Microchip electrophoresis for specific gene detection of the pathogenic bacteria V. cholerae by circle-to-circle amplification.
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 degrees 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.
Mahmoudian
Laili
L
Department of Applied Chemistry, Graduates School of Engineering, Nagoya University, Furo, Chikusa, Nagoya, Japan. mahmoudian2@yahoo.com
Melin
Jonas
J
Mohamadi
Mohamad Reza
MR
Yamada
Keiko
K
Ohta
Michio
M
Kaji
Noritada
N
Tokeshi
Manabu
M
Nilsson
Mats
M
Baba
Yoshinobu
Y
D004269:DNA, Bacterial / Q000032:analysis* / Q000737:chemistry / Q000235:genetics; D004270:DNA, Circular / Q000737:chemistry*; D046209:Electrophoresis, Microchip / Q000295:instrumentation / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013997:Time Factors; D014734:Vibrio cholerae / Q000737:chemistry* / Q000235:genetics
D004269:DNA, Bacterial; D004270:DNA, Circular