Microarray synthesis and assembly of gene-length polynucleotides

Abstract

There is disclosed a process for in vitro synthesis and assembly of long, gene-length polynucleotides based upon assembly of multiple shorter oligonucleotides synthesized in situ on a microarray platform. Specifically, there is disclosed a process for in situ synthesis of oligonucleotide fragments on a solid phase microarray platform and subsequent, “on device” assembly of larger polynucleotides composed of a plurality of shorter oligonucleotide fragments.

1 . (canceled) 
     
     
         2 . A method for producing a polynucleotide comprising a target sequence, the method comprising:
 (a) providing a plurality of double-stranded oligonucleotides, each double-stranded oligonucleotide comprising:
 (i) an internal sequence identical to a portion of the target sequence, wherein the internal sequence, at one or both ends, comprises an overlapping sequence region that:
 is identical to an overlapping sequence region of another double-stranded oligonucleotide in the plurality of double-stranded oligonucleotides; and 
 comprises a Type II restriction endonuclease cleavage site; and 
 
 (ii) one or more flanking sequences, wherein each flanking sequence comprises a Type II restriction endonuclease recognition site corresponding to the Type II restriction endonuclease cleavage site of the internal sequence; 
   (b) digesting the plurality of double-stranded oligonucleotides with a Type II restriction endonuclease that recognizes the Type II restriction endonuclease recognition site; and   (c) ligating the digested oligonucleotides to produce the polynucleotide comprising the target sequence.   
     
     
         3 . The method of  claim 2 , wherein each double-stranded oligonucleotide in (a) comprises a flanking sequence at each end. 
     
     
         4 . The method of  claim 2 , wherein the plurality of double-stranded oligonucleotides are digested in (b) with a Type IIS restriction endonuclease. 
     
     
         5 . The method of  claim 2 , wherein the Type IIS restriction endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI, BmrI, BtsI, and Fok1. 
     
     
         6 . The method of  claim 2 , wherein the method does not comprise PCR amplification prior to step (c). 
     
     
         7 . The method of  claim 2 , further comprising:
 purifying the digested oligonucleotides prior to ligation in (c);   amplifying the polynucleotide comprising the target sequence; or   a combination thereof.   
     
     
         8 . A method for producing a polynucleotide comprising a target sequence, the method comprising:
 (a) synthesizing on a surface a plurality of single-stranded oligonucleotides, each single-stranded oligonucleotide comprising:
 (i) an internal sequence identical to or complementary to a portion of the target sequence, wherein the internal sequence, at one or both ends, comprises an overlapping sequence region that is identical to or complementary to an overlapping sequence region of another single-stranded oligonucleotide in the plurality of single-stranded oligonucleotides; and 
 (ii) flanking sequences, one at each end of the internal sequence, each flanking sequence comprising a primer binding site, and at least one flanking sequence further comprising a Type II restriction endonuclease recognition site; 
   (b) amplifying the plurality of single-stranded oligonucleotides;   (c) digesting the amplified oligonucleotides with a Type II restriction endonuclease that recognizes the Type II restriction endonuclease recognition site; and   (d) ligating the digested oligonucleotides to produce the polynucleotide comprising the target sequence.   
     
     
         9 . The method of  claim 8 , wherein each single-stranded oligonucleotide in the plurality of single-stranded oligonucleotides comprises flanking sequences, one at each end of the internal sequence, each flanking sequence comprising: (i) a primer binding site; and (ii) a Type II restriction endonuclease recognition site. 
     
     
         10 . The method of  claim 8 , wherein the plurality of single-stranded oligonucleotides has been synthesized on a surface. 
     
     
         11 . The method of  claim 10 , wherein the surface is a microarray or wherein the surface comprises beads. 
     
     
         12 . The method of  claim 8 , wherein the amplified oligonucleotides are digested in (c) with a Type IIS restriction endonuclease. 
     
     
         13 . The method of  claim 12 , wherein the Type IIS restriction endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI, BmrI, BtsI, and Fok1. 
     
     
         14 . The method of  claim 8 , further comprising:
 purifying the digested oligonucleotides prior to ligation in (d);   amplifying the polynucleotide comprising the target sequence; or   a combination thereof.   
     
     
         15 . A method for producing a polynucleotide comprising a target sequence, the method comprising:
 (a) providing a plurality of oligonucleotides, each oligonucleotide comprising:
 (i) an internal sequence identical to or complementary to a portion of the target sequence, wherein the internal sequence, at one or both ends, comprises an overlapping sequence region that is identical to or complementary to an overlapping sequence region of another oligonucleotide in the plurality of oligonucleotides; and 
 (ii) flanking sequences, one at each end of the internal sequence, each flanking sequence comprising a primer binding site, and at least one flanking sequence further comprising a Type II restriction endonuclease recognition site; 
   (b) amplifying the plurality of oligonucleotides;   (c) digesting the amplified oligonucleotides with a restriction endonuclease that recognizes the Type II restriction endonuclease recognition site; and   (d) ligating the digested oligonucleotides to produce the polynucleotide comprising the target sequence.   
     
     
         16 . The method of  claim 15 , wherein each oligonucleotide in the plurality of oligonucleotides comprises flanking sequences, one at each end of the internal sequence, each flanking sequence comprising: (i) a primer binding site; and (ii) a Type IIS restriction endonuclease recognition site. 
     
     
         17 . The method of  claim 15 , wherein the plurality of oligonucleotides has been synthesized on a surface. 
     
     
         18 . The method of  claim 17 , wherein the surface is a microarray or comprises beads. 
     
     
         19 . The method of  claim 15 , further comprising:
 purifying the digested oligonucleotides prior to ligation in (d);   amplifying the polynucleotide comprising the target sequence; or   a combination thereof.   
     
     
         20 . The method of  claim 15 , wherein the amplified oligonucleotides are digested in (c) with a Type IIS restriction endonuclease. 
     
     
         21 . The method of  claim 20 , wherein the Type IIS restriction endonuclease includes at least one of MylI, BspMI, BsaXI, BsrI, BmrI, BtsI, and Fok1.