荧光实时定量PCR 技术初探

摘要：
少量的DNA 序列的准确定量曾经是一件很难很累的事情，不过real timePCR 的出现把它变为和普通PCR 差不多一样容易。原理是利用能特异标记PCR产物的荧光物质，显示PCR 产物的动态累积，得到S 型的扩增曲线；假设该曲线的前期PCR 符合指数性扩增，于是在单纯指数方程的基础上，通过比较产物积累的速度（时间）来间接比较（进而确定）初始模板的分子数。我们试验了标准曲线的制作，以及对免疫相关的目的基因IL-4 转录水平的定量，而且用HPRT持家基因对IL-4 进行归一化。结果与RT-PCR 相近。灵敏度，误差降低，可重复性有望更上一层楼。

关键词：
real time PCR 定量 荧光SYBR 扩增曲线 标准曲线 cDNA

Abstract:
It had been difficult and tiring to quantitate a small amount of DNA until the invention of real time PCR technique, which is as easy as regular PCR.It produces and displays the kinetic accumulation of PCR product (oramplicon)----called amplification curve. Assuming that amplicons are amplified exponentially during the earlier cycles, we make use of the simple equation y=axto compare the start copy number of a gene in different samples, by measuring the threshold cycle (Ct). And with some standard, copy number of a gene can be
quantified. In this experiment we tried making some standard curves. Then we quantified IL-4 cDNA in 3 subsets of T helper cell, normalized by HPRT housekeeping gene. The result correlates with RT-PCR. However, sensitivity,deviation and precision still need to be improved.

Key word:
real time PCR quantitation fluorescence SYBR Green I amplification curve standard curve cDNA

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