Methods and Devices for In Situ Nucleic Acid Synthesis

Abstract

Disclosed are compositions, methods and devices for the in situ synthesis of nucleic acids. In an exemplary embodiment, a support-bound oligonucleotide is elongated by addition of one or more nucleotides by hybridization of a partially double-stranded oligonucleotide, ligation and removal of unwanted nucleotides.

1 . A composition for synthesizing a plurality of target nucleic acids on a surface of a solid support, the composition comprising a plurality of partially double-stranded oligonucleotides, each having a first strand and a second strand such that each partially double-stranded oligonucleotide comprises a double-stranded portion and a 5′ overhang on the second strand, wherein the double-stranded portion comprises at least one predetermined addition nucleotide at the 3′ end of the first strand, and wherein the double-stranded portion has a predefined sequence that is identical across the plurality of partially double-stranded oligonucleotides. 
     
     
         2 . The composition of  claim 1  wherein the double-stranded portion comprises a restriction enzyme binding site. 
     
     
         3 . The composition of  claim 1  wherein the shorter strand comprises at least one ribonucleotide upstream of the at least one predetermined addition nucleotide. 
     
     
         4 . The composition of  claim 1  wherein the 5′ overhang comprises a degenerate sequence. 
     
     
         5 . The composition of  claim 1 , further comprising a support having a plurality of features, each feature comprising a plurality of single-stranded support-bound oligonucleotides that can be ligated with the partially double-stranded oligonucleotides at the 3′ end of the predetermined addition nucleotide. 
     
     
         6 . The composition of  claim 5 , wherein the plurality of single-stranded support-bound oligonucleotides each have sequence complementarity to the 5′ overhang. 
     
     
         7 . The composition of  claim 5 , further comprising an ink jet programmed to provide the partially double-stranded oligonucleotides at a select feature. 
     
     
         8 . A composition for synthesizing a plurality of target nucleic acids on a surface of a solid support, the composition comprising:
 a support having a plurality of features, each feature comprising a plurality of single-stranded support-bound oligonucleotides; and   a first partially double-stranded oligonucleotide comprising a first strand and a second strand forming a double-stranded portion and a 5′ overhang on the second strand, wherein the first strand comprises a first predetermined ligatable addition nucleotide at the 3′ end;   wherein the first partially double-stranded oligonucleotide can be ligated to a first support-bound oligonucleotide at the first predetermined ligatable addition nucleotide, thereby generating a first ligation product comprising the first predetermined addition nucleotide; and wherein the first ligation product is cleavable thereby generating a first elongated support-bound oligonucleotide comprising the first predetermined addition nucleotide.   
     
     
         9 . The composition of  claim 8  further comprising a second partially double-stranded oligonucleotide comprising a second predetermined ligatable addition nucleotide. 
     
     
         10 . The composition of  claim 9  wherein the first and second partially double-stranded oligonucleotides comprise in the double-stranded portion an identical predefined sequence. 
     
     
         11 . The composition of  claim 8  wherein the double-stranded portion comprises a restriction enzyme binding site. 
     
     
         12 . The composition of  claim 8  wherein the first ligation product is cleavable by a restriction enzyme. 
     
     
         13 . The composition of  claim 8  wherein the shorter strand comprises at least one ribonucleotide upstream of the first predetermined ligatable addition nucleotide. 
     
     
         14 . The composition of  claim 13  wherein the first ligation product is cleavable by an RNase. 
     
     
         15 . The composition of  claim 8  wherein the 5′ overhang comprises a degenerate sequence. 
     
     
         16 . The composition of  claim 8 , wherein the plurality of single-stranded support-bound oligonucleotides each have sequence complementarity to the 5′ overhang. 
     
     
         17 . The composition of  claim 8 , further comprising an ink jet programmed to provide the first partially double-stranded oligonucleotide at a select feature. 
     
     
         18 . The composition of  claim 8  wherein the first partially double-stranded oligonucleotide is prepared by hybridizing the first strand to the second strand, wherein the first strand comprises at its 5′ end a predefined sequence and at its 3′ end the first predetermined ligatable addition nucleotide, and wherein the second strand comprises at its 3′ end a sequence complementary to the predefined sequence and a nucleotide complementary to the first predetermined ligatable addition nucleotide. 
     
     
         19 . The composition of  claim 8  wherein the first partially double-stranded oligonucleotide comprises a detectable label. 
     
     
         20 . The composition of  claim 8  further comprising an imaging system.