Compositions, methods and apparatus for oligonucleotides synthesis

Abstract

Aspects of the invention relate to methods, compositions for synthesizing oligonucleotides having a predefined sequence.

1 - 17 . (canceled) 
     
     
         18 . A method of parsing a target nucleic acid sequence to obtain nucleic acid sequences for a plurality of construction oligonucleotides, the method comprising:
 receiving, by a processor of a computing device, a sequence of the target nucleic acid;   applying, by the processor of the computing device, one or more parameters to the sequence of the target nucleic acid, wherein the one or more parameters is/are maximum length of a payload of a fragment, minimum length of a payload of a fragment, maximum length of a payload of each construction oligonucleotide, minimum length of a payload of each construction oligonucleotide, number of junctions, length of a junction, sequence of each junction, number of fragments, length of a fragment, sequence of each fragment, maximum GC content of each fragment, maximum GC content of each construction oligonucleotide, and mean junction difference;   generating, by the processor of the computing device, a parse of the sequence of the nucleic acid, wherein the parse comprises nucleic acid sequences for a plurality of construction oligonucleotides that together comprise the target nucleic acid sequence, and   displaying, by the processor of the computing device, the parse on a monitor or display.   
     
     
         19 . The method of  claim 18 , comprising generating a plurality of alternative parses of the target nucleic acid sequence, wherein the sequence of at least one construction oligonucleotide differs from a first parse to a second parse of the plurality of alternative parses. 
     
     
         20 . The method of  claim 18 , wherein the plurality of construction oligonucleotides together forms two or more fragments, and wherein the two or more fragments together form the target nucleic acid sequence. 
     
     
         21 . The method of  claim 19 , further comprising selecting a parse from the plurality of alternative parses. 
     
     
         22 . The method of  claim 21 , wherein the selected parse comprises the smallest number of fragments compared to the number of fragments formed using any of the other parses. 
     
     
         23 . The method of  claim 21 , wherein the selected parse comprises the smallest number of junctions compared to the number of junctions formed using any of the other parses. 
     
     
         24 . The method of  claim 18 , wherein the generating step comprises:
 analyzing the construction nucleotides of the parse for one or more interfering sequences, wherein the one or more interfering sequences is/are a sequence with low GC content, a sequence that forms a secondary structure, and a sequence comprising a restriction enzyme recognition site.   
     
     
         25 . The method of  claim 24 , wherein the sequence with low GC content comprises less than 30% GC over a length of 10 bases. 
     
     
         26 . The method of  claim 24 , wherein the secondary structure is a stem-loop structure. 
     
     
         27 . The method of  claim 18 , further comprising adding, by the processor of the computing device, padding sequences to the sequence of one or more construction oligonucleotides. 
     
     
         28 . The method of  claim 27 , wherein each padding sequence is of a different length from another padding sequence. 
     
     
         29 . The method of  claim 18 , further comprising adding, by the processor of a computing device, flanking sequences at the 3′ and/or 5′ end of the sequence of each of the construction oligonucleotides. 
     
     
         30 . The method of  claim 29 , wherein each flanking sequence comprises a primer recognition site and a restriction enzyme recognition site. 
     
     
         31 . The method of  claim 30 , wherein the restriction enzyme recognition site is a Type IIs restriction enzyme recognition site. 
     
     
         32 . The method of  claim 30 , wherein a padding sequence is positioned between the primer recognition site and the restriction enzyme recognition site within the  5 ′ flanking sequence. 
     
     
         33 . A method of assembling a target nucleic acid from a plurality of construction oligonucleotides, the method comprising:
 providing a plurality of construction oligonucleotides, wherein the plurality of construction oligonucleotides together comprise the target nucleic acid, and wherein the sequence of each of the plurality of construction oligonucleotides has been obtained by parsing the sequence of the target nucleic acid using one or more of the following parameters: maximum length of a payload of a fragment, minimum length of a payload of a fragment, maximum length of a payload of each construction oligonucleotide, minimum length of a payload of each construction oligonucleotide, number of junctions, length of a junction, sequence of each junction, number of fragments, length of a fragment, sequence of each fragment, maximum GC content of each fragment, maximum GC content of each construction oligonucleotide, and mean junction difference;   assembling the plurality of construction oligonucleotides into the target nucleic acid.   
     
     
         34 . The method of  claim 33 , wherein the parsing is performed by a processor of a computing device. 
     
     
         35 . The method of  claim 33 , wherein each fragment is longer than each construction oligonucleotide. 
     
     
         36 . The method of  claim 33 , wherein each of the construction oligonucleotides comprises a flanking sequence at the 3′ and/or 5′ end. 
     
     
         37 . The method of  claim 36 , wherein each flanking sequence comprises a primer recognition site and a restriction enzyme recognition site. 
     
     
         38 . The method of  claim 37 , wherein the restriction enzyme recognition site is a Type IIs restriction enzyme recognition site. 
     
     
         39 . The method of  claim 33 , further comprising amplifying the plurality of construction oligonucleotides to produce a plurality of amplified oligonucleotides. 
     
     
         40 . The method of  claim 39 , further comprising exposing the plurality of amplified oligonucleotides to a restriction enzyme under conditions suitable for digestion. 
     
     
         41 . The method of  claim 33 , further comprising subjecting the plurality of amplified oligonucleotides to error removal prior to or after exposing the plurality of amplified oligonucleotides to a restriction enzyme under conditions suitable for digestion. 
     
     
         42 . The method of  claim 39 , wherein the plurality of amplified oligonucleotides is contacted with a mismatch binding agent, wherein the mismatch binding agent selectively binds and cleaves double-stranded oligonucleotides comprising a mismatch. 
     
     
         43 . The method of  claim 33 , wherein the plurality of construction oligonucleotides is assembled into two or more fragments, and wherein the two or more fragments are assembled into the target nucleic acid.