Abid Hassan Ali, Lin Eric Shen, Ong Jian Wern, Minifie Tristan, Song Zhixiong, Liew Oi Wah, Ng Tuck Wah
Laboratory for Optics and Applied Mechanics, Department of Mechanical and Aerospace Engineering, Monash University, 17 Alliance Lane, Clayton, Victoria3800, Australia.
Centre for Translational Medicine, Cardiovascular Research Institute, Yong Loo Lin School of Medicine, National University of Singapore, National University Health System, 14 Medical Drive, Singapore 117599.
Rev Sci Instrum. 2020 Oct 1;91(10):104105. doi: 10.1063/5.0007879.
A thermal cycling method, whereby capillary tubes holding polymerase chain reactions are subjected to programmed tilt displacements so that they are moved using gravity over three spatial regions (I, II, and III) kept at different constant temperatures to facilitate deoxyribonucleic acid (DNA) denaturation, annealing, and extension, is described. At tilt speeds in excess of 0.2 rad/s, the standard deviation of static coefficient of friction values was below 0.03, indicating in sync movement of multiple capillary tubes over the holding platform. The travel time during the acceleration phase and under constant velocity between adjacent regions (I to II and II to III) and distant regions (III to I) was 0.03 s and 0.31 s, respectively. The deviations in temperature did not exceed 0.05 °C from the average at the prescribed denaturing, annealing, and extension temperatures applied. DNA amplification was determined by optical readings, the fluorescence signal was found to increase twofold after 30 thermal cycles, and 1.16 × 10 DNA copies/μl could be detected. The approach also overcomes problems associated with thermal inertia, sample adhesion, sample blockage, and handling of the reaction vessels encountered in the other thermal cycling schemes used.
描述了一种热循环方法,即对装有聚合酶链反应的毛细管进行程序倾斜位移,使其利用重力在保持不同恒定温度的三个空间区域(I、II和III)上移动,以促进脱氧核糖核酸(DNA)的变性、退火和延伸。在倾斜速度超过0.2 rad/s时,静摩擦系数值的标准偏差低于0.03,表明多个毛细管在固定平台上同步移动。在加速阶段以及相邻区域(I至II和II至III)和远距离区域(III至I)之间以恒定速度移动时的行程时间分别为0.03 s和0.31 s。在所施加的规定变性、退火和延伸温度下,温度偏差不超过平均值0.05℃。通过光学读数确定DNA扩增,发现荧光信号在30个热循环后增加了两倍,并且可以检测到1.16×10个DNA拷贝/μl。该方法还克服了与其他热循环方案中遇到的热惯性、样品粘附、样品堵塞以及反应容器处理相关的问题。