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In Vitro Assembly of Multiple DNA Fragments Using Successive Hybridization

Authors
Journal
PLoS ONE
1932-6203
Publisher
Public Library of Science
Publication Date
Volume
7
Issue
1
Identifiers
DOI: 10.1371/journal.pone.0030267
Keywords
  • Research Article
  • Biology
  • Biochemistry
  • Enzymes
  • Metabolism
  • Nucleic Acids
  • Biotechnology
  • Genetics
  • Microbiology
  • Synthetic Biology
Disciplines
  • Biology

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.

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