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US 6,165,793 AGranted
Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
AssigneeMaxygen, Inc.
InventorWillem P. C. Stemmer
Life of this patent
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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.
Claims62
A method for producing a variant polynucleotide, comprising:
- (a) conducting a polynucleotide extension process under conditions resulting in incomplete extension of synthesized polynucleotides to produce a plurality of polynucleotides in various stages of synthesis wherein at least 20% of the amplified strands are incompletely extended;
- (b) denaturing the plurality of polynucleotides;
- (c) incubating the denatured plurality of polynucleotides with a polymerase under conditions which result in annealing of single-stranded polynucleotides at regions of complementarity between the single stranded polynucleotides and formation of variant double stranded polynucleotide chains;
- (d) repeating (b) and (c);
- (e) screening or selecting a plurality of variant polynucleotides or expression products thereof to identify a variant polynucleotide having a useful functional activity.
2 more elements
A method of producing mutant polynucleotides, comprising:
- (a) conducting a polynucleotide extension process under conditions resulting in incomplete extension of synthesized polynucleotides wherein at least 20% of the amplified strands are incompletely extended;
- (b) subjecting amplification products to further amplification to produce a recombinant variant of the polynucleotide.
A method for producing mutant polynucleotides, comprising:
- (a) conducting a polynucleotide extension process under conditions resulting in incomplete extension of synthesized polynucleotides the process producing a plurality of polynucleotides in various stages of synthesis or amplification wherein at least 20% of the amplified strands are incompletely extended;
- (b) denaturing the plurality of polynucleotides;
- (c) incubating the denatured plurality of polynucleotides with a polymerase under conditions which result in annealing of the single-stranded polynucleotides at regions of identity between the single stranded polynucleotides and formation of mutagenized double stranded polynucleotide chains;
- (d) repeating (b) and (c);
- (e) expressing a plurality of mutant polypeptides from a plurality of mutagenized polynucleotide chains;
- (f) screening or selecting the plurality of mutant polypeptides to identify at least one mutant polypeptide having a useful functional activity.
3 more elements
A method of producing a variant polynucleotide, comprising:
- (a) subjecting a plurality of variants of a polynucleotide to a template-directed amplification process under conditions resulting in incompletely extended amplification products wherein at least 20% of the amplified strands are incompletely extended;
- (b) denaturing the amplification products;
- (c) annealing the denatured amplification products at regions of complementarity and incubating with a polymerase under conditions for template-directed amplification;
- (d) repeating (b) and (c) until the amplification products include a plurality of recombinant variants of the polynucleotide;
- and
- (e) screening or selecting a plurality of recombinant variants or expression products thereof to identify a recombinant variant having a desired functional property.
3 more elements
A method of producing a recombinant polynucleotide, comprising:
- (a) choosing a plurality of existing polynucleotide variants to be shuffled;
- (b) subjecting the plurality of polynucleotide variants to a template-directed polynucleotide extension process under conditions resulting in incomplete extension of the polynucleotide variants wherein at least 20% of the amplified strands are incompletely extended to produce a plurality of single-stranded overlapping segments having sequences from the plurality of polynucleotide variants;
- (c) conducting a multi-cyclic polynucleotide extension process on pairs of the single stranded overlapping segments annealed to each other at region so overlap under conditions whereby one segment serves as a template for extension of the other to generate a population of recombinant variants of the polynucleotides;
- (d) screening or selecting the recombinant variants or expression products thereof to identify a recombinant variant having a desired functional property.
1 more element
A method of producing a shuffled polynucleotide, comprising:
- (a) choosing a plurality of existing polynucleotide variants to be shuffled;
- (b) generating partial length copies of the plurality of variants by PCR amplification under conditions in which at least 20% of products are partial length products the partial length copies constituting a plurality of single-stranded overlapping segments having sequences form the plurality of polynucleotide variants;
- (c) conducting a multi-cyclic polynucleotide extension process on pairs of the single stranded overlapping segments annealed to each other at region so overlap under conditions whereby one segment serves as a template for extension of the other to generate a population of shuffled polynucleotides;
- (d) screening or selecting the shuffled variant polynucleotides or expression products thereof to identify a shuffled variant polynucleotide or expression product thereof having a desired functional property.
1 more element
A method of evolving a variant polynucleotide for acquisition of a desired property, comprising
- conducting a polynucleotide amplification reaction on initial substrates comprising a plurality of variants of a polynucleotide, wherein at least one cycle of the amplification reaction is an incomplete amplification cycle performed under conditions which produce amplification products comprising incompletely extended variants of the polynucleotide wherein at least 20% of the amplified strands are incompletely extended, which amplification products are denatured to component strands, which are reannealed in different pairings to form recombined amplification products, which form the substrates for a subsequent cycle of amplification until the amplification products include evolved recombinant variants of the polynucleotide;
- selecting or screening the evolved recombinant variants of the polynucleotide to identify an evolved recombinant variant having a desired functional property.
A method of evolving a variant polynucleotide for acquisition of a desired property, comprising
- conducting a polynucleotide amplification reaction on initial substrates comprising a plurality of variants of a polynucleotide, wherein at least one cycle of the amplification reaction is an incomplete amplification cycle performed under conditions which produce amplification products comprising incompletely extended variants of the polynucleotide, which amplification products are denatured to component strands, which are reannealed in different pairings to form recombined amplification products, which form the substrates for a subsequent cycle of amplification until the amplification products include evolved recombinant variants of the polynucleotide;
- selecting or screening the evolved recombinant variants of the polynucleotide to identify a recombinant variant having a desired functional property
- wherein the polynucleotide amplification reaction is conducted for 10-20 cycles of incomplete amplification and for 10-20 cycles under conditions permitting complete extension of the variants of the polynucleotide.
Dependencies are inferred from the claim text.