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In vitro assembly of multiple DNA fragments using successive hybridization.

ABSTRACT: Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditional methods, SHA eliminates the need for restriction enzymes, DNA ligases and recombinases, and is sequence-independent. We first demonstrated its feasibility by constructing plasmids from 4, 6 and 8 fragments with high efficiencies, and then applied it to constructing a customized vector and two artificial pathways. As SHA is robust, easy to use and can tolerate repeat sequences, we expect it to be a powerful tool in synthetic biology.

SUBMITTER: Jiang X

PROVIDER: S-EPMC3266897 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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In vitro assembly of multiple DNA fragments using successive hybridization.

Jiang Xinglin X Yang Jianming J Zhang Haibo H Zou Huibin H Wang Cong C Xian Mo M

PloS one 20120126 1

Construction of recombinant DNA from multiple fragments is widely required in molecular biology, especially for synthetic biology purposes. Here we describe a new method, successive hybridization assembling (SHA) which can rapidly do this in a single reaction in vitro. In SHA, DNA fragments are prepared to overlap one after another, so after simple denaturation-renaturation treatment they hybridize in a successive manner and thereby assemble into a recombinant molecule. In contrast to traditiona ...[more]

PMID: 22291927

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