Gene site saturation mutagenesis (GSSM) method
AssigneeBASF SE
InventorsJared Dennis, Xuqiu Tan, Robin Cai
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Abstract
Disclosed herein is an improved Gene Site Saturation Mutagenesis (GSSM) method for producing a plurality of modified polynucleotides and/or polypeptides, creating specific changes to a gene, and reassembling mutations or changes at one or more sites.
Claims42
A method of producing a plurality of modified polynucleotides from a parent polynucleotide comprising the coding region of a subject protein, wherein each of the plurality of modified polynucleotides encode a modified polypeptide that comprises an amino acid substitution at a first amino acid position of interest in the protein of interest, the method comprising:
- (a) providing a plurality of primers capable of binding to a desired region of the parent polynucleotide, wherein the desired region comprises the codon for the amino acid at the first position of interest of the subject protein, wherein at least N of the plurality of primers each comprise a first codon for a different alternative amino acid at the first amino acid position of interest, wherein N is the number of desired amino acid substitutions at the first position of interest;
- (b) contacting the plurality of primers with the parent polynucleotide to form a reaction mixture;
- and
- (c) subjecting the reaction mixture to a polymerase extension reaction to generate the plurality of modified polynucleotides;
- wherein the plurality of primers does not comprise degenerate primers.
A method of producing a plurality of modified polypeptides of a parent polypeptide comprising the coding region of a subject protein, wherein each of the plurality of modified polypeptides comprises an amino acid substitution at a first amino acid position of interest, the method comprising:
- providing a plurality of primers capable of binding to a desired region of a template polynucleotide encoding the parent polypeptide, wherein the desired region comprises the codon for the amino acid at the first amino acid position of interest in the parent polypeptide, wherein at least N of the plurality of primers each comprise a first codon for a different alternative amino acid for the amino acid at the first amino acid position of interest, wherein N is the number of desired amino acid substitutions at the first position of interest;
- contacting the plurality of primers with the template polynucleotide to form a reaction mixture;
- subjecting the reaction mixture to a polymerase extension reaction to generate the plurality of modified polynucleotides;
- and
- introducing the modified polynucleotides to a recombinant expression system to produce the plurality of modified polypeptides;
- wherein the plurality of primers does not comprise degenerate primers.
Dependencies are inferred from the claim text.