Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
First Claim
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1. A method of evolving a polynucleotide toward a desired functional property comprising:
- (a) providing a plurality of polynucleotides comprising two or more species variants;
(b) shuffling said plurality of polynucleotides to form a population of recombinant polynucleotides;
(c) selecting or screening said population of recombinant polynucleotides for recombinant polynucleotides that have evolved toward the desired functional property, (d) repeating steps (b) and (c) with the plurality of polynucleotides in step (b) comprising the recombinant polynucleotides selected or screened in step (c) until a recombinant polynucleotide is obtained which has acquired the desired functional property, wherein at least one shuffling cycle comprises conducting a mult-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the plurality of polynucleotides, the plurality of polynucleotides having regions of similarity and regions of heterology with each other and the partially annealed polynucleotide strands being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate said recombinant polynucleotides.
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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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Citations
73 Claims
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1. A method of evolving a polynucleotide toward a desired functional property comprising:
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(a) providing a plurality of polynucleotides comprising two or more species variants;
(b) shuffling said plurality of polynucleotides to form a population of recombinant polynucleotides;
(c) selecting or screening said population of recombinant polynucleotides for recombinant polynucleotides that have evolved toward the desired functional property, (d) repeating steps (b) and (c) with the plurality of polynucleotides in step (b) comprising the recombinant polynucleotides selected or screened in step (c) until a recombinant polynucleotide is obtained which has acquired the desired functional property, wherein at least one shuffling cycle comprises conducting a mult-cyclic polynucleotide extension process on partially annealed polynucleotide strands having sequences from the plurality of polynucleotides, the plurality of polynucleotides having regions of similarity and regions of heterology with each other and the partially annealed polynucleotide strands being partially annealed through the regions of similarity, under conditions whereby one strand serves as a template for extension of another strand with which it is partially annealed to generate said recombinant polynucleotides. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39)
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40. A method of obtaining a recombinant polynucleotide having a desired functional property comprising:
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a) treating a sample comprising species variants of a polynucleotide wherein the species variants of the polynucleoytide contain regions of similarity, under conditions whereby overlapping double-stranded segments of the species variants of the polynucleotide are formed;
b) denaturing the resultant overlapping double-standed segments of the species variants of the polynucleotide into single-stranded segments;
c) incubating said single-stranded segments under conditions which provide for the annealing of the single-stranded segments at the areas of simlarity to form pairs of annealed segments and extending the annealed segments with a polymerase, wherein said areas of similarity are sufficient for one member of a pair to prime replication of the other thereby forming recombinant double-stranded polynucleotide sequences;
d) repeating steps b) and c) for at least two cycles wherein the resultant mixture in step b) of a cycle comprises the recombinant double-stranded polynucleotide sequences formed in step c) of the previous cycle and the further cycle forms further recombinant sequences whereby the average length of the recombinant polynucleotide sequences increases in each cycle; and
e) screening or selecting recombinant polynucleotide sequences formed in step d) to identify a recombinant polynucleotide having the desired functional property. - View Dependent Claims (41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73)
a) introducing the recombinant polynucleotide to be screened or selected into a population of cells;
b) expressing the the recombinnt polynucleotides in said population of cells; and
c) selecting or screening the population of cells for the desired functional property.
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70. The method of claim 40 further comprising formulating the recombinant polynucleotide having the desired property or an expression product thereof as pharmaceutical.
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71. The method of claim 40 wherein the size of the double-stranded overlapping segments is from 5 base pairs to 5 kilobases.
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72. The method of claim 71 wherein the size of the double-stranded overlapping segments is from 10 to 1000 base pairs.
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73. The method of claim 72 wherein the size of the double-stranded overlapping segments is from 20 to 500 base pairs.
Specification