Methods for making character strings, polynucleotides and polypeptides having desired characteristics

• US 6,917,882 B2
• Filed: 01/18/2000
• Issued: 07/12/2005
• Est. Priority Date: 01/19/1999
• Status: Expired due to Term

• 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): Brusca, John
• Assistant Examiner(s): Zhou, Shubo
• Application Number:
  US09/494,282
• Publication Number:
  US 20020183934A1
• Time in Patent Office: 2,002 Days
• Field of Search: 435/4, 435/6, 435/15, 435/91.2, 435/9, 435/262, 435/23.1, 536/23.1, 536/24.1, 702/19, 702/20
• 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 method of identifying a set of oligonucleotides for use in a physical recombination procedure, the method comprising:

(a) 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) aligning the parental character strings to identify one or more regions of pairwise homology;

(c) manipulating the two or more parental character strings by applying a crossover operator to a point selected outside an identified pairwise homology region to thereby computationally generate one or more derivative character strings; and

(d) from the one or more derivative character strings, computationally selecting 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 pareptal character strings are separated by a crossover point; and

(e) synthesizing the set of oligonucleotides selected from the derivative character strings to be used for a physical recombination procedure.

2. The method of claim 1, 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.

3. The method of claim 1, further comprising, applying a genetic operator to the two more parental character strings, wherein the genetic operator is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

4. The method of claim 1, wherein the at least one oligonucleotide comprising a chimeric nucleic acid sequence is at least about 10 nucleotides in length.

5. The method of claim 1, further comprising analyzing the one or more derivative character strings with an energy expression.

6. A method of identifying a set of oligonucleotides for use in a physical recombination procedure, the method comprising:

(a) 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) aligning the parental character strings to identify one or more reaicms of pairwise homology;

(c) manipulating the two or more parental character strings by applying a crossover operator to a point selected within an identified pairwise homology region to thereby computationally generate one or more derivative character strings; and

(d) from the one or more derivative character strings, computationally selecting 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

(e) synthesizing the set of oligonucleotides selected from the derivative character strings to be used for a physical recombination procedure.

7. The method of claim 6, 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.

8. The method of claim 6, further comprising, applying a genetic operator to the two or more parental character strings, wherein the genetic operator is selected from the group consisting of multiplication, mutation, fragmentation, and ligation.

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

10. The method of claim 6, further comprising analyzing the one or more derivative character strings with an energy expression.

11. A method of identifying a set of oligonucleotides for use in a physical recombination procedure, the method comprising:

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

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

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) synthesizing the set of oligonucleotides selected from the derivative character strings to be used for a physical recombination procedure.