Combined automated parallel synthesis of polynucleotide variants

Abstract

The present disclosure relates to methods for efficient synthesis, cloning, transformation and screening of large diverse libraries of polynucleotide variants comprising well-defined nucleotide differences relative to a reference polynucleotide.

What is claimed is: 
     
         1 . A method of screening an addressable library of polynucleotide variants encoding polypeptides, the method comprising:
 (a) combining members of the addressable library of polynucleotide variants into a pool;   (b) cloning the pool of polynucleotide variants into an expression vector;   (c) transforming cells with the expression vector;   (d) plating the transformed cells to generate a plurality of separate clones comprising the library of polynucleotide variants; and   (e) screening the clones for an improved property relative to a reference polypeptide.   
     
     
         2 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 200 different polynucleotide variants. 
     
     
         3 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 1000 different polynucleotide variants. 
     
     
         4 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 200 different polynucleotide variants each comprising a different defined nucleotide difference at one of at least 10 different selected positions. 
     
     
         5 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 400 different polynucleotide variants each comprising a different defined nucleotide difference at one of at least 20 different selected positions. 
     
     
         6 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 800 different polynucleotide variants each comprising a different defined nucleotide difference at one of at least 40 different selected positions. 
     
     
         7 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 46 different polynucleotide variants each comprising one of 23 different codons at one of 2 different selected positions. 
     
     
         8 . The method of  claim 7 , wherein at least 75% of the addressable library of polynucleotide variants comprise the correct sequence. 
     
     
         9 . The method of  claim 1 , wherein the addressable library of polynucleotide variants comprises at least 460 different polynucleotide variants each comprising one of 23 different codons at one of 20 different selected positions. 
     
     
         10 . The method of  claim 9 , wherein at least 75% of the addressable library of polynucleotide variants comprise the correct sequence.