Claims
- 1. A method of walk-through mutagenesis of a nucleic acid encoding a polypeptide of interest, comprising:
a) selecting one or more target region(s) of prototype amino acids in the polypeptide of interest encoded by the nucleic acid; b) for each of the target region(s), selecting one or more predetermined amino acid(s) to be incorporated into the target region in lieu of the prototype amino acids; and c) synthesizing a mixture of oligonucleotides comprising a nucleotide sequence for each target region, wherein each oligonucleotide contains, at each sequence position in the target region, either a prototype nucleotide that is required for synthesis of the prototype amino acid of the polypeptide, or a predetermined nucleotide that is required for synthesis of the predetermined amino acid, wherein during synthesis, the ratio of available prototype nucleotides, to available predetermined nucleotides, is greater than 1:1.
- 2. The method of claim 1, further comprising generating an expression library of nucleic acids comprising said oligonucleotides.
- 3. The method of claim 1, wherein the ratio is equal to or greater than 4:1.
- 4. The method of claim 1, wherein the ratio is equal to or greater than 7:1.
- 5. The method of claim 1, wherein the ratio is equal to or greater than 9:1.
- 6. The method of claim 1, wherein the target region comprises a functional domain of the polypeptide.
- 7. The method of claim 1, wherein the target region comprises a catalytic site of an immunoglobulin.
- 8. A method of claim 1, wherein the target region comprises a hypervariable region of an antibody.
- 9. A method of claim 1, wherein the predetermined amino acid is Ser, Thr, Asn, Gin, Tyr, Cys, His, Glu, Asp, Lys or Arg.
- 10 A method of claim 1, wherein the the ratio of available prototype nucleotides, to available predetermined nucleotides, is determined using a binomial distribution that takes into consideration the length of the target region and a desired degree of success in incorporating nucleotides that encode the predetermined amino acid.
- 11. A library of nucleic acids prepared by the method of claim 1.
- 12. A library of polypeptides prepared by expressing the nucleic acids of claim 11.
RELATED APPLICATION
[0001] This application claims the benefit of U.S. Provisional Application No. 60/373,686, filed Apr. 17, 2002. The entire teachings of the above application is incorporated herein by reference.
Provisional Applications (1)
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Number |
Date |
Country |
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60373686 |
Apr 2002 |
US |