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1、荧光 PCR 原理及应用周煜ZHOU YuApplication Specialist ABI公司及其荧光PCR仪器3美国ABI 公司荧光定量PCR仪发展历史1995世界上第一台定量PCR仪7700型1997向医院用户推出5700型定量PCR仪2000推出7900型384孔孔荧光定量PCR仪20017900型 96孔荧光定量PCR仪2001推出7000型荧光定量PCR仪取得巨大成功2003获得了荧光定量PCR仪专利确立ABI在业界内的独特地位2004第3代荧光定量PCR仪7300和7500型2007第4代荧光定量PCR仪StepOne和StepOnePlus公认最高端定量公认最高端定量PCR仪
2、仪4ABI 公司荧光PCR仪器5ABI 公司荧光PCR仪器Applied Biosystems 7900HT Fast Real-Time PCR SystemApplied Biosystems 7500 Fast Real-Time PCR SystemApplied Biosystems 7300 Real-Time PCR SystemApplied Biosystems 7500 Real-Time PCR SystemApplied Biosystems StepOne Real-Time PCR System6各种荧光PCR仪器荧光PCR的化学8两种荧光化学原理Fluorogen
3、ic 5 Nuclease Assay An established and proven chemistry for high specificity nucleic acid real-time quantitation The introduction of TaqMan MGB (Minor Groove Binder) probes has produced a turn-key homogeneous chemistry for the detection of Single Nucleotide Polymorphisms (SNPs)SYBR Green 1 Double St
4、randed DNA Binding Dye Assay Ideal for target identification (screening) assays A lower-cost alternative when high specificity is not required9RQ53535353ExcitationRQ QRQRExcitationTaqMan探针10TaqMan MGB探针GCTACACAGTCCGGAACTCTCTATAGCATCACACAGGCCTTGAGAGATATR|QAGGCCTTGAGAGATATRQ(non-fluorescent quencher)N
5、FQQMGB (minor groove binder)提高探针提高探针Tm值值NFQMGBR报告荧光无荧光淬灭基团小沟结合物报告荧光无荧光淬灭基团小沟结合物11MGB探针的优点提高淬灭效率提高淬灭效率减少背景、提高信噪比减少背景、提高信噪比提高探针提高探针Tm值值 长长15碱基，提高碱基，提高 18C缩短探针长度缩短探针长度 最佳范围最佳范围13-21 碱基碱基12TaqMan MGB探针鉴别等位基因FAMFAMMGB完全配对，有信号完全配对，有信号一个碱基不配对，没有信号一个碱基不配对，没有信号分辨分辨1个碱基的精确度个碱基的精确度RQMGBNFQ5 3 FAMFAMMGB13SYBR G
6、reen染料SYBR Green I 与与dsDNA 结合结合与DNA结合时游离时光与DNA结合时游离时光14融解曲线(Dissociation curve)Easily check for specificity of your SYBR assays immediately after PCRThermal conditions (after last cycle of PCR): Hold at 95C for 20 sec Hold at 60C for 20 sec Ramp and collect fluorescent data to 95C over course of 20
7、minutesSoftware automatically generates melt curve profiles15融解曲线(Dissociation curve)导数图谱原始图谱导数图谱原始图谱16高分辨溶解曲线（HRM, High Resolution Melting ）Different from a regular SYBRGreen dye melt curve: Chemistry: Saturating and brighter dsDNA binding dyes LCGreenEvaGreenTMSYTO9 Instrument: More data points ar
8、e collected Software: New fluorescent normalization algorithms and plots17Now can be performed on the ABI 7500 Fast SystemThe analysis is based on the difference in curve shape as well as Tm.HRM can identify mutations that cause Tm changes as minor as 0.1C.18染料法的特点成本低兼容熔解曲线特异性问题不能进行多重扩增19荧光PCR的防污染技术
9、Uracil N-glycosylaseUUUUChemical method to destroy contaminating amplicons.All amplification chemistries use dUTP.UNG does not function above 55oC20荧光参比Passive Reference Dye: ROXROX以固定的浓度配在以固定的浓度配在Master Mix中，不参与中，不参与PCR扩增扩增ROX的功能：的功能：Improves precision.Compensates for small fluorescent fluctuations
10、 that can occur from well-to-wellCan be useful for diagnosing troublesRn = Normalization = Reporter / ReferenceReporterReference21ROX Normalizer36 Replicates analyzed with ROX passive reference dye36 Replicates analyzed without ROX passive reference dye定量PCR的数学原理23实时定量与终点定量终点处产物量不恒定 Ct值则极具重现性!终点处产物量
11、不恒定 Ct值则极具重现性!24PCR动力学曲线和四个阶段平台期 线性增长期基线期指数增长期平台期 线性增长期基线期指数增长期对数图谱对数图谱平台期线性增长期基线期指数增长期平台期线性增长期基线期指数增长期线性图谱线性图谱25什么是什么是CT值？值？荧光信号 穿过荧光信号 穿过阈值阈值线时的循环次数线时的循环次数threshold cycleCT值值对数图谱对数图谱CT值值线性图谱线性图谱26PCR扩增的数学模型扩增产物的总数量可以下式表示：扩增产物的总数量可以下式表示： Yn=X(1+E)nYn为为PCR产物的分子数量产物的分子数量 n为周期数为周期数 E为扩增效率（为扩增效率（0E1）理想
12、条件下理想条件下 浓度增加浓度增加 1倍倍 CT值减小值减小1个单位个单位 浓度增加浓度增加10倍倍 CT值减小值减小3.321928027斜率与截距CT= - k lgX0+ b Slope=-1/log(1+E) If E=1, Slope= -3.3219280 Normally, slope is about -3.5-4.2 Intercept Normally, intercept is about 304028CT值对DNA0作图CT值值lg 起始起始DNACT = - k lgX0+ b29成功的定量PCR数据30实时荧光PCR技术的主要优点可进行定量PCR检测，定量范围(Dy
13、namic Range)宽；操作方便无复杂的PCR后分析步骤；全封闭式检测减少污染；检测灵敏度及特异性高；多通道分析；熔点曲线分析(Dissociation curve)荧光PCR技术的应用32主要应用Quantitative PCRReal timePlus/minus AssaysAllele Discrimination+ + + + - -+ +End point33定量PCR Quantitative PCRHow many? 绝对定量 Absolute QuantitationHow much? 相对定量 Relative Quantitation34Results from RN
14、ase P Instrument Verification PlateStandard curve amplificationsshowing 20K, 10K, 5K, 2.5Kand 1.25K copy populationsin replicates of four“Unknown” amplificationsshowing 10K and 5K copypopulations in replicates of 3635Results from RNase P Instrument Verification Plate36Calculating DNA Quantity of a S
15、ampley = -1.2908Ln(x) + 27.187R2 = 0.9972022242628303234360.0010.0100.1001.00010.000100.000(Log N) initial concentrationSampleCT37Data Report Table38Dynamic Range Data Amplification of serial dilutions of IL-10 target in replicates of 8Standard curve showing 7 logs of linear dynamic range39相对定量Relative gene expression: Compare treated samples vs. untreated samples (calibrator samples) RNAi validation Array validation40相对定量Ct 相对量 2 -CT 目标序列和内参序列的扩增效率相等（且接近100）The Relative Standard Curve Method41Relative Expression42等位基因分析Allelic Discrimination (SNP Detection)43等位基因鉴定机理野生型野生型 (wt)突变型突变型
