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Chemical‐Reaction‐Based Site‐Selective DNA Cutter for PCR‐Free Gene Manipulation
Author(s) -
Yamamoto Yoji,
Uehara Akihiko,
Watanabe Akira,
Aburatani Hiroyuki,
Komiyama Makoto
Publication year - 2006
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.200500402
Subject(s) - wwox , dna , gene , fusion protein , green fluorescent protein , fluorescence , fusion gene , computational biology , biology , microbiology and biotechnology , open reading frame , recombinant dna , chemistry , genetics , biochemistry , peptide sequence , physics , quantum mechanics , suppressor
An artificial restriction DNA cutter (ARCUT), recently developed by the authors, was used to construct a fusion protein. The gene of WW‐domain‐containing oxidoreductase ( WWOX ) was cut by ARCUT just before its stop codon, and ligated to fuse the gene of enhanced green fluorescent protein ( EGFP ). The reading frames of two genes were adjusted to coincide each other. Throughout the manipulation, no PCR was employed. The fluorescent fusion protein was successfully expressed in mammalian cells, and showed entirely different subcellular localization from EGFP itself. Apparently, the DNA was kept completely intact during the manipulation. The man‐made tool ARCUT has promising features for future biotechnology and molecular biology.