The present invention is directed to compositions and methods for producing one or more polynucleotides from smaller oligonucleotide segments within an emulsion. In methods of the present invention, a support having one or more capture oligo-nucleotides is contacted with two or more corresponding tile oligonucleotides. Upon hybridization of the tile oligonucleotides to the capture oligonucleotides, a capture complex is formed. This capture complex is emulsified, optionally with reaction reagents or other additives. The emulsion is then incubated at a temperature regimen sufficient for an adjoining extension reaction to occur, such that a polynucleotide may be formed from the tile oligonucleotides that hybridized to a particular support. A particular advantage of this method is that many different polynucleotides may be produced in parallel with surprising efficiency.

The present invention is directed to compositions and methods for producing one or more polynucleotides from smaller oligonucleotide segments within an emulsion. In methods of the present invention, a support having one or more capture oligo-nucleotides is contacted with two or more corresponding tile oligonucleotides. Upon hybridization of the tile oligonucleotides to the capture oligonucleotides, a capture complex is formed. This capture complex is emulsified, optionally with reaction reagents or other additives. The emulsion is then incubated at a temperature regimen sufficient for an adjoining extension reaction to occur, such that a polynucleotide may be formed from the tile oligonucleotides that hybridized to a particular support. A particular advantage of this method is that many different polynucleotides may be produced in parallel with surprising efficiency.

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Formation of a modified oligonucleotide by treating four or more oligonucleotide raw material fragments in total in the presence of an oligonucleotide ligase; the four or more oligonucleotide raw material fragments in total corresponding to oligonucleotide raw material fragments that are obtained by dividing the modified oligonucleotide at a fragment linking site that satisfies following conditions (i) to (v): (i) one or more fragment linking sites are present in the complementary portion in each strand side, and two or more fragment linking sites in total are present in the modified oligonucleotide; (ii) when the modified oligonucleotide is divided at the fragment linking site, a sticky end is formed in the complementary portion, in which the sticky end has 1 to 10 nucleotide length; (iii) at least one oligonucleotide raw material fragment has a modified nucleotide; (iv) four oligonucleotide raw material fragments out of the four or more oligonucleotide raw material fragments in total include the complementary portion having 5 to 25 nucleotide length; and (v) total nucleotide length of the oligonucleotide raw material fragments corresponding to the complementary portions in each strand side is 11 to 27, is useful for efficiently producing an oligonucleotide comprising a complementary portion, such as an siRNA and a heteroduplex oligonucleotide.

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

Methods and apparatus of some aspects of the invention relate to the synthesis of high fidelity polynucleotides. In particular, aspects of the invention relate to concurrent enzymatic removal of amplification sequences and ligation of processed oligonucleotides into nucleic acid assemblies. According to some embodiments, the invention provides a method for producing a target nucleic acid having a predefined sequence. In some embodiments, the method comprises the step of providing a plurality of oligonucleotides, wherein each oligonucleotides comprises (i) an internal sequence identical to a different portion of a sequence of a target nucleic acid, (ii) a 5′ sequence flanking the 5′ end of the internal sequence and a 3′ flanking sequence flanking the 3′ end of the internal sequence, each of the flanking sequence comprising a primer recognition site for a primer pair and a restriction enzyme recognition site.

The disclosure generally relates to compositions and methods for the production of nucleic acid molecules. In some aspects, the invention allows for the microscale generation of nucleic acid molecules, optionally followed by assembly of these nucleic acid molecules into larger molecules. In some aspects, the invention allows for efficient production of nucleic acid molecules (e.g., large nucleic acid molecules such as genomes).

Aspects of the invention relate to methods, compositions and algorithms for designing and producing a target nucleic acid. The method can include: (1) providing a plurality of blunt-end double-stranded nucleic acid fragments having a restriction enzyme recognition sequence at both ends thereof; (2) producing via enzymatic digestion a plurality of cohesive-end double-stranded nucleic acid fragments each having two different and non-complementary overhangs; (3) ligating the plurality of cohesive-end double-stranded nucleic acid fragments with a ligase; and (4) forming a linear arrangement of the plurality of cohesive-end double-stranded nucleic acid fragments, wherein the unique arrangement comprises the target nucleic acid. In certain embodiments, the plurality of blunt-end double-stranded nucleic acid fragments can be provided by: releasing a plurality of oligonucleotides synthesized on a solid support; and synthesizing complementary strands of the plurality of oligonucleotides using a polymerase based reaction.

Aspects of the invention relate to methods, compositions for designing and producing a target nucleic acid. In particular, aspects of the invention relate to the multiplex synthesis of target polynucleotides.

Compositions and methods are provided herein for high throughput synthesis and assembly of nucleic acid molecules. Specific aspects include methods and rules for designing, grouping and pooling of nucleic acid molecules for efficient multiplex assembly, amplification, processing and analysis to obtain error-free assembly products. Provided compositions and methods allow for miniaturization, parallelization, high throughput production and scaling, and cost reduction in gene synthesis workflows.