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 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 ∼80% fidelity and a 31 kb plasmid from five fragments at ∼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.
Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore 138669, Singapore.
Jing
J
Liang
Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore 138669, Singapore.
Zihe
Z
Liu
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Xi Z
XZ
Low
Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore 138669, Singapore.
Ee L
EL
Ang
Metabolic Engineering Research Laboratory, Science and Engineering Institutes, Agency for Science, Technology and Research, Singapore 138669, Singapore.|NUS High School of Mathematics and Science, Singapore 129957, Singapore.
Huimin
H
Zhao
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics*; D004926:Escherichia coli; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction
D017931:DNA Primers