Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
First Claim
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1. A method for evolving a green fluorescent protein (GFP) protein and a polynucleotide encoding same, comprising(1) providing a population of DNA segments encoding GFP at least one of which is in cell-free form;
- (2) shuffling the population of DNA segments encoding GFP whereby the segments recombine to form recombinant segments,(3) selecting or screening for recombinant segments having evolved toward a desired property;
repeating (2) and (3) with the recombinant segments in (3) providing the population of DNA segments in (1) at each cycle of repetition until a recombinant segment has acquired the desired property.
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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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22 Claims
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1. A method for evolving a green fluorescent protein (GFP) protein and a polynucleotide encoding same, comprising
(1) providing a population of DNA segments encoding GFP at least one of which is in cell-free form; -
(2) shuffling the population of DNA segments encoding GFP whereby the segments recombine to form recombinant segments, (3) selecting or screening for recombinant segments having evolved toward a desired property; repeating (2) and (3) with the recombinant segments in (3) providing the population of DNA segments in (1) at each cycle of repetition until a recombinant segment has acquired the desired property. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8)
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9. A method for evolving a polynucleotide for acquisition of a desired property, comprising:
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(1) providing a population of variants of said polynucleotide, at least one of which is in cell-free form; (2) shuffling said variants of said polynucleotide to form recombinant polynucleotides; (3) selecting or screening for recombinant polynucleotides that have evolved toward the desired property; and (4) repeating steps (2) and (3) with the recombinant polynucleotides selected in step (3) until a recombinant polynucleotide has acquired the desired property. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22)
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Specification