Bio-Rad定量PCR说明书经典知识.pptVIP

Cycle Log Relative Fluorescence Texas Red FAM Hex Cy5 Multiplexing 定量PCR仪应该具备的特点：开放性的系统 可兼容的定量方法： SYBR Green I、Taqman探针、Beacon探针、FRET探针 可兼容的突变检测方法： 熔点曲线功能、MGB探针 可兼容的试剂种类： 所有国产与进口试剂 iQ5 Data Analysis Software iQ5 Data Analysis Software 定量PCR仪应该具备的特点： 分析软件功能强大简洁直观 点击您所需要的程序名称，该程序所包含的所有内容都一目了然。方便您在众多的程序中快速找到您所需要的一个。 黄色提示会警告您在编辑的程序中存在逻辑错误的数据。您可重新审查、修改。 在运行开始之前，警示框提示数据设置的残缺。 异常数据或情况提示 标准曲线 定量计算 自动数据分析 多种报告单模式可供选择----尤其重要 基因表达分析 Introduction to Quantitative PCR PCR仪 + 监测、分析系统 + 荧光标记 l l l l 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 Quantitative PCR Detection Chemistries DNA Binding Dyes 3’ 5’ BD BD 5’ BD BD BD Extension 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Extension Continued Apply Excitation Wavelength 5’ 3’ 5’ 3’ 5’ 5’ Taq Taq 3’ 5’ 3’ Taq Taq 5’ 5’ Repeat DNA binding dyes BD BD BD BD BD BD BD BD BD BD l l l l l DNA Binding Dyes Advantages! Inexpensive compared to hybridization probes No additional design work than the primer used for PCR reaction However… Not template specific, will bind ALL double stranded DNA inducing primer-dimer and unspecific amplicon formation Multiplex assays not possible SYBR Green I Experiment Typical “first step” experiment: Evaluate Primer Specificity Using Melt Curve Analysis Evaluate Primer Pair Efficiencies By running serial dilutions of template as standards Identify Sub-Optimal aspects of assay Optimize further with thermal gradient, etc. Cleavage Probes (TaqManTM) 5’ 3’ Q F Taq 5’ q 3’ l 3’ 5’ 3’ 5’ F Q 杂交探针的互补区在引物间 5’端连有一个荧光reporter, 通常是荧光素 3’端连有一个Quencher,通常是TAMRA 荧光素被488 nm光激发，发射光为520 nm 520 nm 光能激发TAMRA，发射光为570 nm TaqMan Cleavage-based assay: TaqManTM 5’ 5’ 3’ 3’ d.NTPs Thermal Stable DNA Polymerase Primers 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ Add Master Mix and Sample Denaturation 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3’ 5’ 3