Analytical Sciences

Abstract − Analytical Sciences, 24(3), 327 (2008).

Microchip Electrophoresis for Specific Gene Detection of the Pathogenic Bacteria V. cholerae by Circle-to-Circle Amplification
Laili MAHMOUDIAN,*1 Jonas MELIN,*2,*3 Mohamad Reza MOHAMADI,*4 Keiko YAMADA,*5 Michio OHTA,*5 Noritada KAJI,*1,*6 Manabu TOKESHI,*1,*6 Mats NILSSON,*2 and Yoshinobu BABA*1,*6,*7,*8,*9
*1 Department of Applied Chemistry, Graduates School of Engineering, Nagoya University, Furo, Chikusa, Nagoya 464-8603, Japan
*2 Department of Genetic and Pathology, Uppsala University, Rudbeck Laboratory, se-75185, Uppsala, Sweden
*3 Biomedical Diagnostic Institute, Dublin City University, Dublin, Ireland
*4 Laboratory of Macromolecules and Microsystems in Biology and Medicine (MMBM), Department of Physical Chemistry, Institute Curie, UMR 168, Paris, France
*5 Department of Molecular Biology, Graduate School of Medicine, Nagoya University, 65 Tsurumai, Showa, Nagoya 466-8550, Japan
*6 MEXT Innovative Research Center for Preventive Medical Engineering, Nagoya University, Furo, Chikusa, Nagoya 464-8603, Japan
*7 Plasma Nanotechnology Research Center, Nagoya University, Furo, Chikusa, Nagoya 464-8603, Japan
*8 Health Technology Research Center, National Institute of Advanced Industrial Science and Technology (AIST), Takamatsu 761-0395, Japan
*9 Institute for Molecular Science, National Institutes of Natural Sciences, Okazaki 444-8585, Japan
We have developed a new method for a fast and precise analysis of circle-to-circle amplification (C2CA) product for specific gene detection by microchip electrophoresis. In this method, we have added a new enzymatic step to the C2CA reaction, which could be carried out isothermally at 37°C. Compared to the original single-stranded DNA, the double-stranded DNA that is produced by this enzymatic reaction is more reliable for analysis by microchip electrophoresis. C2CA product was detected within 55 s with high reproducibility by this method which was successfully applied to the detection of 10-ng genomic DNA of the pathogenic bacteria Vibrio. cholerae within 110 s. Purification was found to be an indispensable step for the analysis of the C2CA product of genomic DNA samples.