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1、Realtime PCR Detection System实时定量PCR技术及其应用,南 京 农 业 大 学 陈 溥 言,定量PCR技术的产生1992年由Higuchi等人第一次报告：使用EB内插染料法插至双链DNA，经改装的带有冷CCD的PCR仪检测样品的荧光强度PCR循环=双链DNA=染料=荧光后来用与双链DNA有更强结合力的SYBR Green I取代EB,iCycler iQ Real-Time PCR Detection System,荧光定量 PCR仪荧光定量PCR仪是一种带有激发光源和荧光信号检测系统的PCR仪，通常配有电脑系统及相应的分析软件。通过荧光染料或荧光标记的特异性的探
2、针，对PCR产物进行标 记跟踪，实时在线监控反应过程，结合相应的软件可以对结果进行分析，计算待测样品的初始模板量。,Realtime PCR detection system working procedure,荧光定量实时PCR与普通PCR的比较实时在线监控 对样品扩增的整个过程进行实时监控，能够实时地观察到产物的增加，直观地看到反应的对数期降低反应的非特异性 使用引物和荧光探针同时与模板特异性结合，提高了PCR反应的特异性增加定量的精确性 全程监控，准确的算法进行定量结果分析更加快捷方便，无需跑胶,Realtime PCR&Regular PCR Comparasion II,Princi
3、ple I:Primer-Fluoresent,Principle II:Primers&Probe(Beacon)-Fluoresent,Principle IIISYBR Green I dye,探针设计的一般原则：扩增片段的长度不应太大,一般小于300bp探针不能和任一引物互补.探针保证特异性的前提下尽可能的短，长度不超过30bp.探针的Tm值至少比引物的Tm值高5度探针应尽可能的靠近引物如用于检测多态位点,多态位点应尽可能的靠近探针中部.探针5端不能是G，G有淬灭作用.,Pealtime PCR Machine Working Principle,Perform Realtime PC
4、R 1.,Perform Realtime PCR 2.-Plate set Up,Perform Realtime PCR 3.Plate Set Up II.,Rat-Chro.1,Mouse-Chro.19,Human-Chro.11,Exon-X,340bp,193bp,Exon1a-1,159bp,701bp,GC Rich,Exon1,125bp,26bp,1,173bp,Exon2,118bp,25,854bp,Exon3,154bp,ExonX,158bp,430bp,Exon1a,Unkown Seq.,293bp,74bp,525bp,Rat GC Rich,/Exon1,
5、125/26bp,1,198bp,Rat Exon2,121bp,24,494bp,Exon3,154bp,Exon-1a,162bp,837bp,Rat/Mouse Exon1a Region,225bp,Exon-1b,254bp,26,288bp,Exon2,154bp,*,:Exons that could be detected from realtime PCR.,:Exons that couldnt be detected from realtime PCR.,:Unkown Seq.that could be detected from realtime PCR.,Rat E
6、xon2,121bp,ATG,ATG,ATG,804 bp,标准曲线由系列稀释的已知浓度的样品做标准曲线计算待测样品的初始模板浓度,PCR Amp/Cycle Graph for FAM-490,PCR Amp/Cycle Graph for FAM-490,Melt Curve Graph for FAM-490,一.实时监测产物量，计算初始模板量1.基础研究的基因表达分析研究2.临床病原体检测3.食品卫生检疫,2.临床病原体检测 a.病人体液中目标基因的检测.病毒,寄生虫,细菌 市场上有各种开发好的试剂盒 3.食品卫生检疫 a.转基因食品的检疫 根据转入基因的特异性区段设计探针 b.食品中
7、病原微生物的检测,二.基因型分析1.利用扩增信号的种类来分型2.根据熔解曲线的不同来分型,双Taqman探针法检测野生型和突变型 在基因型分析中，可采用两种不同的Taqman探针（分别针对野生型和突变型)，即一个探针以一种荧光素（Flr）标记，而另一个探针则用不同的荧光物（Tet）标记。如果只有1种信号被扩增出来，则样本为对应的基因型（野生型或突变型）的纯合子；如二者都被有效地扩增出来，则样本为杂合型。,实时监测产物量，计算初始模板量1.基础研究的基因表达分析研究2.临床病原体检测3.食品卫生检疫,基础研究的基因表达分析研究 定量PCR和Array技术的结合 Oncogene 2002 Jul
8、 4;21(29):4587-94 Expression profiling of microdissected pancreatic adenocarcinomas.定量PCR和LCM技术的结合 特定细胞中基因表达变化 Proc.Natl.Acad.Sci.USA,Vol.99,Issue 4,2234-2239,February 19,2002 Laser capture microdissection analysis of gene expression in macrophages from atherosclerotic lesions of apolipoprotein E-de
9、ficient mice,临床病原体检测.病人体液中目标基因的检测.病毒,寄生虫,细菌 市场上有各种开发好的试剂盒,食品卫生检疫 a.转基因食品的检疫 根据转入基因的特异性区段设计探针 b.食品中病原微生物的检测,system that fits directly above the iCycler thermal cycler.It covers the widest range of excitation/emission wavelengths available,facilitating the greatest array of fluorescent PCR strategies.
10、With this upgrade the iCycler thermal cycler expands the advantages PCR brings to your research.Features:Real-time analysis can be viewed online,during the PCR Range of fluorophore excitation and emission from 400700 nm 4 different fluorophores can be multiplexed per sample tube 96 samples can be tr
11、acked simultaneously,maximizing throughput Sample data can be reanalyzed at any time Raw data files are user accessible Easy,intuitive software speeds setup and data presentation Storage of hundreds of protocols Standard PCR plate format accommodates existing protocols and minimizes costs User inter
12、face offers easy yet powerful options for data analysis Real-time quantitative PCR provides accurate quantitation,accelerating lead identification in drug discovery and improving viral load assessment iCycler iQ software is designed to automate analysis options,including quantitative and melt-curve
13、analysis;you can also choose to reanalyze raw data at any point post-run Monitor the thermal protocol in real time,saving time and effort in your analysis Closed-tube analysis reduces contamination concerns and speeds time to resultsPractice of the patented polymerase chain reaction(PCR)process requ
14、ires a license.The iCycler iQ system includes a licensed thermal cycler and may be used with PCR licenses available from Applied Biosystems.Its use with authorized reagents also provides a limited PCR license in accordance with the label rights accompanying such reagents.Some applications may require licenses from other parties.,双Taqman探针法检测野生型和突变型 在基因型分析中，可采用两种不同的Taqman探针（分别针对野生型和突变型)，即一个探针以一种荧光素（Flr）标记，而另一个探针则用不同的荧光物（Tet）标记。如果只有1种信号被扩增出来，则样本为对应的基因型（野生型或突变型）的纯合子；如二者都被有效地扩增出来，则样本为杂合型。,
