Twin-primer non-enzymatic DNA assembly: an efficient and accurate multi-part DNA assembly method

摘要

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 up-coming wave in molecular biological automation demands a rethinking of how we perform DNA assembly. To streamline automation workflow andminimize 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 polymerase chain reaction-amplified fragments into a plasmid without the use of enzymes. TPA is capable of assembling a 7 kb plasmid from 10 fragments at similar to 80% fidelity and a 31 kb plasmid from five fragments at similar to 50% fidelity. TPA cloning is scarless and sequence independent. 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.