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 - 46 . (canceled)  
     
     
         47 . A method for automated synthesis of a target polynucleotide sequence, comprising: a) providing a user with an opportunity to communicate a desired target polynucleotide sequence; b) allowing the user to transmit the desired target polynucleotide sequence to a server; c) providing the user with a unique designation; d) obtaining the transmitted target polynucleotide sequence provided by the user.  
     
     
         48 . The method of  claim 47 , further comprising: f) identifying at least one initiating polynucleotide present in the target polynucleotide of e), 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; g) identifying a second polynucleotide present in the target polynucleotide of e), wherein the second polynucleotide is contiguous with the initiating polynucleotide 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 polynucleotide; h) identifying a third polynucleotide present in the target polynucleotide of e), 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 polynucleotide which is not complementary to an overhang of the second polynucleotide; i) contacting the initiating polynucleotide of f) with the second polynucleotide of g) and the third polynucleotide of h) 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; j) in the absence of primer extension, optionally contacting the mixture of i) with a ligase under conditions suitable for ligation; and k) optionally repeating f) through k) to sequentially add double-stranded polynucleotides to the extended initiating polynucleotide through repeated cycles of annealing and ligation, whereby a target polynucleotide is synthesized.  
     
     
         49 . The method of  claim 47 , further comprising: f) identifying at least one initiating polynucleotide present in the target polynucleotide of e), wherein the initiating polynucleotide comprises at least one plus strand oligonucleotide annealed to at least one minus strand oligonucleotide; g) contacting the initiating polynucleotide under conditions suitable for primer annealing with a first oligonucleotide having partial complementarity to the 3′ portion of the plus strand of the initiating polynucleotide, and a second oligonucleotide having partial complementarity to the 3′ portion of the minus strand of the initiating polynucleotide; h) catalyzing under conditions suitable for primer extension: 1) polynucleotide synthesis from the 3′-hydroxyl of the plus strand of the initiating polynucleotide; 2) polynucleotide synthesis from the 3′-hydroxyl of the annealed first oligonucleotide; 3) polynucleotide synthesis from the 3′-hydroxyl of the minus strand of the initiating polynucleotide; and 4) polynucleotide synthesis from the 3′-hydroxyl of the annealed second oligonucleotide, wherein the initiating sequence is extended bi-directionally thereby forming a nascent extended initiating polynucleotide; i) contacting the extended initiating polynucleotide of h) under conditions suitable for primer annealing with a third oligonucleotide having partial complementarity to the 3′ portion of the plus strand of the extended initiating polynucleotide, and a fourth oligonucleotide having partial complementarity to the 3′ portion of the minus strand of the extended initiating polynucleotide; j) catalyzing under conditions suitable for primer extension: 1) polynucleotide synthesis from the 3′-hydroxyl of the plus strand of the extended initiating polynucleotide; 2) polynucleotide synthesis from the 3′-hydroxyl of the annealed third oligonucleotide; 3) polynucleotide synthesis from the 3′-hydroxyl of the minus strand of the extended initiating polynucleotide; and 4) polynucleotide synthesis from the 3′-hydroxyl of the annealed fourth oligonucleotide, wherein the extended initiating sequence is extended bi-directionally thereby forming a nascent extended initiating polynucleotide; and k) optionally repeating f) through j) as desired, resulting in formation of the target polynucleotide sequence.  
     
     
         50 . A method for automated synthesis of a polynucleotide, comprising: a) providing a user with a mechanism for communicating a model polynucleotide sequence; b) optionally providing the user with an opportunity to communicate at least one desired modification to the model sequence if desired; c) allowing the user to transmit the model sequence and desired modification to a server; d) providing user with a unique designation; e) obtaining the transmitted model sequence and desired modification provided by the user; f) inputting into a programmed computer, through an input device, data including at least a portion of the model polynucleotide sequence; g) determining, using the processor, the sequence of the model polynucleotide sequence containing the desired modification; h) further determining, using the processor, at least one initiating polynucleotide sequence present in the model polynucleotide sequence i) selecting, using the processor, a model for synthesizing the modified model polynucleotide sequence based on the position of the initiating sequence in the model polynucleotide sequence; and j) outputting, to the output device, the results of the at least one determination.  
     
     
         51 - 52 . (canceled)