Self-assembly cloning: a rapid construction method for recombinant molecules from multiple fragments

Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructe...

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Bibliographic Details
Published inBioTechniques Vol. 51; no. 1; pp. 55 - 56
Main Authors Matsumoto, Akira, Itoh, Taichi Q
Format Journal Article
LanguageEnglish
Published Natick, MA Future Science Ltd 01.07.2011
Eaton
Subjects
Animals
Base Pairing
Biological and medical sciences
Cell Line
Cloning, Molecular - methods
Diverse techniques
DNA - genetics
Fundamental and applied biological sciences. Psychology
Gene Expression
Green Fluorescent Proteins - genetics
in-frame fusion gene
Insecta - cytology
ligase-free cloning
Luminescent Proteins - genetics
Molecular and cellular biology
multiple fragments ligation
Plasmids - genetics
Recombinant Fusion Proteins - genetics
self-assembly
Time Factors
Self assembly
Molecules
Construction
Method
Molecular cloning
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Summary:Enzyme-free cloning (EFC) can rapidly produce an in-frame fusion gene with multiple fragments. To practically apply EFC, we investigated the extent and sequence of complementary staggered overhangs necessary to direct self-assembly of multiple fragments as well as a size limitation of the constructed DNA molecule. Six-base pair overhangs with 50% GC content were sufficient to direct self-assembly. A functional plasmid that exceeded 10 kb, which includes an in-frame fusion domain, was efficiently constructed from four PCR fragments in one step by our improved method.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0736-6205
1940-9818
DOI:10.2144/000113705