of oligonucleotides for use in a physical recombination procedure, the program code comprising:

(a) code for providing two or more parental character strings representing two or more polynucleotides or polypeptides;

(b) code for manipulating the two or more parental character strings by applying a genetic operator to generate one or more derivative character strings; and

(c) code for selecting a set of character sub strings from the one or more derivative character stings, which character substrings have sequences that identify the set of oligonucleotides for in vitro recombination, and

(d) code for outputting the selected set of character substrings to a computer or a user.

2. The computer program product of claim 1, further comprising:

code for identifying one or more frame shift mutations or premature terminations among the derivative character strings; and

code for removing or repairing derivative character strings possessing the one or more frame shift mutations or premature terminations.

3. The computer program product of claim 1, wherein the genetic operator is selected from the group consisting of multiplication, mutation, fragmentation, crossover, and ligation.

4. The computer program product of claim 1, wherein at least one of the one or more derivative character strings comprises a chimeric nucleic acid sequence that is at least about 10 nucleotides in length.

5. The computer program product of claim 1, further comprising code for analyzing the one or more derivative character strings with an energy expression.

6. A computer program product comprising a machine readable medium on which is provided program code for identifying a set of oligonucleotides for use in a physical recombination procedure, the program code comprising:

(a) code for providing two or more parental character strings representing two or more polynucleotides or polypeptides, which character strings, when aligned for maximum identity, comprise at least one region of heterology;

(b) code for aligning the parental character stings;

(c) code for manipulating the two or more parental character strings by applying a crossover operator to computationally generate one or more derivative character strings; and

(d) code for computationally selecting, from the one or more derivative character strings, a set of character subsirings having sequences that identify the set of oligonucleotides for in vitro recombination,

wherein the selected set of character sub strings identifies at least one oligonucleotide comprising a chimeric nucleic acid sequence that comprises subsequences from at least two of the parental character strings,

wherein the subsequences from the at least two parental character strings are separated by a crossover point, and

wherein the crossover point is selected from a region of identified pairwise homology, and

(e) code for outputting the selected set of character substrings to a computer or a user.

7. The computer program product of claim 6, further comprising:

code for identifying one or more frame shift mutations or premature terminations among the derivative character strings; and

code for removing or repairing derivative character stings possessing the one or more frame shift mutations or premature terminations.

8. The computer program product of claim 6, further comprising code for applying a genetic operator to the one or more parental character strings, wherein the genetic operator is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

9. The computer program product of claim 6, wherein the at least one oligonucleotide comprising a chimeric nucleic add sequence is at least about 10 nucleotides in length.

10. The computer program product of claim 6, fUrther comprising code for analyzing the one or more derivative character strings with an energy expression.

11. A computer program product comprising a machine readable medium on which is provided program instructions for identifying a set of oligonucleotides for use in a physical recombination procedure, the program code comprising:

(a) code for providing two or more parental character strings representing two or more polynucleotides or polypeptides, which character strings, when aligned for maximum identity, comprise at least one region of heterology;

(b) code for manipulating the two or more parental character strings by applying a crossover operator to computationally generate one or more derivative character strings; and

(c) code for computationally selecting, from the one or more derivative character strings, a set of character substrings having sequences that identify the set of oligonucleotides for in vitro recombination,

wherein the selected set of character substrings identifies at least one oligonucleotide comprising a chimeric nucleic acid sequence that comprises subsequences from at least two of the parental character strings, and

wherein the subsequences from the at least two parental character strings are separated by a crossover point, and

(d) code for outputting the selected set of character substrings to a computer or a user.

12. A method of generating a set of oligonucleotide variants, the method comprising:

(a) providing two or more parental character strings representing two or more polynucleotides or polypeptides;

(b) manipulating the two or more parental character strings by applying a genetic operator to computationally generate one or more derivative character strings;

(c) from the one or more derivative character strings, computationally selecting a set of character substrings;

(d) providing a set of oligonucleotides corresponding in sequence to the set of character substrings; and

(e) recombining the set of oligonucleotides in vitro to generate a set of polynucleotide variants.

13. The method of claim 12, further comprising:

identifying one or more frame shift mutations or premature terminations among the derivative character strings; and

removing or repairing derivative character strings possessing the one or more frame shift mutations or premature terminations.

14. The method of claim 12, wherein the genetic operator is selected from the group consisting of multiplication, mutation, fragmentation, crossover, and ligation.

15. The method of claim 12, wherein (e) comprises assembling a library of recombinant nucleic acids by assembly PCR from the set of oligonucleotides corresponding in sequence to the set of character substrings.

16. The method of claim 15, further comprising:

(f) selecting or screening the library for one or more recombinant polynucleotide or polypeptide having a desired property.

17. The method of claim 15, further comprising reiterative recombination or selection of the library of recombinant nucleic acids.

18. The method of claim 12, further comprising:

(f) deconvoluting one or more of said set of character substrings to produce one or more variant character strings corresponding to the sequences of the set of character substrings; and

(g) performing (a)-(e) using the one or more variant character strings as the parental character strings.

19. The method of claim 12, wherein the set of oligonuoleotides comprises one or more oligonucleotide member between about 20 and about 60 nucleotides in length.

20. The method of claim 12, wherein the polynucleotide variants are recombinant nucleic acids and wherein (e) comprises assembling a library of the recombinant nucleic acids in parallel.

21. The method of claim 12, wherein the polynucleotide variants are recombinant nucleic acids and wherein (e) comprises assembling a library of the recombinant nucleic acids by ligation of the oligonucleotides.