Twin-Primer Non-Enzymatic DNA Assembly: An Efficient and Accurate Multi-Part DNA Assembly Method

DNA assembly forms the cornerstone of modern synthetic biology. Despite the numerous available methods, scarless multi-fragment assembly of large plasmids remains challenging. Furthermore, the upcoming wave in molecular biological automation demands a rethinking of how we perform DNA assembly. To streamline automation workflow and minimize operator intervention, a non-enzymatic assembly method is highly desirable. Here, we report the optimization and operationalization of a process called Twin-Primer Assembly (TPA), which is a method to assemble PCR-amplified fragments into a plasmid without the use of enzymes.

Each fragment used in TPA requires two PCR products, amplified separately using two sets of primers. One product has an overlap to the fragment that comes before and the other has an overlap to the fragment that comes after. The length of the overlap region is determined by the melting temperature (T_M) of the overlap region, and all overlaps used in an assembly are designed to have the same T_M. The two PCR products are mixed and fully denatured at high temperature before they are allowed to re-anneal slowly, generating intermediates that have overhangs on both sides. These intermediates are then allowed to hybridize at an elevated temperature to form a circular nicked plasmid. Upon E. coli transformation, the cells phosphorylate and ligate the nicks to create the final product plasmid.

The TPA non-enzymatic DNA assembly method is scarless and generally sequence independent. It is capable of assembling a 7 kb plasmid from 10 fragments at close to 80% fidelity, and a 31kb plasmid from 5 fragments at around 50% fidelity. Assembling more than 10 fragments is likely possible for constructing smaller plasmids, while assembling a plasmid larger than 31 kb is likely possible using fewer fragments. Even without the use of enzymes, the performance of TPA is on par with some of the best in vitro assembly methods currently available. TPA should be an invaluable addition to a synthetic biologist’s toolbox.