QPCR原理.ppt

Klicken Sie fr das Titelformat,Klicken Sie,um die Formate des Vorlagentextes zu bearbeiten,Zweite Ebene,Dritte Ebene,Vierte Ebene,Fnfte Ebene,兼顾速度与可靠性,Eppendorf PCR,系统,20,Years of PCR,Kary Mullis,PCR,只是一个概念，所发生的奇迹是：,PCR,概念变成了可操作的实验系统，变成了一项成熟的技术，后者又上升成为新的概念。,它最大的特点就是能不断推出新形式。,摘自,Making PCR:A Story of Biotechnology by Paul Rabinow,T,T,T,T,T,A,A,A,C,C,C,C,C,A,A,A,A,T,T,T,C,C,C,C,C,C,T,A,A,G,G,G,G,G,G,G,G,G,G,G,Denaturation,94C,Annealing,55C,Elongation,72C,Pol,Pol,PCR,5,3,5,3,Separation of strands,Annealing of primer,Elongation by polymerase,PCR,后分析结果,PCR products in different sizes,3,kb,500bp,300bp,为什么终点法定量不准确?,Ct,Endpoints,同一样品尽管平台期,DNA,拷贝数波动很大，,CT,值却是相对固定的。,典型的,PCR,四阶段,半对数图谱,线性图谱,平台期,典型的,PCR,四阶段,线性增长期,基线期,指数增长期,PCR,理论方程,lg DNA,循环数,线性期,平台期,y=x(1+e),n,指数期,N=N,0,x(1+e),n,N:,产物分子数,N,0:,起始分子数,e：,扩增效率,n:,循环次数,PCR,理论方程只在指数期成立,PCR,指数增长期的规律,PCR,扩增为指数扩增，每一扩增周期后产物的量可以下式表达：,Y,n,=X(1+E),n,0E1,其中,E,表示扩增效率，,Y,n,表示在,n,个周期后,PCR,产物的分子数量，,X,为,n-1,个周期后,PCR,产物的分子数量。,等式仅在限定的扩增周期数（通常为20或30）内成立。超过此周期数，扩增过程即由指数扩增降低至稳定的扩增速率，最终达到平台，不再扩增.,定量,PCR,测定的测定点为,PCR,扩增的指数扩增期;,而定性,PCR,测定的测定点则多为,PCR,扩增的平台期,为什么终点法定量不准确?,Ct,Endpoints,同一样品尽管平台期,DNA,拷贝数波动很大，,CT,值却是相对固定的。,C,t,(Threshold Cycle),-The cycle number where the reaction fluorescence exceeds background fluorescence.,Cycle#,Fluorescence,C,t,C,t,C,t,Threshold,Threshold,-,The peak of background fluorescence,as defined by,the platform or the user.,Background Fluorescence,Amplification Plot,定量分析原理,Y,ct,=X(1+E),n,=2,n,X,Ect=1,CT,值的定义是,PCR,扩增过程中，荧光信号开始由本底进入指数增长阶段的拐点所对应的循环次数。,CT,值与起始模板浓度成,负相关关系,不同,CT,值的,模板起始浓度相差,2,n,倍,n,为,CT,间差值,为什么,C,T,值,起始,DNA,浓度,?,当循环次数,n=C,T,值时：,R,CT,=R,B,+X,0,(1+e),CT,R,s,lg(R,CT,-R,B,)=lg X,0,+C,T,lg(1+e)+lg R,s,C,T,lg(1+e)=-lg X,0,+lg(R,CT,-R,B,)lg R,s,即,C,T,=-k lg X,0,+b,（,线性方程）,R,n,=R,B,+X,0,(1+e),n,R,s,定量,PCR,的实质,模板定量,找到,PCR,的指数增长期,定量数据归一化,real-time PCR,应用范围,科研的主要工具,mRNA,与基因表达的研究,DNA,拷贝数的检测,单核苷酸多态性（,SNPs）,的测定,DNA,芯片结果认证,医学方面应用前景更是令人鼓舞,极微量的基因表达定量,病,原体检测,绝对定量,Unknown,通常用标准品作为,外参照,制作标准曲线.,注意保持与目的基因的同源性,标准曲线的制作,什么是阈值,基线,阈 值,标准偏差,基线信号的标准偏差,x 10,C,T,值,基 线,阈 值,基线,阈值,C,T,值,I,3,125,I,6,250,I,12,500,I,25,000,I,50,000,I,100,000,Absolute amount.(copies),EXACT scale,20-,19-,18-,17-,16-,15-,C,T,A,B,C,C,T,16,18,19,Absolute amount,50,000,copies,12,500,copies,6,250,copies,Sample A,Sample B,Sample C,标准曲线方法,通过,C,T,值测定起始,DNA,量,浓度增加,1,倍，,C,T,值减小,1,个单位,浓度增加,10,倍，,C,T,值减小,3.3,个单位,相对定量,C,t,Sample,C,t,Non treated sample,Housekeeping Gene,C,t,C,t,C,t2,-,Ct,=Change in Expression Level,Gene of Interest,相对定量,D,Ct=9.70,D,Ct=-1.7,D,Ct=target-ref,D,Ct=target-ref,Difference =,D,Ct-,D,Ct,=,DD,Ct,=9.70-(-1.7),=11.40,IL1-b con,RPLP0 con,av=19.93,av=29.63,control,IL1-b vit,RPLP0 vit,av=19.80,av=18.03,experiment,-2,-,Ct,=-2(C,target,-C,calibrator,)-(C,target,-C,calibrator,),DNA,芯片结果,复,证,DNA,甲基化检测,Before Bisulfite Treatment:,Wild-Type DNA,5.G,C,GGA,CC,G,CG,.,After Bisulfite Treatment:,Unmethylated DNA,5.G,U,GGA,UU,G,U,G.,Methylated DNA,5.G,C,GGA,U,C,G,C,G.,高通量,SNP,筛查,Amplifluor Primer(UniPrimer),Quencher,PCR,SNP,Real Time PCR,技术的诞生,1992,年，,Higuchi R,等第一个,报导了实时定量,PCR,技术,Higuchi R,Fockler C,etal.Kinetic PCR analysis:real-time monitoring of DNA amplification reactions.Biotechnology,1993,11(9):1026-1030.,所谓,实时定量,PCR,技术 是指在,PCR,反应体系中加入荧光基因,利用荧光信号累积实时监控整个,PCR,进程,最后通过标准曲线对未知模板进行定量分析的方法.,实时定量,PCR,技术是,PCR,技术和荧光检测技术的结合,Fluorescent Dyes,荧光分子或荧光染料,Exitation peak,Emission peak,Reaction,Amplification Plot,Time(Cycle#),Fluorescence,荧光信号累积与扩增曲线,荧光分子或染料的检测方法,荧光分子内参式检测（,DNA Specific Dyes,）,SYBR Green I,荧光标记的特异探针,水解探针:,Taqman,杂交探针:,FRET,（,Dual Probes,）,分子信标:,Molecular Beacons,荧光标记引物:,Sc,orpion,和,Amplifuor,其它类:,BD,QZyme,DNA specific Dyes,e.g.SYBR Green I,Excitation 494 nm,SYBR Green I,的检测原理,Emission 521 nm,熔解曲线分析引物二聚体,Primer dimers,Product of Interest,UNG,预防污染,52,C 2:00 UNG treatment,95,C 10:00 enzyme activation,60,C 1:00 annealing/extension,95,C 0:15 denat.,UNG,酶的作用原理是降解含有,dU,的双链或单链,DNA。,它在50,C,激活，95,C,灭活.用于预防非特异性,PCR,扩增和污染,。,在定量,PCR,开始前增加50,C,的保温步骤，,UNG,酶即可将已有的,PCR,产物降解破坏，防止可能造成的污染,仪器热启动,Innovative technical feature for device-supported HotStart PCR,Perfect combination:Mastercycler ep S and HotMaster,Enhances the PCR to nearly the maximum,脉冲,PCR,=,超快的升温速度,+,极大缩短初始升温所需时间,Block temperature C,Time,100,80,60,40,20,6,C/sec,8,C/sec,TaqMan,探针法的核心:,利用,Taq,酶的53外切核酸酶活性，切断探针，产生荧光信号,FRET,荧光共振能量转移,F,luorescence,R,esonance,E,nergy,T,ransfer,TaqMan,探针原理,T,A,G,C,C,T,G,C,A,G,A,G,R,Q,Reporter,Quencher,Real-Time QPCR,常用染料,Alx350,FAM,TET,HEX,JOE,ROX,Cy5,Cy3,TAM,TxRd,T,T,T,T,T,A,A,A,C,C,C,C,C,A,G,G,G,G,G,G,T,T,T,T,T,A,A,A,C,C,C,C,C,A,G,G,G,G,G,G,T,T,T,A,A,C,CC,C,G,G,G,T,A,G,C,C,T,G,C,A,G,A,G,R,Q,TaqMan,探针原理,当探针完整的时候，报告基团所发射的荧光能量被淬灭基团吸收，,仪器检测不到信号。随着,PCR,的进行，,Taq,酶在链延伸过程中遇到与模板结合的探针，,其,53外切核酸酶活性就会将探针切断,，报告基团远离淬灭基团，,其能量不能被吸收，即产生荧光信号,A,G,C,C,A,C,C,T,T,T,T,T,T,T,G,G,G,C,C,C,C,A,A,A,A,G,C,C,G,A,T,R,多探针的反应体系,Two or more Probes and Dyes in one Tube,T,A,G,C,C,T,G,C,A,G,A,G,R,1,Q,T,A,G,C,C,T,G,C,A,G,A,G,R,2,Q,Target 1,Target 2,微卫星系统,Mastercycler ep,-新一代,PCR,仪的代表,超快的升/降温速度,脉冲,PCR,独立直观的控制面板,ESP,电子样品保护热盖,M,astercycler ep,系列-多种模块选择,铝块,铝块,银块,E,ppendorf,在定量,PCR,领域厚积薄发,M,astercycler ep realplex,实时定量,PCR,仪的开发,秉承了,Eppendorf,既往的经验.,是将经验融入最新技术的精品之作,回顾,Eppendorf 60,年的发展历史，当之无愧为高品质实验室仪器的供应商。,Eppendorf,拥有55年生产光学仪器的经验，,近10年来，热循环仪已成为,Eppendorf,产品家族中的重要组成,。,。,real-time PCR,仪器,构造,Exitation light source,Filter,Detector,热,循环仪,光学元件,软件分析,Mastercycler ep,realplex,A 4-colour detection unit in a space saving design,Thermal Module,Sliding Optics Module,Mastercycler ep,升级为定量,PCR,仪,ep Gradient/S,两通道,ep RealPlex,2,四通道,ep RealPlex,4,六通道,ep RealPlex,6,COMING SOON!,在,Mastercycler ep,梯度,PCR,的基础上，整合一个可靠而高度灵敏的光学检测系统，就构成了最新的,Mastercycler ep realplex,实时荧光定量,PCR,系统。,Mastercycler ep,realplex-,模块化设计,A.Mastercycler ep,realplex,2,96 LED Array excitation at 470 nm,1,channel photomultiplier(CPM),2,detection wavelenghts:520 nm and 550 nm,B.Mastercycler ep,realplex,4,96 LED Array excitation at 470 nm,2,channel photomultipliers(CPM),4,detection wavelengths:520 nm,550 nm,580 nm,605 nm,Two Thermo modules,Aluminum block(ep gradient):4C/s heating,3C/s cooling,Silver block(ep gradient S):6C/s heating,4.5C/s cooling,impulse PCR,Mastercycler ep realplex,2,：,光路特征,96,LED Array,CPM Detector,每个,CPM,上带两个滤光片,96孔激发模式,由96个,LED（,光电二级管）组成的光阵列，依一种一一对应的关系将每个反应管中的荧光染料激发，,SYBR,Green,FAM,VIC,TET,HEX,ROX,JOE and TAMRA,优点:,设计紧凑，无可移动部件,低,能耗,无光密度损失,相对于传统的卤素灯，寿命更长,检测器-通道式光电倍增管,相对于传统的光电倍增管，新颖的通道光电倍增管（,CPM）,结构更坚固，更不易被磁场所干扰,快速数据获取:15秒检测4种染料,RT CCD,iCycler,Cooled CCD,ABI 7000,ABI 7900,7300,PMT,RotoGene,Sensitivity,CPM,realplex ep,敏感性,-,Photon cascade creates exponential signal amplification,-“Gain”of PMT adjusts strength of amplification,hv,hv,e,-,e,-,CCD,Mirror,CCD,vs,PMT,检测性能对比,CCD,无法克服检测的边缘效应,快速,qPCR,新理念,非常快速的温度控制速度，,配合快速的检测、直观的编程与软件分析，缩短了,实时定量,PCR,的整体时间。,兼顾速度与可靠性,优化参数实现快速,PCR,试剂与耗材均为开放,快速,Q PCR,的实现,主要因素:,快速升/降温,Heating rate:6,o,C/s,Cooling rate:4.5,o,C/s,on average,a time saving of 15-20 min.,减少,holding,时间,快速数据获取,减少反应体积,MasterCycler ep RealPlex,Software,Mastercycler ep realplex Software,The software offer the following possibilities for evaluating or viewing the recorded data:,Raw data,Absolute Quantification,Relative quantification,Melting point determination,Endpoint determination,+/-assays,gene identification,fluorimeter,Online analysis of raw data as they appear,Software-Absolute Quantification,Software Modules:Plate Layout for Absolute Quantification,Red boxes-standards,Blue boxes-unknowns,Yellow boxes-“-”control,Choose a dye,Auto Series function,Creating dilution series for absolute quantification is easily done using the“Auto Series”function,Mastercycler ep,realplex-,SoftwareUser can create sub assays to perform different experiments in the same plate,provided that the experiments share the same PCR protocol.,Mastercycler ep,realplex-,Software,Software Modules:PCR Program,Melting curve protocol,PCR protocol,1,to 5 step protocol,temp function,pause,melting curve,end point,sound,hold,Measuring point,Measuring point for melting curve analysis,Mastercycler ep,realplex-,Software,ep Gradient S:,1-24,o,C(silver block),freely programmable within 30 to 99,o,C,useful for protocol optimization,a way to develop fast protocols,Mastercycler ep,realplex-,Software,Software Modules:Monitoring,Mastercycler ep,realplex-,Software,Data Analysis:Quantification,Mastercycler ep,realplex-,Software,Data Analysis:Melting Curve Analysis,Mastercycler ep,realplex-,Software,Data Analysis:End Point Detection,Mastercycler ep,realplex-,Software,Data Analysis:+/-Assays,