Open AccessUsefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on‐chip enzymatic digestion
Author(s) -
Kataoka Masatoshi,
Inoue Sonoko,
Kajimoto Kazuaki,
Sinohara Yasuo,
Baba Yoshinobu
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04161.x
Subject(s) - capillary electrophoresis , dna , endonuclease , chromatography , reproducibility , electrophoresis , chemistry , restriction enzyme , microbiology and biotechnology , sizing , biology , biochemistry , organic chemistry
The Hitachi SV1100 utilizes capillary electrophoresis on a microchip that is capable of rapidly sizing DNA fragments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNA fragments of 100 and 800 bp. The range of DNA sizing for this microchip is between 100 and 800 bp, and accuracy in sizing of a 322 bp DNA fragment of a pUC118 Pvu II digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng·µL −1 , error increased in a dose‐dependent manner. Furthermore, the feasibility of sequential analysis with this microchip was shown by the reproducibility of successive electrophoreses of the internal control in one channel. When the pUC118 Pvu II digest was treated with endonuclease Kpn I on the microchip for 10 min, sequential analysis showed that the 322 bp fragment completely disappeared and two peaks corresponding to the 130 and 192 bp fragments appeared. This analysis was performed within 4 min, and the peaks were estimated as 127 and 183 bp, respectively. These results indicate the potential of on‐microchip endonuclease treatment of plasmid DNA with sequential analysis, offering high resolution in a short time.