Methods for identifying sets of oligonucleotides for use in an in vitro recombination procedure

Abstract

In silico nucleic acid recombination methods, related integrated systems utilizing genetic operators and libraries made by in silico shuffling methods are provided.

1 . (canceled) 
     
     
         2 . A method of identifying improved polypeptide or nucleic acid sequences, the method comprising:
 (a) receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;   (b) generating a multivariate sequence activity model from said sequences and associated activities of said parental polypeptides or parental nucleic acids;   (c) using the multivariate sequence activity model to identify one or more oligonucleotides for recombination;   (d) recombining the one or more oligonucleotides identified in (c) to produce recombined oligonucleotides;   (e) screening at least some of the recombined oligonucleotides produced in (d) to select one or more of the recombined oligonucleotides; and   (f) performing (a)-(e) recursively using selected recombined nucleic acids from (e) as identified sequences in (a) to identify an improved polypeptide or nucleic acid sequences.   
     
     
         3 . The method of  claim 2 , wherein using the multivariate sequence activity model to identify one or more oligonucleotides for recombination comprises using the model to identify select one or more cross-overs. 
     
     
         4 . The method of  claim 2 , wherein using the multivariate sequence activity model to identify one or more oligonucleotides for recombination comprises using the model to extrapolate new sequences. 
     
     
         5 . The method of  claim 2 , wherein the generating the multivariate sequence activity model comprises performing PLS. 
     
     
         6 . The method of  claim 2 , wherein the multivariate sequence activity model is a neural network. 
     
     
         7 . The method of  claim 2 , wherein the one or more oligonucleotides identified in (c) comprise a plurality of overlapping oligonucleotides. 
     
     
         8 . The method of  claim 2 , wherein the two or more parental polypeptides or parental nucleic acids comprise naturally occurring polypeptides or naturally occurring nucleic acids that encodes a polypeptides. 
     
     
         9 . The method of  claim 2 , wherein at least one of the parental polypeptides or parental nucleic acids is an evolutionary or artificial intermediate. 
     
     
         10 . The method of  claim 2 , wherein at least one of the parental polypeptides or parental nucleic acids is a designed polypeptide or designed nucleic acid. 
     
     
         11 . The method of  claim 2 , wherein the screening performed in (e) comprises selecting the one or more recombinant nucleic acids for a desired trait or property using an in vitro or an in vivo selection assay. 
     
     
         12 . The method of  claim 2 , wherein one or more oligonucleotides identified for recombination in (c) comprises one or more oligonucleotides between about 20 and about 60 nucleotides in length. 
     
     
         13 . A computer program product comprising a non-transitory machine readable medium having instructions for identifying improved polypeptide or nucleic acid sequences, the instructions comprising:
 (a) code for receiving data identifying sequences of two or more parental polypeptides or parental nucleic acids that encode the polypeptides;   (b) code for generating a multivariate sequence activity model from said sequences and associated activities of said parental polypeptides or parental nucleic acids;   (c) code for using the multivariate sequence activity model to identify one or more oligonucleotides for recombination;   (d) code for receiving data identifying one or more of the recombined oligonucleotides produced from the one or more oligonucleotides identified in (c) and selected by screening; and   (e) performing (a)-(d) recursively using identified recombined nucleic acids from (d) as identified sequences in (a) to identify an improved polypeptide or nucleic acid sequences.   
     
     
         14 . The computer program product of  claim 13 , wherein the code for using the multivariate sequence activity model to identify one or more oligonucleotides for recombination comprises code for using the model to identify select one or more cross-overs. 
     
     
         15 . The computer program product of  claim 13 , wherein the code for using the multivariate sequence activity model to identify one or more oligonucleotides for recombination comprises code for using the model to extrapolate new sequences. 
     
     
         16 . The computer program product of  claim 13 , wherein the code for generating the multivariate sequence activity model comprises code for performing PLS. 
     
     
         17 . The computer program product of  claim 13 , wherein the multivariate sequence activity model is a neural network. 
     
     
         18 . The computer program product of  claim 13 , wherein code for using the multivariate sequence activity model comprise code for identifying a plurality of overlapping oligonucleotides. 
     
     
         19 . The computer program product of  claim 13 , wherein code for using the multivariate sequence activity model comprise code for identifying one or more oligonucleotides between about 20 and about 60 nucleotides in length.