Current State-of-the-Art

Oligonucleotides are assembled into double-stranded DNA fragments up to 6000 base pairs long using in vitro techniques (e.g., polymerase cycling assembly, ligation cycling) as well as in vivo techniques (yeast-mediated homologous recombination), producing non-clonal DNA fragments.¹Li, M. Z., & Elledge, S. J. (2007). Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods, 4(3), 251–256. View publication.

Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. View publication.

Smith, H. O., Hutchison, C. A., Pfannkoch, C., & Venter, J. C. (2003). Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15440–15445. View publication.

Richardson, S. M., Mitchell, L. A., Stracquadanio, G., Yang, K., Dymond, J. S., DiCarlo, J. E., … Bader, J. S. (2017). Design of a synthetic yeast genome. Science, 355(6329), 1040–1044. View publication. Clonal (isogenic) fragments are then identified using a combination of enzyme-based removal of mismatched base pairs (e.g., MutS) and DNA sequencing (Sanger or NGS). Multiple verified DNA fragments are then assembled together into longer fragments (10,000 to 100,000 base pairs long) using hierarchical approaches employing DNA assembly techniques (e.g., Gibson assembly, ligation cycling reaction, Golden Gate). Megabase length DNA is then assembled from 100,000 base pair fragments using yeast-mediated homologous recombination. More detailed descriptions of commonly used techniques follow:

Polymerase Cycling Assembly (PCA) is a method to assemble larger DNA constructs from shorter oligonucleotides.²Smith, H. O., Hutchison, C. A., Pfannkoch, C., & Venter, J. C. (2003). Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15440–15445. View publication. PCA is an efficient method for assembling constructs between 200 to 1,000 base pairs in length. The process is similar to PCR, but utilizes a set of overlapping “seed” oligonucleotides that are designed to hybridize to one another leaving gaps that are then filled in using a thermostable DNA polymerase. The oligonucleotides are generally 50 to 100 nucleotides in length to ensure uniqueness in the hybridization with their complement. The reactions are cycled from ~60 and ~95 Co for 15 to 30 cycles. The full-length assembled product is then usually amplified by PCR using two terminal-specific primers. PCA is an efficient method for assembling constructs between 200 and 1,000 base pairs in length and can be performed in individual tubes or multiplexed using microtiter well plates.

Emulsion PCA is a method developed by Sriram Kosuri for highly multiplexing the assembly of larger constructs from small amounts of shorter DNA fragments.³Plesa, C., Sidore, A. M., Lubock, N. B., Zhang, D., & Kosuri, S. (2018). Multiplexed gene synthesis in emulsions for exploring protein functional landscapes. Science, 359(6373), 343–347. View publication. In this method, the oligos required for a given construct are designed with a unique barcode on the terminus which specifically hybridizes with a complementary barcoded attached to a bead from a complex pool of oligonucleotides. The bead mixture is then emulsified into picoliter-sized droplets containing a Type IIs restriction endonuclease (RE), dNTPs, and a thermostable DNA polymerase. The oligonucleotides are released from the bead by the Type II RE and then assembled by PCA through thermal cycling of the emulsion. Using this method, thousands of specific constructs can be assembled in a single emulsion tube depending upon the number uniquely barcoded beads.

Ligase Cycling Assembly (LCA) is a method to assemble larger DNA constructs from shorter oligonucleotides or double-stranded DNA fragments.⁴de Kok, S., Stanton, L. H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., … Chandran, S. S. (2014). Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synthetic Biology [Electronic Resource], 3(2), 97–106. View publication. LCA is an efficient method for assembling constructs between 500 and 10,000 base pairs in length. LCA assembly uses shorter, single-stranded bridging oligonucleotides that are complementary to the termini of adjacent DNA fragments that are to be joined using a thermostable ligase. Like PCA, LCA utilizes multiple temperature cycling to denature, re-anneal, and then ligate the fragments to assemble the larger DNA construct, and can be performed in individual tubes or multiplexed using microtiter well plates.

Gibson Assembly is a method to assemble larger DNA constructs from shorter oligonucleotides or double-stranded DNA fragments.⁵Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. View publication. Gibson assembly is an efficient method for assembling constructs up to many tens of kilobase-pairs in length. This method, which is isothermal, utilizes up to 15 double-stranded DNA fragments having around 20 to 40 base pair overlaps with the adjacent DNA fragments. The DNA fragments are first incubated with 5’ to 3’ exonuclease, resulting in single-stranded regions on the adjacent DNA fragments that can anneal in a base pair-specific manner. The gaps are then filled in with a DNA polymerase and the final nicks closed with a DNA ligase. This method can be performed in individual tubes or multiplexed using microtiter well plates.

Importantly, the final fidelity (error rate) of the assembled constructs using the above methods are at the mercy of the quality of the input oligonucleotides. These methods usually incorporate some type of error reduction or correction methods which include removing errored duplexes (mismatches and insertions) with the MutS protein after denaturation and reannealing of the construct, or degradation of the error containing DNA using T7 or CEL endonuclease. (For review please see: Ma, S., Saaem, I., & Tian, J. (2012). Error correction in gene synthesis technology. Trends in Biotechnology, 30(3), 147–154. View publication.)

Breakthrough Capabilities & Milestones

Predictive design of DNA sequences for improved assembly of longer, more information-rich DNA fragments.

Methods for one-step, simultaneous assembly and sequence-verification of long DNA fragments.

Pipelined synthesis, assembly, and functional testing of engineered genetic systems.

Footnotes

1. Li, M. Z., & Elledge, S. J. (2007). Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC. Nature Methods, 4(3), 251–256. View publication.; Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. View publication.; Smith, H. O., Hutchison, C. A., Pfannkoch, C., & Venter, J. C. (2003). Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15440–15445. View publication.; Richardson, S. M., Mitchell, L. A., Stracquadanio, G., Yang, K., Dymond, J. S., DiCarlo, J. E., … Bader, J. S. (2017). Design of a synthetic yeast genome. Science, 355(6329), 1040–1044. View publication.
2. Smith, H. O., Hutchison, C. A., Pfannkoch, C., & Venter, J. C. (2003). Generating a synthetic genome by whole genome assembly: phiX174 bacteriophage from synthetic oligonucleotides. Proceedings of the National Academy of Sciences of the United States of America, 100(26), 15440–15445. View publication.
3. Plesa, C., Sidore, A. M., Lubock, N. B., Zhang, D., & Kosuri, S. (2018). Multiplexed gene synthesis in emulsions for exploring protein functional landscapes. Science, 359(6373), 343–347. View publication.
4. de Kok, S., Stanton, L. H., Slaby, T., Durot, M., Holmes, V. F., Patel, K. G., … Chandran, S. S. (2014). Rapid and reliable DNA assembly via ligase cycling reaction. ACS Synthetic Biology [Electronic Resource], 3(2), 97–106. View publication.
5. Gibson, D. G., Young, L., Chuang, R.-Y., Venter, J. C., Hutchison, C. A., & Smith, H. O. (2009). Enzymatic assembly of DNA molecules up to several hundred kilobases. Nature Methods, 6(5), 343–345. View publication.

Ma, S., Saaem, I., & Tian, J. (2012). Error correction in gene synthesis technology. Trends in Biotechnology, 30(3), 147–154. View publication.