Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
First Claim
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1. A method for selecting from or screening a library of recombinant proteins to identify a recombinant protein having a desired functional property, said method comprising:
- a) randomly fragmenting a template double-stranded DNA into a plurality of double-stranded fragments of a desired size;
b) adding to said plurality of double-stranded fragments one or more single- or double-stranded oligonucleotides, wherein said single- or double-stranded oligonucleotides comprise areas of identity and areas of heterology to the template double-stranded DNA;
c) denaturing said plurality of double-stranded fragments and said oligonucleotides to form a population of single-stranded fragments;
d) incubating said population of single-stranded fragments from step c) with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other member of the pair, thereby forming mutagenized double-stranded DNA molecules;
e) repeating steps c) and d) for a desired number of cycles, wherein repeated step c) further comprises denaturing the mutagenized double-stranded DNA molecules formed in step d) in previous cycles, thereby forming a library of mutagenized double-stranded DNA molecules;
f) expressing a library of recombinant proteins encoded by said library of mutagenized double-stranded DNA molecules from step e); and
g) selecting or screening said library of recombinant proteins to identify a recombinant protein with a desired functional 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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12 Claims
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1. A method for selecting from or screening a library of recombinant proteins to identify a recombinant protein having a desired functional property, said method comprising:
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a) randomly fragmenting a template double-stranded DNA into a plurality of double-stranded fragments of a desired size; b) adding to said plurality of double-stranded fragments one or more single- or double-stranded oligonucleotides, wherein said single- or double-stranded oligonucleotides comprise areas of identity and areas of heterology to the template double-stranded DNA; c) denaturing said plurality of double-stranded fragments and said oligonucleotides to form a population of single-stranded fragments; d) incubating said population of single-stranded fragments from step c) with a polymerase under conditions which result in the annealing of said single-stranded fragments at said areas of identity to form pairs of annealed fragments, said areas of identity being sufficient for one member of a pair to prime replication of the other member of the pair, thereby forming mutagenized double-stranded DNA molecules; e) repeating steps c) and d) for a desired number of cycles, wherein repeated step c) further comprises denaturing the mutagenized double-stranded DNA molecules formed in step d) in previous cycles, thereby forming a library of mutagenized double-stranded DNA molecules; f) expressing a library of recombinant proteins encoded by said library of mutagenized double-stranded DNA molecules from step e); and g) selecting or screening said library of recombinant proteins to identify a recombinant protein with a desired functional property. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)
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Specification