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

Abstract:

: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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Liang J,Liu Z,Low XZ,Ang EL,Zhao H

doi

10.1093/nar/gkx132

subject

Has Abstract

pub_date

2017-06-20 00:00:00

pages

e94

issue

11

eissn

0305-1048

issn

1362-4962

pii

3045848

journal_volume

45

pub_type

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