定量PCR原理及PCR仪光学原理.ppt

基本概念 定量PCR的数学原理 定量PCR的化学原理 等位基因鉴定原理 定量PCR仪光学原理 什么是PCR 定量PCR的数学原理 CT值与起始DNA浓度的关系 为什么CT ? 起始DNA浓度? 为什么CT ? 起始DNA浓度? 软件自动的数据分析方法 定量PCR的化学原理 两种定量化学 染料染色 SYBR? Green I 溴乙锭(Ethidium Bromide) 探针标记 TaqManTM TaqMan MGB Dual-oligo FRET pairs 分子信标(Molecular beacons) ScorpionsTM/AmplifluorTM TaqMan探针的结构 TaqMan探针定量原理 TaqMan探针的共振能量转移 Taq酶的5’→3’外切活性 TaqMan MGB探针的好处 两种探针比较 探针法小结 染料法小结 多重定量 多色荧光技术 荧光定量需要ROX校正和IPC校正 检材和对照可以在同一管中定量 仪器须有多荧光检测能力 推荐的4色组合 FAM 目的基因 VIC 第二个目的基因或者IPC TAMRA 淬灭基团 ROX 参比荧光 荧光染料功能分类 7000：4色荧光 FAM? / SYBR? Green I VIC? / JOE? TAMRA? / NED? ROX? (参比荧光passive reference) 等位基因鉴定原理 两种探针加入同一管 等位基因鉴定实验数据 定量PCR仪光学原理及性能 光学结构简图 定量PCR仪功能一览 绝对定量(Absolute Quantification) 自动计算基线、阈值、CT值、浓度、百分比 相对定量(Relative Quantification) 等位基因分型(Allelic Discrimination) 阴阳性鉴定(Plus/Minus with IPC) FC’s only provide FAM, VIC and TET target gene: FAM house keeping gene: VIC TAMRA will gradually getting weak during reaction, so it can not be reference, that is why Rox added into the reagent mix----confidential 7700: 6- 8 sec scan one plate 5700: 15 sec for one plate scan Probe Tm: 68 to 70 degree. Under this high temperature , Taq DNA polymerase need to cleave DNA when probe is not perfect match( ex: AD) Taq DNA polymerase just kick out the probe The first principle that the 5’ Nuclease chemistry takes advantage of is FRET … or When the reporter dye (in green) is in close proximity to the quencher dye (in red), the emission energy from the reporter is donated to the quencher, resulting in a low signal from the reporter. When they are no longer in close proximity, they are free to fluoresce Want to compare the fold difference in expression of one treatment condition to another or one tissue type to another Samples are extrapolated off of the standard curve, and result in data with either total NA concentration or dilution factor. Some what meaningless. 分子信标(Molecular beacon) TaqMan探针的衍生形式 杂交双探针 变 性 退 火 延 伸 完 成 No Blots! No Gels! 既灵敏又特异：PCR与分子杂交的结合 双倍放大：P