Methods for generating polynucleotides having desired characteristics by iterative selection and recombination

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.

1 - 40 . (canceled)  
     
     
         41 . A method of producing a progeny polynucleotide set by subjecting a double-stranded circular parental polynucleotide molecule to mutagenesis, said method comprising synthesizing by means of a polymerase-catalyzed amplification reaction a first progeny polynucleotide strand comprised of said first primer and a second progeny polynucleotide strand comprised of said second primer, wherein the first progeny polynucleotide strand and the second progeny polynucleotide strand form a double-stranded mutagenized circular polynucleotide product, and wherein at least one of said primers contains a non-stochastic mutagenic cassette with respect to the parental polynucleotide molecule, thereby producing a progeny polynucleotide set.  
     
     
         42 . The method of  claim 41 , wherein said non-stochastic mutagenic cassette contained in said at least one primer is degenerate in nature.  
     
     
         43 . The method of  claim 42 , wherein a degenerate progeny polynucleotide set is produced.  
     
     
         44 . A method of producing a set of progeny polypeptides, in which a non-stochastic range of single amino acid substitutions is represented at each amino acid position, from a template polypeptide set, said method comprising subjecting a codon-containing template polynucleotide to polymerase-based amplification using a degenerate oligonucleotide for each codon to be mutagenized.  
     
     
         45 . The method of  claim 44 , wherein said method generates from at least one to twenty different amino acids at each amino acid site along a parental polypeptide template.  
     
     
         46 . The method of  claim 44 , wherein said degenerate oligonucleotides is comprised of a first homologous sequence and a degenerate trinucleotide cassette.  
     
     
         47 . The method of  claim 44 , wherein said degenerate oligonucleotide is comprised of a first homologous sequence, a degenerate trinucleotide cassette, and a second homologous sequence.  
     
     
         48 . The method of  claim 44 , wherein said degenerate trinucleotide cassette is comprised of a first mononucleotide cassette selected from the group consisting of: 
 a degenerate A/C mononucleotide cassette,    a degenerate A/G mononucleotide cassette,    a degenerate A/T mononucleotide cassette,    a degenerate C/G mononucleotide cassette,    a degenerate C/T mononucleotide cassette,    a degenerate G/T mononucleotide cassette,    a degenerate C/G/T mononucleotide cassette,    a degenerate A/G/T mononucleotide cassette,    a degenerate A/C/T mononucleotide cassette,    a degenerate A/C/G mononucleotide cassette, and    a degenerate A/C/G/T mononucleotide cassette.    
     
     
         49 . The method of  claim 48 , wherein said degenerate trinucleotide cassette is further comprises a second and a third mononucleotide cassette, each selected from the group consisting of: 
 a degenerate A/C mononucleotide cassette,    a degenerate A/G mononucleotide cassette,    a degenerate A/T mononucleotide cassette,    a degenerate C/G mononucleotide cassette,    a degenerate C/T mononucleotide cassette,    a degenerate G/T mononucleotide cassette,    a degenerate C/G/T mononucleotide cassette    a degenerate A/G/T mononucleotide cassette,    a degenerate A/C/T mononucleotide cassette,    a degenerate A/C/G mononucleotide cassette,    a degenerate A/C/G/T mononucleotide cassette,    a non-degenerate A mononucleotide cassette, and    a non-degenerate C mononucleotide cassette,    a non-degenerate G mononucleotide cassette,    and a non-degenerate T mononucleotide cassette.    
     
     
         50 . The method of  claim 44 , where said degenerate trinucleotide cassette is selected from the group consisting of: 
 a degenerate N,N,N trinucleotide cassette,    a degenerate N,N,G/T trinucleotide cassette,    a degenerate N,N,G/C trinucleotide cassette,    a degenerate N,N,A/C/G trinucleotide cassette,    a degenerate N,N,A/G/T trinucleotide cassette, and    a degenerate N,N,C/G/T trinucleotide cassette.    
     
     
         51 . The method of  claim 44 , wherein said degenerate oligonucleotide is comprised of a first homologous sequence and a plurality of trinucleotide cassettes.  
     
     
         52 . The method of  claim 51 , wherein said method generates a progeny polypeptide having a plurality of concurrent single amino acid changes, at each amino acid site, with respect to a parental polypeptide template.  
     
     
         53 . The method of  claim 44 , wherein each of said degenerate trinucleotide cassettes is comprised of a first mononucleotide cassette selected from the group consisting of: 
 a degenerate A/C mononucleotide cassette,    a degenerate A/G mononucleotide cassette,    a degenerate A/T mononucleotide cassette,    a degenerate C/G mononucleotide cassette,    a degenerate C/T mononucleotide cassette,    a degenerate G/T mononucleotide cassette,    a degenerate C/G/T mononucleotide cassette,    a degenerate A/G/T mononucleotide cassette,    a degenerate A/C/T mononucleotide cassette,    a degenerate A/C/G mononucleotide cassette, and    a degenerate A/C/G/T mononucleotide cassette.    
     
     
         54 . The method of  claim 53 , wherein each of said degenerate trinucleotide cassettes further comprises a second and third mononucleotide cassette, each selected from the group consisting of: 
 a degenerate A/C mononucleotide cassette,    a degenerate A/G mononucleotide cassette,    a degenerate A/T mononucleotide cassette,    a degenerate C/G mononucleotide cassette,    a degenerate C/T mononucleotide cassette,    a degenerate G/T mononucleotide cassette,    a degenerate C/G/T mononucleotide cassette    a degenerate A/G/T mononucleotide cassette,    a degenerate A/C/T mononucleotide cassette,    a degenerate A/C/G mononucleotide cassette,    a degenerate A/C/G/T mononucleotide cassette,    a non-degenerate A mononucleotide cassette,    a non-degenerate C mononucleotide cassette,    a non-degenerate G mononucleotide cassette, and    a non-degenerate T mononucleotide cassette.    
     
     
         55 . The method of  claim 51 , where said degenerate trinucleotide cassette is selected from the group consisting of: 
 a degenerate N,N,N trinucleotide cassette,    a degenerate N,N,G/T trinucleotide cassette,    a degenerate N,N,G/C trinucleotide cassette,    a degenerate N,N,A/C/G trinucleotide cassette,    a degenerate N,N,A/G/T trinucleotide cassette, and    a degenerate N,N,C/G/T trinucleotide cassette.    
     
     
         56 . The method of  claim 44 , wherein said degenerate oligonucleotide is comprised of a first homologous sequence, and a plurality of trinucleotide cassettes, and a second homologous sequence.  
     
     
         57 . The method of  claim 44 , further comprising screening the progeny polypeptides to identify those that display a desirable change with respect to at least one molecular property as compared with its parental polypeptide.  
     
     
         58 . A method of producing a plurality of mutagenized polypeptides from a template polypeptide, comprising: 
 providing a set of degenerate oligonucleotides comprising degenerate codons encoding all 20 amino acid residues at at least one amino acid residue position of the template polypeptide to be mutated, wherein said oligonucleotide comprises an area of identity to a template polynucleotide encoding the template polypeptide;    amplifying the template polynucleotide using the set of degenerate oligonucleotides to generate a plurality of mutagenized polynucleotides; and    expressing the plurality of mutagenized polypeptides from the plurality of mutagenized polynucleotides.    
     
     
         59 . The method of  claim 58 , further comprising screening the plurality of mutagenized polypeptides to identify those having an enhanced desired property compared to the template polypeptide.