Computer-directed assembly of a polynucleotide encoding a target polypeptide

Abstract

The present invention outlines a novel approach to utilizing the results of genomic sequence information by computer-directed polynucleotide assembly based upon information available in databases such as the human genome database. Specifically, the present invention may be used to select, synthesize and assemble a novel, synthetic target polynucleotide sequence encoding a target polypeptide. The target polynucleotide may encode a target polypeptide that exhibits enhanced or altered biological activity as compared to a model polypeptide encoded by a natural (wild-type) or model polynucleotide sequence.

1 - 38 . (cancelled)  
     
     
         39 . A method of synthesizing a target polynucleotide comprising: a) providing a target polynucleotide sequence derived from a model sequence; b) chemically synthesizing a plurality of single-stranded oligonucleotides each of which is partially complementary to at least one oligonucleotide present in the plurality, wherein the sequence of the plurality of oligonucleotides is a contiguous sequence of the target polynucleotide; c) contacting the partially complementary oligonucleotides of b) under conditions and for such time suitable for annealing, the contacting resulting in a plurality of partially double-stranded polynucleotides, wherein each double-stranded polynucleotide is comprised of a 5′ overhang and a 3′ overhang; d) identifying at least one initiating polynucleotide derived from the model sequence, wherein the initiating polynucleotide is present in the plurality of double-stranded polynucleotides set forth in c); e) in the absence of primer extension, subjecting a mixture comprising the initiating polynucleotide and 1) a double-stranded polynucleotide that will anneal to the 5′ portion of said initiating and sequence; 2) a double-stranded polynucleotide that will anneal to the 3′ portion of the initiating polynucleotide; and 3) a DNA ligase under conditions suitable for annealing and ligation, wherein the initiating polynucleotide is extended bi-directionally; f) sequentially annealing double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing, whereby the target polynucleotide is produced.  
     
     
         40 . The method of  claim 39 , wherein the oligonucleotides are produced by synthesis on an automated DNA synthesizer.  
     
     
         41 - 52 . (cancelled)