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US 6,319,713 B1Granted
Methods and compositions for polypeptide engineering
AssigneeMaxygen, Inc.
InventorsPhillip A. Patten, Willem P. C. Stemmer
Life of this patent
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Abstract
Methods are provided for the evolution of proteins of industrial and pharmaceutical interest, including methods for effecting recombination and selection. Compositions produced by these methods are also disclosed.
Claims53
A method for making recombined nucleic acids, the method comprising:
- (a) providing at least one single-stranded polynucleotide;
- (b) providing one or more nucleic acids, at least one of which differs from the single-stranded polynucleotide(s) in at least one nucleotide, and fragmenting the one or more nucleic acids to produce a plurality of non-identical nucleic acid fragments that are capable of hybridizing to the single-stranded polynucleotide(s);
- (c) contacting the single-stranded polynucleotide(s) with the plurality of nucleic acid fragments, thereby producing annealed nucleic acid products;
- (d) contacting the products of (c) with a polymerase;
- and,
- (e) contacting the products of (d) with a ligase, thereby producing recombined nucleic acids annealed to the single-stranded polynucleotide(s).
3 more elements
A method for making a modified or recombinant nucleic acid, the method comprising:
- (a) providing a selected single-stranded template nucleic acid;
- (b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of:
- (i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;
- (ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,
- (iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,
- (iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,
- (v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or
- (vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product;
- and
- (c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand, wherein the template nucleic acid comprises uracil and the method further comprises degrading the template nucleic acid.
7 more elements
A method for making a recombined nucleic acid, the method comprising:
- (a) providing a selected single-stranded template nucleic acid;
- (b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of:
- (i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;
- (ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,
- (iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,
- (iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,
- (v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or
- (vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product;
- and
- (c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand, wherein the template nucleic acid comprises uracil and the method further comprises degrading the template nucleic acid and releasing the resulting cleaved template nucleic acid from the annealed nucleic acid.
7 more elements
A method for making a recombined nucleic acid, the method comprising:
- (a) providing a selected single-stranded template nucleic acid;
- (b) contacting the selected single-stranded template nucleic acid with a population of nucleic acid fragments, wherein the population of nucleic acid fragments comprises one or more of:
- (i) nucleic acid fragments which comprise nucleic acid sequences which are homologous to the single-stranded template nucleic acid;
- (ii) nucleic acid fragments resulting from digestion of at least first substrate molecules with a DNase,
- (iii) nucleic acid fragments which comprise nucleic acid sequences produced by mutagenesis of a parental nucleic acid,
- (iv) nucleic acid fragments comprising at least one nucleic acid sequence which is homologous to the single-stranded template nucleic acid, which sequence is present in the population at a concentration of less than 1% by weight of the total population of nucleic acid fragments,
- (v) nucleic acid fragments comprising at least one hundred nucleic acid sequences which are homologous to the template, or
- (vi) nucleic acid fragments comprising sequences of at least 50 nucleotides, thereby producing an annealed nucleic acid product;
- (c) contacting the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined nucleic acid strand;
- and
- (d) transforming the recombined nucleic acid into a host, wherein the host is a mutS host.
8 more elements
Dependencies are inferred from the claim text.