Methods for generating polynucleotides having desired characteristics by iterative selection and recombination
View Patent ↗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.
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.
2. The method of claim 1 wherein the concentration of a specific double-stranded fragment in the plurality of double-stranded fragments in step b) is less than 1% by weight of the total amount of DNA in the combined plurality of double-stranded fragments and single- or double-stranded oligonucleotides in step b).
3. The method of claim 1 wherein the number of different specific double-stranded fragments in step a) comprises at least about 100.
4. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 5 bp to 5 kb.
5. The method of claim 1 wherein the size of the mutagenized double-stranded DNA molecules in the library of mutagenized double-stranded DNA molecules in step e) is from about 50 bp to 100 kb.
6. The method of claim 1 wherein the template double-stranded DNA encodes a wild-type protein.
7. The method of claim 1 , wherein the polymerase is Taq.
8. The method of claim 1 , wherein the polymerase is Klenow polymerase.
9. The method of claim 1 , wherein the template double-stranded DNA is from 50 bp to 50 kb.
10. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 10 bp to 1000 bp.
11. The method of claim 1 wherein the size of the double-stranded fragments in step a) is from about 20 bp to 500 bp.
12. The method of claim 1 , wherein the template double-stranded DNA in step a) is obtained from DNA associated with a displayed antibody that has been screened for affinity for binding a predetermined ligand, and wherein the library of recombinant proteins in step f) is expressed as an antibody display library.