TAILIEUCHUNG - Báo cáo khoa học: 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 DNAfrag-ments. Reproducibility of electrophoresis in different channels was shown by comparing the migration times of the internal controls, DNAfragments of 100 and 800 bp. The range of DNAsizing 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, inde-pendent of DNAconcentration. Although relatively good quantification of this fragment was observed with a DNA concentration of ngÆlL )1 , error increased in a dose-dependent manner. . | Eur. J. Biochem. 271 2241-2247 2004 FEBS 2004 doi Usefulness of microchip electrophoresis for reliable analyses of nonstandard DNA samples and subsequent on-chip enzymatic digestion Masatoshi Kataoka1 Sonoko Inoue2 Kazuaki Kajimoto1 Yasuo Sinohara1 2 3 and Yoshinobu Baba1 2 4 Division of Gene Expression Institute for Genome Research The University of Tokushima Japan 2Faculty of Pharmaceutical Science University of Tokushima Japan 3 Single-Molecule Bioanalysis Laboratory National Institute of Advanced Industrial Science and Technology Takamatsu Japan 4CREST Japan Science and Technology Corporation Tokushima Japan 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 framnenss of 100 and 800 bp. The range of DNAsizing for this microchip is between 100 and 800 bp and accuracy in sizing of a 322 bp DNA fragment of a pUC118 PvulI digest was observed independent of DNA coneentration. .lXIlt cghl1 relatively good quantification of this fragment was observed with a DNA concentration of ng-pL-1 error increased in a dosedependent 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 Kpnl 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 DNAwith sequential analysis offering high resolution in a short time. Keywords electrophoresis microchip plasmid .

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