[生物学]Bio-Rad定量PCR说明书

1、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 PCRReal Time PCR and Conventional PCR技术背景 本来，本来，PCRPCR技术是为了将样本中微量的技术是为了将样本中微量的DNADNA模版放大模版放大以便研究模版特性以便研究模版特性 随着研究的深入，需要了解样本中基因的表

2、达模式随着研究的深入，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与疾病的关系，这就要了解标本中的DNADNA原始拷贝原始拷贝数。数。DenaturationPrimer AnnealingElongation53535353535355TaqTaqRepeat常规常规 PCR ProcessIn theory, product accumulation is proportional to 2n, where n is the number of amplification cycle repeatsnA linear increase follows exponential

3、nEventually plateausCycle #TheoreticalReal LifeLog Target DNAReality vs. TheoryAmplification is exponential, but the exponential increase is limited:常规常规PCR方法的局限性分析：方法的局限性分析：无法对起始模板准确定量无法对起始模板准确定量, ,只能对终产物进行只能对终产物进行分析分析对终产物分析，受对终产物分析，受PCRPCR过程平台效应的干扰，过程平台效应的干扰，定量准确度难以提高相对误差定量准确度难以提高相对误差1000%1000%；存在

4、扩增产物的污染问题。存在扩增产物的污染问题。必须在扩增后用电泳方法分析必须在扩增后用电泳方法分析, ,费时费力而且费时费力而且EBEB有毒有毒无法对扩增反响实时检测无法对扩增反响实时检测 C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointPCR: Theory vs. Reality 实时定量PCR技术，是指在PCR反响体系中参加荧光基团，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

5、进行定量的方法。 Real-Time PCR Quantitative PCR 或或 Real time PCR 是确定样品中是确定样品中DNA (或或 cDNA) 拷贝拷贝数最敏感、最准确的方法数最敏感、最准确的方法Quantitative PCR /Real time PCR C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointC(t)PCR: Theory vs RealityQuantitative information comes from mo

6、nitoring the early stages of amplification.常用的定量方法常用的定量方法 相对定量：相对于另一参照样本的量；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绝对定量：用标准品作标准曲线来推算未知的样本的量。样本的量。Quantitative PCR和常规和常规PCR技术的区别技术的区别 常规常规PCR是通过电泳对扩增反响的最终产物进是通过电泳对扩增反响的最终产物进行定性分析定量不准确；行定性分析定量不准确； Quantitative PCR是在是在PCR反响体系中参加荧反响体系中参加荧光基团，利用荧光信号积累实时监测整个光基

7、团，利用荧光信号积累实时监测整个PCR进程，使每一个循环变得进程，使每一个循环变得“可见可见，通过，通过Ct值值和标准曲线对样品中的和标准曲线对样品中的DNA (or cDNA) 的起始的起始浓度进行定量的方法准确定量浓度进行定量的方法准确定量 。Introduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 温度梯度温度梯度选择可以选择可以允许使用者在同一允许使用者在同一次扩增中优化复性次扩增中优化复性温度。温度。采用多点温控和传采用多点温控和传感技术，可以实现感技术，可以实现梯度梯度PCR功能功能采用的离心管采用的离心管,

8、 排管和排管和 96孔孔PCR反响板。反响板。 降低消耗品本钱降低消耗品本钱。同时扩增和检测同时扩增和检测96个样品个样品Introduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 DetectorEmissionFilterLight SourceExcitationFilter53FQTubulin53HQGapdH53TXQb b-actin53Cy5QCyclophilinMultiplexing-CycleLog Relative FluorescenceTexas RedFAMHexCy510100100014

9、71013161922252831343740434649TubulinCyclophilinbeta ActinGAPDHMultiplexingv可兼容的定量方法：可兼容的定量方法： SYBR Green I、Taqman探针、探针、Beacon探针、探针、FRET探针探针v可兼容的突变检测方法：可兼容的突变检测方法： 熔点曲线功能、熔点曲线功能、MGB探针探针v可兼容的试剂种类：可兼容的试剂种类： 所有国产与进口试剂所有国产与进口试剂 iQ5 Data Analysis Software iQ5 Data Analysis Software 标准曲线标准曲线定量计算定量计算基因表达分析I

10、ntroduction to Quantitative PCRPCR仪仪 + 监测、分析系统监测、分析系统 + 荧光标记荧光标记 l ll ll ll lDetection ChemistriesNon-specific DNA binding dyesSYBR Green ISYBR GoldEthidium Bromide Specific Hybridization ProbesCleavage Probes (TaqManTM)Molecular beaconsScorpionsTMAmplifluorTMLUXTMdual-oligo FRET pairsQuantitative P

11、CR Detection ChemistriesDNA Binding Dyes35BDBD5BDBDBDExtension53535353Extension ContinuedApply ExcitationWavelength535355TaqTaq353TaqTaq55RepeatDNA binding dyesBDBDBDBDBDBDBDBDBDBDlllllDNA Binding Dyes Advantages!Inexpensive compared to hybridization probesNo additional design work than the primer u

12、sed for PCR reaction However Not template specific, will bind ALL double stranded DNA inducing primer-dimer and unspecific amplicon formationMultiplex assays not possibleTypical “first step experiment: Evaluate Primer SpecificityUsing Melt Curve Analysis Evaluate Primer Pair EfficienciesBy running s

13、erial dilutions of template as standards Identify Sub-Optimal aspects of assayOptimize further with thermal gradient, etc.Cleavage Probes (TaqManTM)53QFTaq5q3l l3535FQCleavage-based assay:TaqManTM5533d.NTPsThermal Stable DNA PolymerasePrimers5353535353535353Add Master Mix and SampleDenaturation53535

14、3535353AnnealingReaction TubeTaql53RQProbe53RQ535353RQExtension Step531. Strand DisplacementTaq3QR5533QTaqR52. Cleavage3. PolymerizationComplete53QTaqR354. Detection533QTaqR5lRCleavage-based assay:TaqManTMCleavage Probes (TaqManTM)Advantages! Generates a robust cumulative fluorescence signal Simple

15、to design and synthesize compared to other hybridization probes (i.e. beacons) Ideal approach for multiplex assays SNP (Single Nucleotide Polymorphism) assay possibleHowever More expensive than DNA binding dyesMolecular BeaconsRQMolecularBeacon535353RQMolecular BeaconsAdvantages! Good for SNP (Singl

16、e Nucleotide Polymorphism) detection Multiplex assays possibleHowever Molecular Beacons Are DIFFICULT to Design and Synthesize Does NOT generate a cumulative fluorescence signal, much weaker signal than the 5 Nuclease Assay (Cleavage probe) Expensive!Hybridization oligos should have Tms 6 12 degrees

17、 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 for:DimerizationSecondary StructureCross Reactivity with Primers Each method has advantages and disadvantages Bio

18、-Rad Real-Time Instrumentation is equipped to handle all chemistries One method may be more appropriate for an application over anotherWhich Method to Use?Outline Real Time PCR and Conventional PCR Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique P

19、rimer Design for Real-Time PCRCt value and Real-Time Quantitative PCR Theory基线基线baseline) baseline) 阈值阈值(threshold)(threshold) 基线基线baselinebaseline的设置以的设置以PCRPCR反响的前反响的前1515个循环的荧光信号作为荧光本底信号个循环的荧光信号作为荧光本底信号 阈值阈值(threshold)(threshold)的设置一般是的设置一般是3-153-15个循个循环的荧光信号的标准偏差的环的荧光信号的标准偏差的1010倍。倍。 PCRPCR扩增信号进

20、入相对稳定对数增长期时扩增信号进入相对稳定对数增长期时的荧光值。的荧光值。阈值循环数阈值循环数CtCt PCRPCR扩增过程中荧光信号开始由本底进入指数增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，也就是荧光信号到长阶段所对应的循环次数，也就是荧光信号到达阈值时的循环次数。达阈值时的循环次数。 从图中的重复实验中可以直观地看到，随着从图中的重复实验中可以直观地看到，随着PCRPCR反反响过程，荧光信号从基线经一个转折点进入指数响过程，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，尽管平台期期、线性期和最终的平台期，尽管平台期DNADNA拷贝拷贝数波动很大，数波动

21、很大，CTCT值却是相对固定的。值却是相对固定的。CtCt值与起始模板的关系值与起始模板的关系Cycle 如果用不同浓度模版如果用不同浓度模版DNADNA作作PCRPCR，可以看出模版，可以看出模版浓度越高，浓度越高，CTCT值越小。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值与模板起始浓度的关系 如果模板浓度增加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 amount3.3 CT D

22、ifference = 10 fold difference in starting template amountProductT=(Template0)2nWhere n=Number of CyclesMathematic ImplicationsIdeal PCRC o p y N u m b e r v s. Ct - Sta n d a r d C u r v ey = -3 .31 9 2x + 3 9 .77 2R2 = 0 .9 9 6 7051 01 52 02 53 03 54 001234567891 01 1Lo g o f co p y nu m b er (1 0

23、n)CtThreshold Cycle, Ct, is a reliable indicator of initial copy number利用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 quantificationQuantificationNormalization of RT-PCR using reference genesDetermines changes in steady state transcription of a

24、 gene or genesExpression of the gene/s of interest is normalized against a reference gene/s (housekeeping gene/s) with no or insignificant expression variationExamples of some reference genes/housekeeping genes used : b-Actin, GAPDH, 18s rRNA b-2 Micro globulin, CyclophilinsBeta-Actin Ornithine deca

25、rboxylase (ODC)S-adenysyl methionine decarboxylase (SAMDC)Human Prostate and ThymusBeta-ActinODCSAMDCProstate Ct : 23.25 Prostate Ct : 23.10 Only possible with DNA Binding dyes (SYBR Green I) and after completed real time PCR Determines the temperature at which 50% of the DNA molecules separate into

26、 two strands - or “melts apartDiscriminates by Melting Temperature (Tm), Tm is dependent on:- sequence (G/C content)- lengthWhat is Melt Curve Analysis?Fluorescence vs. Temperaturechange in fluorescencesingle amplified productTmMelt curve showing two amplified productsTwo amplified productsTmTmMelt

27、CurveCheck specificity of the reactionCollecting 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 AssayOutline 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 PCRLaboratory Technique Do not underestimate the importance of using:Screw