PCR温度特性实验研究与分析

目的：测量PCR温度的准确性和均一性。方法：PCR主程序：变性95℃，30s，退火55℃，40s，延伸72℃，50s，6个循环；热电偶分别放在孔内和管内。结果：每个循环以及每次测量的孔和管内的温度重复性很好；变性阶段，孔的温度和管内液体温度与程序温度偏差较大，温度准确性都较差，但所有孔的温度都达到通常需要的变性温度（变性90℃以上），但是44．4％的孔在90℃以上停留的时间不能达到设置的时间；变性阶段，11.1％管内液体不能达到所需的变性温度（90℃）.结论：这些缺陷可能导致扩增结果假阳性或假阴性。

Experimental Study and analysis of temperature characteristics on PCR Performance

Both temperature accuracy of wells at block and uniformity of well-to-well and hatch-to batch were tested on a standard PCR process. Method： The primary thermal cycling settings were, 6 cycles of 95℃（denaturation）for 30 s,55 ℃（ annealing ）for 40 s,72 ℃ （ extension ）for 50 s. Two thermal couples were embedded in bottom of wells and tubes respectively. Results： The temperatures of not only wells but also liquids in tubes measured had good repeatability cycle-to-cycle and batch-to-batch respectively. Temperatures of wells and in-tube liquids had poor uniformity during denaturation and deviated far from the programmed temperature. Although all wells reach the required denatured temperature （over 90℃）,There are 44.4% wells which can not retain at all programmed times above 90℃ during denaturation.11.1% in-tube liquids cannot reach the common reaction temperature（over 90℃）. Conclusion： False positive or false negative may be induced because of these shortcomings.