Oligonucleotide mediated nucleic acid recombination

Abstract

Methods of recombining nucleic acids, including homologous nucleic acids, are provided. Families of gene shuffling oligonucleotides and their use in recombination procedures, as well as polymerase and ligase mediated recombination methods are also provided.

1 .- 104 . (canceled) 
     
     
         105 . A method of recombining homologous oligonucleotides, the method comprising:
 computationally selecting at least some members of a set of family gene shuffling oligonucleotides;   synthesizing a set of family gene shuffling oligonucleotides including the computationally selected members; and   recombining the set of the family gene shuffling oligonucleotides to provide a population of recombined nucleic acids.   
     
     
         106 . The method of  claim 105 , wherein computationally selecting comprises aligning homologous nucleic acids. 
     
     
         107 . The method of  claim 105 , wherein computationally selecting comprises identifying regions of similarity and regions of diversity. 
     
     
         108 . The method of  claim 107 , further comprising selecting a set of overlapping oligonucleotides that encompasses the regions of similarity and the regions of diversity. 
     
     
         109 . The method of  claim 105 , further comprising receiving input of oligonucleotide selections by a user. 
     
     
         110 . The method of  claim 105 , wherein the recombining comprises:
 (i) hybridizing a chemically synthesized set of family gene shuffling oligonucleotides; and   (ii) elongating the chemically synthesized set of family gene shuffling oligonucleotides.   
     
     
         111 . The method of  claim 110 , wherein the set of family gene shuffling oligonucleotides are overlapping. 
     
     
         112 . The method of  claim 110 , wherein elongating involves using a polymerase or a ligase. 
     
     
         113 . The method of  claim 110 , wherein the chemically synthesized set of family gene shuffling oligonucleotides encodes an evolutionary intermediate nucleic acid. 
     
     
         114 . The method of  claim 105 , the method further comprising:
 (iii) denaturing the population of recombined nucleic acids, thereby providing denatured recombined nucleic acids;   (iv) reannealing the denatured recombined nucleic acids;   (v) extending or ligating the resulting reannealed recombined nucleic acids;   (vi) optionally, repeating (iii)-(v); and,   (vii) optionally, selecting one or more of the resulting recombined nucleic acids for a desired property.   
     
     
         115 . The method of  claim 114 , wherein, prior to performing step (vii), the resulting reannealed recombined nucleic acids are recombined. 
     
     
         116 . The method of  claim 114 , further comprising:
 (viii) recombining the selected recombined nucleic acids.   
     
     
         117 . The method of  claim 116 , further comprising selecting the recombined nucleic acids for a desired trait or property. 
     
     
         118 . The method of  claim 105 , further comprising selecting one or more member of the population of recombined nucleic acids for a desired property. 
     
     
         119 . The method of  claim 118 , wherein a plurality of members of the population of recombined nucleic acids are screened for the desired property and are determined to have the desired property, thereby providing first round screened nucleic acids, the method further comprising:
 hybridizing a second set of family gene shuffling oligonucleotides, which second set of family gene shuffling oligonucleotides are derived from the first round screened nucleic acids; and,   elongating the second set of family gene shuffling oligonucleotides, thereby providing a population of further recombined nucleic acids.   
     
     
         120 . The method of  claim 119 , wherein the second set of family gene shuffling oligonucleotides are overlapping. 
     
     
         121 . The method of  claim 119 , further comprising sequencing the first round screened nucleic acids, wherein the second set of family gene shuffling oligonucleotides is derived from the first round screened nucleic acids by aligning sequences of the first round screened nucleic acids to identify regions of identity and regions of diversity in the first round screened nucleic acids, and synthesizing the second set of family gene shuffling oligonucleotides to comprise a plurality of oligonucleotides, each of which comprise subsequences corresponding to at least one region of diversity. 
     
     
         122 . The method of  claim 119 , wherein the first round screened nucleic acids encode polypeptides of about 50 amino acids or less. 
     
     
         123 . The method of  claim 119 , wherein the second set of family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of the first round screened nucleic acids. 
     
     
         124 . The method of  claim 105 , wherein the set of family shuffling gene oligonucleotides comprise a plurality of oligonucleotide member types which comprise consensus region subsequences derived from a plurality of homologous target nucleic acids. 
     
     
         125 . The method of  claim 105 , wherein the set of family shuffling gene oligonucleotides comprise at least one module shuffling oligonucleotides. 
     
     
         126 . The method of  claim 105 , wherein the set of family shuffling gene oligonucleotides comprise a plurality of module shuffling oligonucleotides, wherein one or more of the plurality of oligonucleotides each comprise at least a first subsequence from a first sequence module and a second subsequence from a second sequence module. 
     
     
         127 . The method of  claim 105 , wherein the set of family shuffling oligonucleotides comprise a plurality of codon-varied oligonucleotides. 
     
     
         128 . The method of  claim 105 , the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprising at least 3 member types. 
     
     
         129 . The method of  claim 105 , the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprising at least 5 member types. 
     
     
         130 . The method of  claim 105 , the set of family shuffling gene oligonucleotides comprising a plurality of oligonucleotide member types comprising at least 10 member types. 
     
     
         131 . The method of  claim 105 , the set of family shuffling gene oligonucleotides comprising a plurality of homolgous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in approximately equimolar amounts. 
     
     
         132 . The method of  claim 105 , wherein the set of family shuffling gene oligonucleotides comprises a plurality of homolgous oligonucleotide member types, wherein the homologous oligonucleotide member types are present in non-equimolar amounts. 
     
     
         133 . An apparatus comprising:
 a computational element comprising
 logic for selecting at least some members of a set of family gene shuffling oligonucleotides; 
 a user interface for receiving user input for selecting oligonucleotides; and 
 an oligonucleotide synthesizer coupled to the computational element and configured to synthesize at least some of the set of family shuffling oligonucleotides based on information provided by the computational element. 
   
     
     
         134 . The apparatus of  claim 133 , wherein the computational element further comprises a display. 
     
     
         135 . The apparatus of  claim 133 , wherein the user interface comprises an input field for inputting oligonucleotide selections by the user.