Analysis of DNA ligation by microchip electrophoresis

作者：Umemoto Yoshihiro; Kataoka Masatoshi^*; Yatsushiro Shouki; Yamamura Shouhei; Ooie Toshihiko; Kido Jun ichi; Yamamoto Takenori; Shinohara Yasuo; Baba Yoshinobu

来源：Journal of Pharmaceutical and Biomedical Analysis, 2010, 52(2): 323-328.

DOI：10.1016/j.jpba.2009.12.023

• Microchip electrophoresis
• DNA ligase
• Endonuclease
• Alkaline phosphatase
• Recombinant DNA

We describe the potential of microchip electrophoresis with a Hitachi SV1100, which can be used to determine DNA sizes between 500 and 5000 by with good quantification (DNA concentration, <8 ng/mu l) within 5 rein, for the analysis of DNA ligation. On analysis of an electropherogram of a ligation mixture of the pTAC1-T vector and a 7896bp PCR-amplified DNA fragment, the presence of recombinant DNA was easily detected by comparison with an electropherogram obtained without ligase. Oil analysis of a ligation mixture of pUC19/Eco RI without alkaline phosphatase treatment and a 667 by 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 by 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.

• 出版日期2010-6-5