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技術背景 本來，本來，PCRPCR技術是為了將樣本中微量的技術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、達模式隨著研究的深入，需要了解樣本中基因的表達模式與疾病的關系，這就要了解標本中的與疾病的關系，這就要了解標本中的DNADNA原始拷貝原始拷貝數。數。DenaturationPrimer AnnealingElongation53535353535355TaqTaqRepeat常規常規 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:常規常規PCR方法的局限性分析：方法的局限性分析：無法對起始模板準確定量無法對起始模板準確定量, ,只能對終產物進行只能對終產物進行分析分析對終產物分析，受對終產物分析，受PCRPCR過程平臺效應的干擾，過程平臺效應的干擾，定量準確度難以提高相對誤差定量準確度難以提高相對誤差1000%1000%；存在

4、擴增產物的污染問題。存在擴增產物的污染問題。必須在擴增后用電泳方法分析必須在擴增后用電泳方法分析, ,費時費力而且費時費力而且EBEB有毒有毒無法對擴增反響實時檢測無法對擴增反響實時檢測 C(t)s from 96 replicates are nearly identical, but the final fluorescence varies.CycleEnd PointPCR: Theory vs. Reality 實時定量PCR技術，是指在PCR反響體系中參加熒光基團，利用熒光信號積累實時監測整個PCR進程，使每一個循環變得“可見，最后通過Ct值和標準曲線對樣品中的未知樣品 的起始濃度

5、進行定量的方法。 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) 拷貝拷貝數最敏感、最準確的方法數最敏感、最準確的方法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：用標準品作標準曲線來推算未知的絕對定量：用標準品作標準曲線來推算未知的樣本的量。樣本的量。Quantitative PCR和常規和常規PCR技術的區別技術的區別 常規常規PCR是通過電泳對擴增反響的最終產物進是通過電泳對擴增反響的最終產物進行定性分析定量不準確；行定性分析定量不準確； Quantitative PCR是在是在PCR反響體系中參加熒反響體系中參加熒光基團，利用熒光信號積累實時監測整個光基

7、團，利用熒光信號積累實時監測整個PCR進程，使每一個循環變得進程，使每一個循環變得“可見可見，通過，通過Ct值值和標準曲線對樣品中的和標準曲線對樣品中的DNA (or cDNA) 的起始的起始濃度進行定量的方法準確定量濃度進行定量的方法準確定量 。Introduction to Quantitative PCRPCR儀儀 + 監測、分析系統監測、分析系統 + 熒光標記熒光標記 溫度梯度溫度梯度選擇可以選擇可以允許使用者在同一允許使用者在同一次擴增中優化復性次擴增中優化復性溫度。溫度。采用多點溫控和傳采用多點溫控和傳感技術，可以實現感技術，可以實現梯度梯度PCR功能功能采用的離心管采用的離心管,

8、 排管和排管和 96孔孔PCR反響板。反響板。 降低消耗品本錢降低消耗品本錢。同時擴增和檢測同時擴增和檢測96個樣品個樣品Introduction to Quantitative PCRPCR儀儀 + 監測、分析系統監測、分析系統 + 熒光標記熒光標記 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： 熔點曲線功能、熔點曲線功能、MGB探針探針v可兼容的試劑種類：可兼容的試劑種類： 所有國產與進口試劑所有國產與進口試劑 iQ5 Data Analysis Software iQ5 Data Analysis Software 標準曲線標準曲線定量計算定量計算基因表達分析I

10、ntroduction to Quantitative PCRPCR儀儀 + 監測、分析系統監測、分析系統 + 熒光標記熒光標記 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基線基線baseline) baseline) 閾值閾值(threshold)(threshold) 基線基線baselinebaseline的設置以的設置以PCRPCR反響的前反響的前1515個循環的熒光信號作為熒光本底信號個循環的熒光信號作為熒光本底信號 閾值閾值(threshold)(threshold)的設置一般是的設置一般是3-153-15個循個循環的熒光信號的標準偏差的環的熒光信號的標準偏差的1010倍。倍。 PCRPCR擴增信號進

20、入相對穩定對數增長期時擴增信號進入相對穩定對數增長期時的熒光值。的熒光值。閾值循環數閾值循環數CtCt PCRPCR擴增過程中熒光信號開始由本底進入指數增擴增過程中熒光信號開始由本底進入指數增長階段所對應的循環次數，也就是熒光信號到長階段所對應的循環次數，也就是熒光信號到達閾值時的循環次數。達閾值時的循環次數。 從圖中的重復實驗中可以直觀地看到，隨著從圖中的重復實驗中可以直觀地看到，隨著PCRPCR反反響過程，熒光信號從基線經一個轉折點進入指數響過程，熒光信號從基線經一個轉折點進入指數期、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拷貝拷貝數波動很大，數波動

21、很大，CTCT值卻是相對固定的。值卻是相對固定的。CtCt值與起始模板的關系值與起始模板的關系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的對數存在線性關系Ct值與模板起始濃度的關系值與模板起始濃度的關系 如果模板濃度增加1倍，Ct值就提前1個循環到達 如果模板濃度減少1倍，Ct值就滯后1個循環到達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利用起始拷貝數的標準品可作出標準曲線，其中橫坐標代表起始拷貝數的對數，縱坐標代表Ct值。因此，只要獲得未知樣品的Ct值，即可從標準曲線上計算出該樣品的起始拷貝數。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