文档介绍：Outline
Real Time PCR and Conventional PCR
Introduction to Quantitative PCR
Ct value and Real-Time Quantitative PCR Theory
Laboratory Technique
Primer Design for Real-Time PCR
Real Time PCR and Conventional PCR
技术背景
本来,PCR技术是为了将样本中微量的DNA模版放大以便研究模版特性
随着研究的深入,需要了解样本中基因的表达模式与疾病的关系,这就要了解标本中的DNA原始拷贝数。
Denaturation
Primer Annealing
Elongation
5’
3’
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Taq
Taq
Repeat
常规 PCR Process
In theory, product accumulation is proportional to 2n,
where n is the number of amplification cycle repeats
A linear increase follows exponential
Eventually plateaus
Cycle #
Theoretical
Real Life
Log Target DNA
Reality vs. Theory
Amplification is exponential, but the exponential increase is limited:
常规PCR方法的局限性分析:
无法对起始模板准确定量,只能对终产物进行分析
对终产物分析,受PCR过程平台效应的干扰,定量准确度难以提高(相对误差>1000%);存在扩增产物的污染问题。
必须在扩增后用电泳方法分析,费时费力而且EB有毒
无法对扩增反应实时检测
C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.
Cycle
End Point
PCR: Theory vs. Reality
实时定量PCR技术,是指在PCR反应体系中加入荧光基团,利用荧光信号积累实时监测整个PCR进程,使每一个循环变得“可见”,最后通过Ct值和标准曲线对样品中的未知样品的起始浓度进行定量的方法。
Real-Time PCR
Quantitative PCR 或 Real time PCR
是确定样品中DNA (或 cDNA) 拷贝
数最敏感、最准确的方法
Quantitative PCR /Real time PCR
C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.
Cycle
End Point
C(t)
PCR: Theory vs Reality