Bio-Rad定量PCR說明書

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技術(shù)背景 本來，本來，PCRPCR技術(shù)是為了將樣本中微量的技術(shù)是為了將樣本中微量的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 隨著研究的深入，需要了解樣本中基因的表

2、達(dá)模式隨著研究的深入，需要了解樣本中基因的表達(dá)模式與疾病的關(guān)系，這就要了解標(biāo)本中的與疾病的關(guān)系，這就要了解標(biāo)本中的DNADNA原始拷貝原始拷貝數(shù)。數(shù)。DenaturationPrimer AnnealingElongation53535353535355TaqTaqRepeat常規(guī)常規(guī) 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:常規(guī)常規(guī)PCR方法的局限性分析：方法的局限性分析：無法對起始模板準(zhǔn)確定量無法對起始模板準(zhǔn)確定量, ,只能對終產(chǎn)物進(jìn)只能對終產(chǎn)物進(jìn)行分析行分析對終產(chǎn)物分析，受對終產(chǎn)物分析，受PCRPCR過程平臺效應(yīng)的干擾，過程平臺效應(yīng)的干擾，定量準(zhǔn)確度難以提高（相對誤差定量準(zhǔn)確度難以提高（相對誤差1000%1000%）

4、；）；存在擴(kuò)增產(chǎn)物的污染問題。存在擴(kuò)增產(chǎn)物的污染問題。必須在擴(kuò)增后用電泳方法分析必須在擴(kuò)增后用電泳方法分析, ,費(fèi)時費(fèi)力而費(fèi)時費(fèi)力而且且EBEB有毒有毒無法對擴(kuò)增反應(yīng)實(shí)時檢測無法對擴(kuò)增反應(yīng)實(shí)時檢測 C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointPCR: Theory vs. Reality 實(shí)時定量實(shí)時定量PCR技術(shù)，是指在技術(shù)，是指在PCR反應(yīng)體系中反應(yīng)體系中加入加入熒光基團(tuán)熒光基團(tuán)，利用熒光信號積累實(shí)時監(jiān)測整個，利用熒光信號積累實(shí)時監(jiān)測整個PCR進(jìn)

5、程，使每一個循環(huán)變得進(jìn)程，使每一個循環(huán)變得“可見可見”，最后通，最后通過過Ct值和標(biāo)準(zhǔn)曲線值和標(biāo)準(zhǔn)曲線對樣品中的未知樣品對樣品中的未知樣品 的起始濃的起始濃度進(jìn)行定量的方法。度進(jìn)行定量的方法。 Real-Time PCR Quantitative PCR 或或 Real time PCR 是確定樣品中是確定樣品中DNA (或或 cDNA) 拷貝拷貝數(shù)最敏感、最準(zhǔn)確的方法數(shù)最敏感、最準(zhǔn)確的方法Quantitative PCR /Real time PCR C(t)s from 96 replicates are nearly identical, but the final fluorescen

6、ce varies.CycleEnd PointC(t)PCR: Theory vs RealityQuantitative information comes from monitoring the early stages of amplification.常用的定量方法常用的定量方法 相對定量：相對于另一參照樣本的量；相對定量：相對于另一參照樣本的量； 絕對定量：用標(biāo)準(zhǔn)品作標(biāo)準(zhǔn)曲線來推算未知的絕對定量：用標(biāo)準(zhǔn)品作標(biāo)準(zhǔn)曲線來推算未知的樣本的量。樣本的量。Quantitative PCR和常規(guī)和常規(guī)PCR技術(shù)的區(qū)別技術(shù)的區(qū)別 常規(guī)常規(guī)PCR是通過電泳對擴(kuò)增反應(yīng)的最終產(chǎn)物進(jìn)是通過電泳對擴(kuò)增反應(yīng)

7、的最終產(chǎn)物進(jìn)行定性分析（行定性分析（定量不準(zhǔn)確定量不準(zhǔn)確）；）； Quantitative PCR是在是在PCR反應(yīng)體系中加入熒反應(yīng)體系中加入熒光基團(tuán)，利用熒光信號積累實(shí)時監(jiān)測整個光基團(tuán)，利用熒光信號積累實(shí)時監(jiān)測整個PCR進(jìn)程，使每一個循環(huán)變得進(jìn)程，使每一個循環(huán)變得“可見可見”，通過，通過Ct值值和標(biāo)準(zhǔn)曲線對樣品中的和標(biāo)準(zhǔn)曲線對樣品中的DNA (or cDNA) 的起始的起始濃度進(jìn)行定量的方法（濃度進(jìn)行定量的方法（準(zhǔn)確定量準(zhǔn)確定量） 。Introduction to Quantitative PCRPCR儀儀 + 監(jiān)測、分析系統(tǒng)監(jiān)測、分析系統(tǒng) + 熒光標(biāo)記熒光標(biāo)記 溫度梯度溫度梯度選擇可以選擇

8、可以允許使用者在同一允許使用者在同一次擴(kuò)增中優(yōu)化復(fù)性次擴(kuò)增中優(yōu)化復(fù)性溫度。溫度。采用多點(diǎn)溫控和傳采用多點(diǎn)溫控和傳感技術(shù)，可以實(shí)現(xiàn)感技術(shù)，可以實(shí)現(xiàn)梯度梯度PCR功能功能采用采用0.2ml的離心管的離心管, 排管和排管和 96孔孔PCR反應(yīng)板。反應(yīng)板。 降低消耗品成降低消耗品成本。本。同時擴(kuò)增和檢測同時擴(kuò)增和檢測96個樣品個樣品Introduction to Quantitative PCRPCR儀儀 + 監(jiān)測、分析系統(tǒng)監(jiān)測、分析系統(tǒng) + 熒光標(biāo)記熒光標(biāo)記 DetectorEmissionFilterLight SourceExcitationFilter53FQTubulin53HQGapdH5

9、3TXQb b-actin53Cy5QCyclophilinMultiplexing-CycleLog Relative FluorescenceTexas RedFAMHexCy51010010001471013161922252831343740434649TubulinCyclophilinbeta ActinGAPDHMultiplexingv可兼容的定量方法：可兼容的定量方法： SYBR Green I、Taqman探針、探針、Beacon探針、探針、FRET探針探針v可兼容的突變檢測方法：可兼容的突變檢測方法： 熔點(diǎn)曲線功能、熔點(diǎn)曲線功能、MGB探針探針v可兼容的試劑種類：可兼容的

10、試劑種類： 所有國產(chǎn)與進(jìn)口試劑所有國產(chǎn)與進(jìn)口試劑 iQ5 Data Analysis Software iQ5 Data Analysis Software 標(biāo)準(zhǔn)曲線標(biāo)準(zhǔn)曲線定量計(jì)算定量計(jì)算基因表達(dá)分析Introduction to Quantitative PCRPCR儀儀 + 監(jiān)測、分析系統(tǒng)監(jiān)測、分析系統(tǒng) + 熒光標(biāo)記熒光標(biāo)記 l ll ll ll lDetection ChemistriesNon-specific DNA binding dyesSYBR Green ISYBR GoldEthidium Bromide Specific Hybridization ProbesClea

11、vage Probes (TaqManTM)Molecular beaconsScorpionsTMAmplifluorTMLUXTMdual-oligo FRET pairsQuantitative PCR Detection ChemistriesDNA Binding Dyes35BDBD5BDBDBDExtension53535353Extension ContinuedApply ExcitationWavelength535355TaqTaq353TaqTaq55RepeatDNA binding dyesBDBDBDBDBDBDBDBDBDBDlllllDNA Binding D

12、yes Advantages!Inexpensive compared to hybridization probesNo 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 amplicon formationMultiplex assays not possibleTypical “first step” experim

13、ent: Evaluate Primer SpecificityUsing Melt Curve Analysis Evaluate Primer Pair EfficienciesBy running serial dilutions of template as standards Identify Sub-Optimal aspects of assayOptimize further with thermal gradient, etc.Cleavage Probes (TaqManTM)53QFTaq5q3l l3535FQCleavage-based assay:TaqManTM5

14、533d.NTPsThermal Stable DNA PolymerasePrimers5353535353535353Add Master Mix and SampleDenaturation535353535353AnnealingReaction TubeTaql53RQProbe53RQ535353RQExtension Step531. Strand DisplacementTaq3QR5533QTaqR52. Cleavage3. PolymerizationComplete53QTaqR354. Detection533QTaqR5lRCleavage-based assay:

15、TaqManTMCleavage Probes (TaqManTM)Advantages! Generates a robust cumulative fluorescence signal Simple 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

16、 binding dyesMolecular BeaconsRQMolecularBeacon535353RQMolecular BeaconsAdvantages! Good for SNP (Single Nucleotide Polymorphism) detection Multiplex assays possibleHowever Molecular Beacons Are DIFFICULT to Design and Synthesize Does NOT generate a cumulative fluorescence signal, much weaker signal

17、 than the 5 Nuclease Assay (Cleavage probe) Expensive!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 for:Dimeri

18、zationSecondary StructureCross 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 anotherWhich Method to Use?Outline Real Time PCR and Conventional PCR

19、 Introduction to Quantitative PCR Ct value and Real-Time Quantitative PCR Theory Laboratory Technique Primer Design for Real-Time PCRCt value and Real-Time Quantitative PCR Theory基線（基線（baseline) baseline) 閾值閾值(threshold)(threshold) 基線（基線（baselinebaseline）的設(shè)置以）的設(shè)置以PCRPCR反應(yīng)的前反應(yīng)的前1515個循環(huán)的熒光信號作為熒光本底信號個循

20、環(huán)的熒光信號作為熒光本底信號 閾值閾值(threshold)(threshold)的設(shè)置一般是的設(shè)置一般是3-153-15個循個循環(huán)的熒光信號的標(biāo)準(zhǔn)偏差的環(huán)的熒光信號的標(biāo)準(zhǔn)偏差的1010倍。倍。 PCRPCR擴(kuò)增信號進(jìn)入相對穩(wěn)定對數(shù)增長期時擴(kuò)增信號進(jìn)入相對穩(wěn)定對數(shù)增長期時的熒光值。的熒光值。閾值循環(huán)數(shù)閾值循環(huán)數(shù)CtCt PCRPCR擴(kuò)增過程中熒光信號開始由本底進(jìn)入指數(shù)增擴(kuò)增過程中熒光信號開始由本底進(jìn)入指數(shù)增長階段所對應(yīng)的循環(huán)次數(shù)，也就是熒光信號達(dá)長階段所對應(yīng)的循環(huán)次數(shù)，也就是熒光信號達(dá)到閾值時的循環(huán)次數(shù)。到閾值時的循環(huán)次數(shù)。 從圖中的重復(fù)實(shí)驗(yàn)中可以直觀地看到，隨著從圖中的重復(fù)實(shí)驗(yàn)中可以直觀地看

21、到，隨著PCRPCR反反應(yīng)過程，熒光信號從基線經(jīng)一個轉(zhuǎn)折點(diǎn)進(jìn)入指數(shù)應(yīng)過程，熒光信號從基線經(jīng)一個轉(zhuǎn)折點(diǎn)進(jìn)入指數(shù)期、線性期和最終的平臺期，盡管平臺期期、線性期和最終的平臺期，盡管平臺期DNADNA拷貝拷貝數(shù)波動很大，數(shù)波動很大，CTCT值卻是相對固定的。值卻是相對固定的。CtCt值與起始模板的關(guān)系值與起始模板的關(guān)系Cycle 如果用不同濃度模版如果用不同濃度模版DNADNA作作PCRPCR，可以看出模版，可以看出模版濃度越高，濃度越高，CTCT值越小。值越小。 模板起始濃度越高，Ct值越小 Ct值與模板起始拷貝數(shù)的對數(shù)存在線性關(guān)系Ct值與模板起始濃度的關(guān)系值與模板起始濃度的關(guān)系 如果模板濃度增加1

22、倍，Ct值就提前1個循環(huán)到達(dá) 如果模板濃度減少1倍，Ct值就滯后1個循環(huán)到達(dá)1 CT Difference = 2 fold difference in starting template amount3.3 CT Difference = 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

23、= -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 0n)CtThreshold Cycle, Ct, is a reliable indicator of initial copy number利用已知起始拷貝數(shù)的標(biāo)準(zhǔn)品可作出標(biāo)準(zhǔn)曲線，其中橫坐標(biāo)代表起始拷貝數(shù)的對數(shù)，縱坐標(biāo)代表Ct值。因此，只要獲得未知樣品的Ct值，即可從標(biāo)準(zhǔn)曲線上計(jì)算出該樣品的起始拷貝數(shù)。Absolute and Relative quantificationQ

24、uantificationNormalization of RT-PCR using reference genesDetermines changes in steady state transcription of a 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 gen

25、es/housekeeping genes used : b-Actin, GAPDH, 18s rRNA b-2 Micro globulin, CyclophilinsBeta-Actin Ornithine decarboxylase (ODC)S-adenysyl methionine decarboxylase (SAMDC)Human Prostate and ThymusBeta-ActinODCSAMDCProstate Ct : 23.25 Thymus Ct : 26.93Prostate Ct : 23.10 Thymus Ct : 25.53Prostate Ct :

26、21.25Thymus Ct : 21.90 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 two strands - or “melts” apartDiscriminates by Melting Temperature (Tm), Tm is dependent on:- sequence (G/C content)-

27、 lengthWhat is Melt Curve Analysis?Fluorescence vs. Temperaturechange in fluorescencesingle amplified productTmMelt curve showing two amplified productsTwo amplified productsTmTmMelt CurveCheck specificity of the reactionCollecting at Higher Temperatures; does that work to avoid detection of primer

28、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 usin