REDUCED CODON MUTAGENESIS
First Claim
1. A method of making a library of polynucleotide variants, the method comprising:
- providing a reference polynucleotide molecule or sequence;
selecting a plurality of codon sites in the reference polynucleotide molecule or sequence to be varied;
selecting a degenerate codon set for the plurality of codon sites to be varied, the degenerate codon set comprising a plurality of degenerate codons;
separately producing a plurality of sets of variant polynucleotide molecules, each variant polynucleotide comprising a member of the degenerate codon set at each of the plurality of sites of codon variation, wherein the set of variant polynucleotide molecules collectively comprises all of the members of the degenerate codon set at each of the sites of codon variation;
optionally pooling the sets of polynucleotide variants; and
,transforming the variants into a population of host cells, thereby producing the library.
1 Assignment
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Accused Products
Abstract
Methods and compositions that reduce complexity of libraries of variant biological molecules, that reduce oversampling of these libraries during screening and that improve screening efficiency are provided. Sets of efficient degenerate codon sets are provided that efficiently encode all, or nearly all canonical amino acids. Degenerate oligonucleotides comprising these codons are provided, as are polynucleotide variants. Variant pooling strategies are used during library construction. Logical filtering is applied to select codon sites for mutagenesis, or to select amino acid sets to be incorporated at such sites. Methods for reducing non-optimal oversampling during screening are provided.
50 Citations
63 Claims
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1. A method of making a library of polynucleotide variants, the method comprising:
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providing a reference polynucleotide molecule or sequence; selecting a plurality of codon sites in the reference polynucleotide molecule or sequence to be varied; selecting a degenerate codon set for the plurality of codon sites to be varied, the degenerate codon set comprising a plurality of degenerate codons; separately producing a plurality of sets of variant polynucleotide molecules, each variant polynucleotide comprising a member of the degenerate codon set at each of the plurality of sites of codon variation, wherein the set of variant polynucleotide molecules collectively comprises all of the members of the degenerate codon set at each of the sites of codon variation; optionally pooling the sets of polynucleotide variants; and
,transforming the variants into a population of host cells, thereby producing the library. - View Dependent Claims (2, 3, 4, 8, 9, 10, 11, 12, 13, 14, 15, 19, 20, 24, 25)
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5-7. -7. (canceled)
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16-18. -18. (canceled)
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21-23. -23. (canceled)
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26-36. -36. (canceled)
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37. A method of making a library of polynucleotide variants, the method comprising:
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providing a reference polynucleotide molecule or reference polynucleotide sequence; selecting at least one site in the reference polynucleotide molecule or reference polynucleotide sequence to be varied; and
,producing a set of variant polynucleotides comprising degenerate codons at the site of interest, wherein the degenerate codons are selected from the group consisting of;
(NDT, VHG), (NDC, VHG), (VWG, NNC), (NNT, VWG), (VMA, NDT), (NDC, VMA), (NDT, VMG), (NDC, VMG), (NNT, VAA), (NNC, VAA), (NNT, VAG), (VAG, NNC), (VMA, NDT, WKG), (NDT, TGG, VHG), (NNT, VWG, TGG), (NDC, TGG, VHG), (NDC, VMA, WKG), (NDT, WKG, VMG), (NDC, WKG, VMG), (VMA, NAT, DKK), (VMA, NAC, DKK), (VMA, DKS, NAT), (VMA, NAC, DKS), (NAT, VMG, DKK), (NAC, VMG, DKK), (DKS, NAT, VMG), (NAC, DKS, VMG), and (TDK, VDT, VVA), thereby providing the library.
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38-39. -39. (canceled)
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40. A composition comprising a set of polynucleotide variants comprising at least one degenerate codon position, with codons at the position being selected from the group consisting of:
- (NDT, VHG), (NDC, VHG), (VWG, NNC), (NNT, VWG), (VMA, NDT), (NDC, VMA), (NDT, VMG), (NDC, VMG), (NNT, VAA), (NNC, VAA), (NNT, VAG), (VAG, NNC), (VMA, NDT, WKG), (NDT, TGG, VHG), (NNT, VWG, TGG), (NDC, TGG, VHG), (NDC, VMA, WKG), (NDT, WKG, VMG), (NDC, WKG, VMG), (VMA, NAT, DKK), (VMA, NAC, DKK), (VMA, DKS, NAT), (VMA, NAC, DKS), (NAT, VMG, DKK), (NAC, VMG, DKK), (DKS, NAT, VMG), (NAC, DKS, VMG), and (TDK, VDT, VVA), wherein the polynucleotide variants collectively comprise all possible variants represented by the degenerate codon position.
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41-44. -44. (canceled)
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45. A library of polynucleotides, comprising:
- a mixture of polynucleotide variant molecules, the variant molecules comprising at least a first degenerate codon position, wherein degenerate codons of the first position encode any encoded amino acid in a selected molar ratio, and wherein the degenerate codons at each of the positions collectively comprise fewer than 32 codons and encode more than 12 different amino acids.
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46-61. -61. (canceled)
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62. A method of making a library of nucleic acid variants, the method comprising:
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providing a reference polynucleotide sequence to be varied, which reference polynucleotide sequence is present as a subsequence of a circular nucleic acid template; amplifying the circular nucleic acid template in a plurality of separate polymerase reactions, wherein each polymerase reaction comprises at least one polymerase primer that comprises one or more variant sequence that is partially complementary to the reference sequence, which primer comprises at least one nucleotide difference as compared to the variant sequence, and each reaction comprises at least one unique variant primer as compared to at least one other polymerase reaction; pooling the resulting variant amplicons; and
,transforming the pooled variant amplicons into a population of host cells.
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63-70. -70. (canceled)
Specification