Chemical-reaction-based site-selective DNA cutter for PCR-free gene manipulation.
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.
Research Center for Advanced Science and Technology, University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo, 153-8904, Japan.
Yoji
Y
Yamamoto
Akihiko
A
Uehara
Akira
A
Watanabe
Hiroyuki
H
Aburatani
Makoto
M
Komiyama
D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D004247:DNA / Q000737:chemistry*; D004262:DNA Restriction Enzymes / Q000737:chemistry*; D004274:DNA, Recombinant / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D008967:Molecular Biology / Q000379:methods; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics; D011118:Polynucleotide Ligases / Q000737:chemistry*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
D004274:DNA, Recombinant; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D004247:DNA; D010088:Oxidoreductases; D004262:DNA Restriction Enzymes; D011118:Polynucleotide Ligases