Analysis of DNA ligation by microchip electrophoresis.
We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 bp with good quantification (DNA concentration, <8 ng/l) within 5 min, for the analysis of DNA ligation. On analysis of an electropherogram of a ligation mixture of the pTAC1-T vector and a 789 bp PCR-amplified DNA fragment, the presence of recombinant DNA was easily detected by comparison with an electropherogram obtained without ligase. On analysis of a ligation mixture of pUC19/Eco RI without alkaline phosphatase treatment and a 667 bp Eco RI-digested fragment of foreign DNA, several peaks observed in the electropherogram corresponded to the formation of monomeric and polymeric insert DNAs, self-ligated vector DNA, and recombinant DNA. On the other hand, several peaks were also observed in the electropherogram of the ligation mixture of pUC19/Eco RI with alkaline phosphatase treatment and the 667 bp Eco RI-digested fragment of foreign DNA, the fluorescence intensity corresponding to recombinant DNA apparently being increased. These results indicate the potential of microchip electrophoresis for the analysis of DNA ligation, it offering high resolution in a short time.
Umemoto
Yoshihiro
Y
Department of Molecular and Pharmaceutical Biotechnology, Graduate School of Pharmaceutical Sciences, University of Tokushima, Shomachi 1-78, Tokushima 770-8505, Japan.
Kataoka
Masatoshi
M
Yatsushiro
Shouki
S
Yamamura
Shouhei
S
Ooie
Toshihiko
T
Kido
Jun-ichi
J
Yamamoto
Takenori
T
Shinohara
Yasuo
Y
Baba
Yoshinobu
Y
D017122:Bacteriophage T4 / Q000737:chemistry; D001483:Base Sequence; D002021:Buffers; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes / Q000378:metabolism; D004270:DNA, Circular / Q000737:chemistry; D004274:DNA, Recombinant / Q000032:analysis* / Q000378:metabolism*; D004279:DNA, Viral / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D046209:Electrophoresis, Microchip / Q000379:methods*; D005240:Feasibility Studies; D046210:Microfluidic Analytical Techniques / Q000379:methods; D008969:Molecular Sequence Data; D012015:Reference Standards
D002021:Buffers; D004270:DNA, Circular; D004274:DNA, Recombinant; D004279:DNA, Viral; D004262:DNA Restriction Enzymes