Bio-Rad定量PCR说明书.pptVIP

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 常规 PCR Process PCR: Theory vs. Reality C(t)s from 96 replicates are nearly identical, but the final fluorescence varies. Quantitative PCR /Real time PCR PCR: Theory vs Reality C(t)s from 96 replicates are nearly identical, but the final fluorescence varies. 常用的定量方法 相对定量：相对于另一参照样本的量； 绝对定量：用标准品作标准曲线来推算未知的样本的量。 Quantitative PCR和常规PCR技术的区别 常规PCR是通过电泳对扩增反应的最终产物进行定性分析（定量不准确）； Quantitative PCR是在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”，通过Ct值和标准曲线对样品中的DNA (or cDNA) 的起始浓度进行定量的方法（准确定量） 。 Introduction to Quantitative PCR 采用半导体加热和制冷 加热速度 3.3°C /秒 ，制冷速度 2°C/秒 升降温速度可调 控温准确性：±0.3℃ 定量PCR仪应该具备的特点：梯度PCR功能 Introduction to Quantitative PCR 定量PCR仪应该具备的特点： 先进的光路设计----多孔样品同时检测 Fluorescence Detection 定量PCR仪应该具备的特点：开放性的系统 Introduction to Quantitative PCR Detection Chemistries Quantitative PCR Detection Chemistries Non-specific DNA binding dyes SYBR? Green I SYBR? Gold Ethidium Bromide Specific Hybridization Probes Cleavage Probes (TaqManTM) Molecular beacons ScorpionsTM AmplifluorTM LUXTM dual-oligo FRET pairs DNA Binding Dyes DNA binding dyes DNA Binding Dyes Cleavage Probes (TaqManTM) Cleavage Probes (TaqManTM) Advantages! Generates a robust cumulative fluorescence signal Simple to design and synthesize compared to other hybridization probes (i.e. beacons) Ideal approach for multiplex assays SNP (Single Nucleotide Polymorphism) assay possible However… More expensive than DNA binding dyes Molecular Beacons Molecular Beacons Advantages! Good for SNP (Single Nucleotide Polymorphism) detection Multiplex assays possible However… Molecular Beacons Are DIFFICULT to Design and Synthesize Does NOT generate a cumulative fluorescence signal, much weaker signal than the 5’ Nuclease Assay (Cleavage probe) Expensive! Outline Real Time PCR and Conventional PCR Introduc