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US 6,355,484 B1Granted
Methods and compositions for polypeptides engineering
AssigneeMaxygen, Inc.
InventorsPhillip A. Patten, Willem P. C. Stemmer
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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.
Claims63
A method of making a recombined nucleic acid that encodes a product having a desired property, the method comprising:
- (a) providing at least one single-stranded polynucleotide;
- (b) hybridizing a plurality of nucleic acid fragments to the single-stranded polynucleotide, which nucleic acid fragments are produced by fragmentation of a plurality of non-identical substrate nucleic acids;
- (c) extending and ligating the resulting hybridized nucleic acid fragments, thereby producing one or more recombined nucleic acid;
- and,
- (d) screening or selecting one or more product encoded by the recombined nucleic acid, or a complementary strand thereto, for the desired property, thereby identifying the recombined nucleic acid that encodes the product having the desired property.
2 more elements
A method of identifying a recombined polynucleotide with a desired functional property, comprising:
- (a) providing at least one single-stranded uracil-containing polynucleotide;
- (b) providing a plurality of non-identical nucleic acid fragments capable of hybridizing to the single-stranded uracil-containing polynucleotide, wherein said plurality of nucleic acid fragments are produced by fragmentation of one or more substrate nucleic acids differing in sequence from the single-stranded uracil-containing polynucleotide;
- (c) contacting the single-stranded uracil-containing polynucleotide with the plurality of nucleic acid fragments, thereby producing an annealed nucleic acid;
- (d) incubating the annealed nucleic acid with a polymerase and a ligase, thereby producing a recombined polynucleotide strand annealed to the uracil-containing polynucleotide;
- (e) amplifying the recombined polynucleotide strand under conditions wherein the uracil-containing polynucleotide is not amplified, thereby producing a population of recombined polynucleotides;
- and,
- (f) screening or selecting the population of recombined polynucleotides for the desired functional property, thereby identifying one or more polynucleotide(s) with the desired functional property.
4 more elements
A method of identifying a recombined DNA molecule encoding a protein with a desired functional property, comprising:
- (a) providing at least one single-stranded uracil-containing DNA molecule, which single-stranded uracil-containing DNA molecule, or a complementary strand thereto, encodes a protein;
- (b) providing a plurality of non-identical DNA fragments capable of hybridizing to the single-stranded uracil-containing DNA molecule, wherein said DNA fragments are produced by fragmentation of one or more substrate DNA molecules encoding at least one additional variant of the protein and wherein the fragmentation is by digestion with DNAse I;
- (c) contacting the single-stranded uracil-containing DNA molecule with the plurality of DNA fragments, thereby producing an annealed DNA molecule;
- (d) incubating the annealed DNA molecule with a polymerase and a ligase, thereby producing a recombined DNA strand annealed to the uracil-containing DNA molecule;
- (e) amplifying the recombined DNA strand under conditions wherein the uracil-containing DNA molecule is not amplified, thereby producing a population of recombined DNA molecules;
- and,
- (f) screening or selecting the population of recombined DNA molecules to identify those that encode a polypeptide having the desired functional property, thereby identifying one or more DNA molecules(s) that encode a polypeptide with the desired functional property.
4 more elements
Dependencies are inferred from the claim text.