Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion.
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 PvuII digest was observed, independent of DNA concentration. Although relatively good quantification of this fragment was observed with a DNA concentration of 1.83 ng.microL(-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 PvuII digest was treated with endonuclease KpnI 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.
Kataoka
Masatoshi
M
Division of Gene Expression, Institute for Genome Research, The University of Tokushima, Kuramotocho 3-18, Tokushima 770-8503, Japan. kataoka@genome.tokushima-u.ac.jp
Inoue
Sonoko
S
Kajimoto
Kazuaki
K
Sinohara
Yasuo
Y
Baba
Yoshinobu
Y
D004247:DNA / Q000032:analysis* / Q000737:chemistry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D010957:Plasmids / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results
D004247:DNA; C025630:CAGCTG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific