Rapid microvolume PCR of DNA confirmed by microchip electrophoresis.
PCR is an indispensable technique used in DNA analysis. However, with the traditional methods, the time spent on amplification and the subsequent analysis of PCR products is generally long. Therefore, it is essential to improve these two steps so that the whole procedure can be made faster. In the present work, with lambda-DNA as the control template, the amplification of 300-bp fragment could be completed within 37 s with capillary reaction chambers of LightCycler, and the following analysis of PCR products could be completed within 120 s with microchip electrophoresis as the detector. Since the high detection sensitivity of microchip electrophoresis, PCR products with template concentration as low as 5 fg/microL could be detected only after 435 s of amplification. In addition, based on additional optimized conditions simulated by CoventorWare, PCR microchips with distinct structure of the reaction chambers have been designed and successfully applied to the amplification of 300-bp fragment. By comparison, those chambers with ellipse and racket shapes were found to offer very high amplification efficiency. All of these results demonstrate the promise of integrating PCR and electrophoresis on microchip for developing easy-carrying instruments for the fast in situ detection of DNA.
Hataoka
Yukari
Y
Department of Molecular and Pharmaceutical Biotechnology, Graduate School of Pharmaceutical Sciences, The University of Tokushima, 21st Century COE, CREST, JST, Shomachi, Tokushima, Japan.
Zhang
Lihua
L
Yukimasa
Tetsuo
T
Baba
Yoshinobu
Y
D004247:DNA / Q000032:analysis / Q000302:isolation & purification*; D046209:Electrophoresis, Microchip; D004867:Equipment Design; D008832:Microchemistry; D016133:Polymerase Chain Reaction / Q000295:instrumentation / Q000379:methods*; D013696:Temperature; D013995:Time
D004247:DNA