Computer-directed assembly of a polynucleotide encoding a target polypetide

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 - 27 . (canceled)  
     
     
         28 . A method for isolating a target polypeptide encoded by a target polynucleotide, comprising: a) providing a target polynucleotide sequence derived from a model sequence; b) identifying at least one initiating polynucleotide present in the target polynucleotide of a), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′ overhang and a 3′ overhang; c) identifying a second polynucleotide present in the target polynucleotide of a), wherein the second polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′ overhang, a 3′ overhang, or a 5′ overhang and a 3′ overhang, wherein at least one overhang of the second polynucleotide is complementary to at least one overhang of the initiating sequence; d) identifying a third polynucleotide present in the target polynucleotide of a), wherein the third polynucleotide is contiguous with the initiating sequence and comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide resulting in a partially double-stranded polynucleotide comprised of a 5′ overhang, a 3′ overhang, or a 5′ overhang and a 3′ overhang, wherein at least one overhang of the third polynucleotide is complementary to at least one overhang of the initiating sequence which is not complementary to an overhang of the second polynucleotide; e) contacting the initiating polynucleotide of b) with the second polynucleotide of c) and the third polynucleotide of d) under conditions and for such time suitable for annealing, the contacting resulting in a contiguous double-stranded polynucleotide, wherein the initiating sequence is extended bi-directionally; f) in the absence of primer extension, optionally contacting the mixture of e) with a ligase under conditions suitable for ligation; g) optionally repeating b) through f) to sequentially add double-stranded polynucleotides to the extended initiating sequence through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized; h) incorporating the target polynucleotide of g) in an expression vector; i) introducing the expression vector of h) into a suitable host cell; j) culturing the cell of i) under conditions and for such time as to promote the expression of the target polypeptide encoded by the target polynucleotide; and k) isolating the target polypeptide.  
     
     
         29 . The method of  claim 28 , wherein the target polypeptide is a chimeric protein.  
     
     
         30 . The method of  claim 28 , wherein the target polypeptide is a fusion protein.  
     
     
         31 . The method of  claim 28 , wherein the expression vector is a bacterial expression vector.  
     
     
         32 . The method of  claim 29 , wherein the expression vector is an animal cell expression vector.  
     
     
         33 . The method of  claim 28 , wherein the expression vector is an insect cell expression vector.  
     
     
         34 . The method of  claim 28 , wherein the expression vector is a retroviral vector.  
     
     
         35 . The method of  claim 29 , wherein the expression vector is contained in a host cell.  
     
     
         36 . The method of  claim 35 , wherein the host cell is a prokaryotic cell.  
     
     
         37 . The method of  claim 35 , wherein the host cell is a eukaryotic cell.  
     
     
         38 - 52 . (canceled)