PCR(聚合酶链式反应)仪温度特性的实验研究与分析

Experimental Study and Analysis on Temperature Characteristics of PCR Processor

该文的目的是测量PCR温度的精确性和PCR仪上孔间以及不同循环和不同次测量的温度均一性。PCR程序:95℃(变性),30 s;55℃(退火),40 s;72℃(延伸),50 s;6个循环。孔和管内的温度重复性都很好;变性阶段,孔和管内液体温度与程序温度偏差较大,孔及管内的温度均一性和重复性均很好;孔内温度不能达到通常所需的温度(90℃以上)的比例为22.2%。退火和延伸阶段孔和管内的温差较小;有2个孔不能达到通常需要的温度。实际温度在各阶段停留的时间分别占设定时间的90%的比例较低。这些缺陷可能导致扩增结果假阳性或假阴性。

Both temperature accuracy of wells at block and uniformity of well-to-well were tested on a standard PCR process. The primary thermal cycling settings were 6 cycles of 95℃ （denaturation） for 30 s, 55 ℃ （annealing） for 40 s, 72 ℃ （extension） for 50 s, The results are the temperatures of wells and liquids in mierotubes measured have good repeatability cycle-to-cycle respectively. Temperatures of wells have poor uniformity at denaturation step and deviated far from the programmed temperature. During denaturation, temperatures of 22.2% wells cannot reach the required denatured temperature （above 91 ℃）, while during annea- ling and extension temperatures, 2 wells cannot reach the required reaction needed temperatures （ annealing at 55 ℃ to 56℃, extension at 70 ℃ to 72 ℃ ） respectively. Percentages of holding time at above three steps temperatures of 90% programmed times are very low. The conclusion is false positive or false negative may be induced because of these shortcomings.