Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
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Abstract
A method for DNA reassembly after random fragmentation, and its application to mutagenesis of nucleic acid sequences by in vitro or in vivo recombination is described. In particular, a method for the production of nucleic acid fragments or polynucleotides encoding mutant proteins is described. The present invention also relates to a method of repeated cycles of mutagenesis, shuffling and selection which allow for the directed molecular evolution in vitro or in vivo of proteins.
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Citations
110 Claims
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1-40. -40. (canceled)
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41. A method for producing a library of nucleic acids encoding a plurality of modified antigen binding sites, wherein the modified antigen binding sites are derived from a first nucleic acid comprising a sequence encoding a first antigen binding site, the method comprising:
- (a) providing a first nucleic acid encoding a first antigen binding site;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
(c) using the set of mutagenic oligonucleotides to generate a set of antigen binding site-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of modified antigen binding sites. - View Dependent Claims (42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70)
- (a) providing a first nucleic acid encoding a first antigen binding site;
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71. A method for producing from a library of variant antibodies from a template antibody, the method comprising:
- (a) providing a first nucleic acid encoding the template antibody;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
c) using the set of mutagenic oligonucleotides to generate a set of antibody-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant antibodies. - View Dependent Claims (72, 73, 74, 75, 76)
- (a) providing a first nucleic acid encoding the template antibody;
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77. A method for producing from a library of variant T cell receptors (TCRs) from a template T cell receptor (TCR), the method comprising:
- (a) providing a first nucleic acid encoding the template T cell receptor;
(b) providing a set of mutagenic oligonucleotides that encode naturally-occurring amino acid variants at a plurality of targeted codons in the first nucleic acid; and
c) using the set of mutagenic oligonucleotides to generate a set of T cell receptor (TCR)-encoding variant nucleic acids encoding a range of amino acid variations at each amino acid codon that was mutagenized, thereby producing a library of nucleic acids encoding a plurality of variant T cell receptors (TCRs). - View Dependent Claims (78)
- (a) providing a first nucleic acid encoding the template T cell receptor;
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79. A method of making a set of nucleic acids encoding a set of antigen binding site variants comprising the steps of:
- (a) providing a template nucleic acid encoding an antigen-binding polypeptide;
(b) providing a plurality of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at a single amino acid residue of the antigen-binding polypeptide; and
(c) generating a set of progeny antigen binding site-encoding variant nucleic acids encoding a non-stochastic range of single amino acid substitutions at each amino acid codon that was mutagenized, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby making a set of nucleic acids encoding a set of antigen binding site variants. - View Dependent Claims (80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100)
- (a) providing a template nucleic acid encoding an antigen-binding polypeptide;
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101. A method of making a set of antibody variants comprising the steps of:
- (a) providing a nucleic acid encoding an antibody;
(b) providing a plurality of oligonucleotides;
(c) generating a non-stochastic range of single amino acid substitutions at each amino acid codon, whereby all 19 possible natural amino acid changes are generated at each amino acid codon mutagenized, thereby generating a set of variant nucleic acids; and
(d) expressing the set of variant nucleic acids such that the antibody variants encoded by the variant nucleic acids are expressed. - View Dependent Claims (102, 103, 104)
- (a) providing a nucleic acid encoding an antibody;
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105. A method of identifying a variant of an antigen binding site comprising the steps of:
- (a) providing a nucleic acid encoding an antigen binding site;
(b) providing a set of oligonucleotides that encode all nineteen naturally-occurring amino acid variants at all residues of the antigen-binding site;
(c) incorporating the sequence of the oligonucleotides of step (b) into the nucleic acid of step (a) to generate a set of variant nucleic acids encoding nineteen amino acid substitution variants at each residue of the antigen binding site;
(d) expressing each of the variant nucleic acids as polypeptides and measuring the variant'"'"'s affinity to the antigen; and
(e) identifying a variant of the antigen binding site by its increased or decreased antigen binding specificity as compared to the antigen binding affinity of the antigen binding site encoded by the nucleic acid of step (a). - View Dependent Claims (106, 107, 108, 109, 110)
- (a) providing a nucleic acid encoding an antigen binding site;
Specification