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1、Bio-rad实时荧光定量PCR仪,BIO-RAD Gene Expression Division,Outline,Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Real Time PCR and Conventional PCR,技术背景,本来，PCR技术是为了将样本中微量的DNA模版放大以便研究模版特性
2、 随着研究的深入，需要了解样本中基因的表达模式与疾病的关系，这就要了解标本中的DNA原始拷贝数。,Denaturation Primer Annealing Elongation,Repeat,常规 PCR Process,In theory, product accumulation is proportional to 2n, where n is the number of amplification cycle repeats,A linear increase follows exponential Eventually plateaus,Cycle #,Reality vs. Th
3、eory,Amplification is exponential, but the exponential increase is limited:,常规PCR方法的局限性分析：,无法对起始模板准确定量,只能对终产物进行分析 对终产物分析，受PCR过程平台效应的干扰，定量准确度难以提高（相对误差1000%）；存在扩增产物的污染问题。 必须在扩增后用电泳方法分析,费时费力而且EB有毒 无法对扩增反应实时检测,C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.,Cycle,End Po
4、int,PCR: Theory vs. Reality,实时定量PCR技术，是指在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得“可见”，最后通过Ct值和标准曲线对样品中的未知样品 的起始浓度进行定量的方法。,Real-Time PCR,Quantitative PCR 或 Real time PCR 是确定样品中DNA (或 cDNA) 拷贝 数最敏感、最准确的方法,Quantitative PCR /Real time PCR,C(t)s from 96 replicates are nearly identical, but the final f
5、luorescence varies.,Cycle,End Point,C(t),PCR: Theory vs Reality,Quantitative information comes from monitoring the early stages of amplification.,Cycle #,Theoretical,Real Life,Log Target DNA,Detector,定量的最佳时期,常用的定量方法,相对定量：相对于另一参照样本的量； 绝对定量：用标准品作标准曲线来推算未知的样本的量。,Quantitative PCR和常规PCR技术的区别,常规PCR是通过电泳对扩
6、增反应的最终产物进行定性分析（定量不准确）； Quantitative PCR是在PCR反应体系中加入荧光基团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得“可见”，通过Ct值和标准曲线对样品中的DNA (or cDNA) 的起始浓度进行定量的方法（准确定量） 。,Introduction to Quantitative PCR,PCR仪 + 监测、分析系统 + 荧光标记,采用半导体加热和制冷 加热速度 3.3C /秒 ，制冷速度 2C/秒 升降温速度可调 控温准确性：0.3,定量PCR仪应该具备的特点：杰出的温度控制,温度梯度选择可以允许使用者在同一次扩增中优化复性温度。,定量P
7、CR仪应该具备的特点：梯度PCR功能,采用多点温控和传感技术，可以实现梯度PCR功能,采用0.2ml的离心管, 排管和 96孔PCR反应板。 降低消耗品成本。,定量PCR仪应该具备的特点： 消耗品成本低，样品容量大,同时扩增和检测96个样品,Introduction to Quantitative PCR,PCR仪 + 监测、分析系统 + 荧光标记,定量PCR仪应该具备的特点： 先进的光路设计-多孔样品同时检测,Fluorescence Detection,Multiplexing-同时检测5色荧光,Cycle,Log Relative Fluorescence,Multiplexing,定量
8、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,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,
9、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,5,Extension,Extension Co
10、ntinued Apply Excitation Wavelength,Repeat,DNA binding dyes,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
11、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 furthe
12、r with thermal gradient, etc.,Cleavage Probes (TaqManTM),5,3,Q,F,Taq,5,q,3,l,3,5,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,d.NTPs,Thermal Stable DNA Polymerase,Primers,Add Master Mix and Sa
13、mple,Denaturation,Annealing,Reaction Tube,l,5,3,Extension Step,1. Strand Displacement,2. Cleavage,3. Polymerization Complete,4. Detection,l,Cleavage-based assay: TaqManTM,Cleavage Probes (TaqManTM),Advantages! Generates a robust cumulative fluorescence signal Simple to design and synthesize compared
14、 to other hybridization probes (i.e. beacons) Ideal approach for multiplex assays SNP (Single Nucleotide Polymorphism) assay possible However More expensive than DNA binding dyes,Molecular Beacons,R,Q,Molecular Beacon,5,3,R,Q,探针有核心的杂交区 探针有自互补末端 在未杂交时探针呈发夹状 报道子, 荧光素在探针的 5 端 猝灭子在探针的3 端,分子 Beacons,当探针是
15、发夹结构时，在报道子和猝灭子间有直接的能量转移,Beacon 构造,Molecular Beacons,Advantages! Good for SNP (Single Nucleotide Polymorphism) detection Multiplex assays possible However Molecular Beacons Are DIFFICULT to Design and Synthesize Does NOT generate a cumulative fluorescence signal, much weaker signal than the 5 Nucleas
16、e Assay (Cleavage probe) Expensive!,A Few Words about Probes,Hybridization oligos should have Tms 6 12 degrees higher than the associated primers. Avoid secondary structure in the complementary region of the probe. Avoid Gs at the 5 end of the probe sequence. Use oligo analysis tools to check probe
17、for: Dimerization Secondary Structure Cross Reactivity with Primers,Each method has advantages and disadvantages Bio-Rad Real-Time Instrumentation is equipped to handle all chemistries One method may be more appropriate for an application over another,Which Method to Use?,Outline,Real Time PCR and C
18、onventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Ct value and Real-Time Quantitative PCR Theory,基线（baseline) 阈值(threshold),基线（baseline）的设置以PCR反应的前15个循环的荧光信号作为荧光本底信号 阈值(threshold)的设置一般是3-15个循环的荧光信号的标准偏
19、差的10倍。 PCR扩增信号进入相对稳定对数增长期时的荧光值。,阈值循环数Ct,PCR扩增过程中荧光信号开始由本底进入指数增长阶段所对应的循环次数，也就是荧光信号达到阈值时的循环次数。,从图中的重复实验中可以直观地看到，随着PCR反应过程，荧光信号从基线经一个转折点进入指数期、线性期和最终的平台期，尽管平台期DNA拷贝数波动很大，CT值却是相对固定的。,Ct值与起始模板的关系,Threshold Cycle, CT,如果用不同浓度模版DNA作PCR，可以看出模版浓度越高，CT值越小。,模板起始浓度越高，Ct值越小 Ct值与模板起始拷贝数的对数存在线性关系,Ct值与模板起始浓度的关系,如果模板浓
20、度增加1倍，Ct值就提前1个循环到达 如果模板浓度减少1倍，Ct值就滞后1个循环到达,1 CT Difference = 2 fold difference in starting template amount 3.3 CT Difference = 10 fold difference in starting template amount,ProductT=(Template0)2n Where n=Number of Cycles,Mathematic Implications,Ideal PCR,Threshold Cycle, Ct, is a reliable indicator
21、 of initial copy number,利用已知起始拷贝数的标准品可作出标准曲线，其中横坐标代表起始拷贝数的对数，纵坐标代表Ct值。因此，只要获得未知样品的Ct值，即可从标准曲线上计算出该样品的起始拷贝数。,Absolute and Relative quantification,Quantification,Normalization of RT-PCR using reference genes,Determines changes in steady state transcription of a gene or genes Expression of the gene/s o
22、f interest is normalized against a reference gene/s (housekeeping gene/s) with no or insignificant expression variation Examples of some reference genes/housekeeping genes used : b-Actin, GAPDH, 18s rRNA b-2 Micro globulin, Cyclophilins,Beta-Actin Ornithine decarboxylase (ODC) S-adenysyl methionine
23、decarboxylase (SAMDC) Human Prostate and Thymus,Real-Time Multiplex PCR: Gene Expression,Real-time Multiplex Gene Expression,Beta-Actin,ODC,SAMDC,Prostate Ct : 23.25 Thymus Ct : 26.93,Prostate Ct : 23.10 Thymus Ct : 25.53,Prostate Ct : 21.25 Thymus Ct : 21.90,Only possible with DNA Binding dyes (SYB
24、R Green I) and after completed real time PCR Determines the temperature at which 50% of the DNA molecules separate into two strands - or “melts” apart Discriminates by Melting Temperature (Tm), Tm is dependent on: - sequence (G/C content) - length,What is Melt Curve Analysis?,Fluorescence vs. Temper
25、ature,Melt curve showing two amplified products,Melt Curve Check specificity of the reaction,Collecting at Higher Temperatures; does that work to avoid detection of primer dimers?,Collecting at 82 C would record specific product only,Primer Dimers Impact on the Assay,Outline,Real Time PCR and Conven
26、tional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCR,Laboratory Technique,Good Laboratory Technique,Do not underestimate the importance of using: Screw cap tubes Aerosol barrier tips Hot-start polymerase Maste
27、r mixes Replicates Serial dilutions And the golden rule: Pipet only once into each well,Same Reagents, Different Hands,Good Technique,Poor Technique,Cycle,Cycle,Outline,Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Techn
28、ique Primer Design for Real-Time PCR,Primer Design for Real-Time PCR,Primer Design for Real-Time PCR,Targets an amplicon length of 75 to 200 bp 50 to 60% overall GC content Limit secondary structure Limit stretch of G or Cs longer than 3 bases No stable interactions between primers (primer/dimer) Pl
29、ace Cs and Gs on ends of primers, but no more than 2 in the last 5 bases on 3 end,Primer Design: Step by Step,Evaluate target sequence for secondary structure. Identify regions of 100 bp as potential amplicons. Scan sequence by eye or use a program to select potential primer pairs.* Use oligo analysis tools to check primers for: Dimerization Secondary Structure Cross Reactivity *Repeat if necessary,谢 谢 ！,