原理應(yīng)用探針設(shè)計和應(yīng)用實例

實時熒光定量PCR原理及應(yīng)用郝近技術(shù)支持fas229上海吉泰生物科技有限公司內(nèi)容提綱熒光定量PCR原理及應(yīng)用熒光定量PCR實驗引物/探針設(shè)計及實驗條件優(yōu)化StratageneMx3000P熒光定量PCR儀常規(guī)核酸定量方法Southern雜交Northern雜交原位雜交傳統(tǒng)RT-PCR半定量PCR存在的問題

靈敏度準(zhǔn)確性特異性時間RealtimePCR定義

利用熒光信號對PCR反應(yīng)的過程進(jìn)行實時監(jiān)控目的

對起始模板進(jìn)行定量

優(yōu)點

靈敏,重復(fù)性,動態(tài)范圍寬,高通量原理C(t)值與起始模板濃度的對數(shù)成比例應(yīng)用

腫瘤，微生物檢測，SNP等熒光化學(xué)原理SYBRGreenITMTaqMan?/TaqMan-MGB?MolecularBeaconsMGBEclipseTMProbesLux?primersScorpionsTM

AmplifluorQ-ZymeOthers...染料法：探針法：ssDNA+freedye(weakfluorophore)dsDNA=Bindsminorgroove(intensefluorophore)SYBRGreenIdye

(dsDNA-bindingdye)TaqMan?ProbesUnboundprobefreeinsolutionProbeandprimerbindtarget,FREToccursLightEmissionorHeatDonordye(Reporter)Acceptordye(Quencher)LightEnergytransferTaqTaqextendsandhydrolyzesprobe,donordyefreetoemitfluorescence-->accumulationofsignalTaqLightEmissionLight什么是C(t)值C(t)Threshold（域值）Non-QuantitativeNatureofPCREndpointProductsCtEndpoints30~40個循環(huán)后，由于擴增效率下降，產(chǎn)物量差異很大反應(yīng)初期，即剛剛檢測到熒光時，每個反應(yīng)管中產(chǎn)物量差異很小96replicatesofB7genemolecularbeacon傳統(tǒng)的定量方法11,500counts/5200counts=2.2folddifferenceReal-time定量Ct18andCt22,2^(4Ctshift)=16folddifference定量PCR是如何定量的？標(biāo)準(zhǔn)曲線

未知樣品的C(t)值

定量熒光定量PCR應(yīng)用特異核酸（基因）定量RNA(cDNA)基因表達(dá)差異分析基因芯片結(jié)果驗證DNA病原體定性與定量GMO(遺傳改良組織)檢測序列特異性等位基因分型,SNP檢測應(yīng)用之一：病原體檢測與定量絕對定量（特異基因的拷貝數(shù)－病原體的多少）標(biāo)準(zhǔn)品（如質(zhì)粒）：梯度稀釋未知樣品陰性對照（NTC）如何確定標(biāo)準(zhǔn)品的量標(biāo)準(zhǔn)品是通過某種方法制備得到的已知拷貝數(shù)的含有目標(biāo)片段的核苷酸序列通常通過紫外定量等方法測得濃度C(ng/uL)通過核苷酸的相對分子量和標(biāo)準(zhǔn)品序列信息估算出標(biāo)準(zhǔn)品的分子量B這樣拷貝數(shù)A(copy/uL)為：

A=C/B*6.02E+14應(yīng)用之一：病原體檢測與定量1432應(yīng)用之一：病原體檢測與定量HCV(FAM)RNAvirus65bpampliconHIV-1(HEX)RNAVirus74bpampliconHBV(CY5)DNAvirus81bpampliconTaqmanTriplexAssayDanielCandotti-Cambridge,UK應(yīng)用之二：基因表達(dá)差異分析相對定量：基因表達(dá)量上升或者表達(dá)被抑制的倍數(shù)兩種樣品：Calibrator（對照，如正常組織）與Sample（待測樣品）兩個基因：目的基因與看家基因應(yīng)用之二：基因表達(dá)差異分析RNA抽提RT定量PCRcDNA產(chǎn)物系列稀釋兩個基因的擴增效率Normalizer看家基因應(yīng)用之二：基因表達(dá)差異分析CalibratorSampleGeneofInterest目的基因CtCtCtCt

Ct1

Ct2應(yīng)用之二：基因表達(dá)差異分析基因表達(dá)差異＝2

/2

Ct1

Ct2＝2

Ct1-

Ct2這個公式的前提是看家基因和待測基因的擴增效率相同且都為1Xn＝X0(1+E)n基因表達(dá)相對定量分析實例采用SYBRGreenI的方法研究Mal基因在食管癌中的表達(dá)狀況；采用看家基因GAPDH作為內(nèi)參樣品采集，新鮮食管癌標(biāo)本和遠(yuǎn)端正常組織抽提總RNA，電泳檢測質(zhì)量通過紫外分光光度計檢測RNA含量取500ng的總RNA參加反應(yīng)合成cDNA總RNADNaseIcDNA總RNART無RTRNaseHcDNARNaseH無檢查是否樣品中有基因組DNA污染真正用于檢測的樣品樣品的準(zhǔn)備和處理腫瘤和正常標(biāo)準(zhǔn)品的準(zhǔn)備選擇某種已知表達(dá)較高的組織或細(xì)胞作為標(biāo)準(zhǔn)品來源提取總RNA，樣本處理方法同前經(jīng)梯度稀釋：1：10；1：100；1：1000；1：10000；1：100000并自己給稀釋的標(biāo)準(zhǔn)品定義數(shù)值：10000，1000，100，10，1等Mal基因引物GAPDH基因引物標(biāo)準(zhǔn)品正常樣品標(biāo)準(zhǔn)品腫瘤樣品正常樣品腫瘤樣品Ct1cbCt2cb擴增效率擴增效率無RT產(chǎn)物無Ct1sCt2s腫瘤樣本和正常樣本的相對表達(dá)差異應(yīng)用之二：基因表達(dá)差異分析GAPDHMal表達(dá)倍數(shù)差異（腫瘤/正常）copy數(shù)相對值Xcopy數(shù)相對值Y校正后Y/X正常樣品5482130560.5570.5570.129腫瘤樣品87251.599970.1140.072結(jié)果計算方法說明在腫瘤樣本中Mal的基因表達(dá)量有顯著下調(diào)，只相當(dāng)于正常樣本的0.129倍兩條探針分別針對不同等位基因ACGTACGT應(yīng)用之三：SNP分析應(yīng)用之三：SNP分析FAMTETFAMTET熒光定量PCR儀的硬件條件激發(fā)熱循環(huán)－加熱、制冷樣品檢測熒光定量PCR儀應(yīng)滿足的要求光源的光譜范圍寬廣，能激發(fā)不同的熒光染料能分開不同熒光素的激發(fā)光與發(fā)射光波長能檢測的靈敏度與動態(tài)檢測范圍準(zhǔn)確的溫度控制和良好的孔間溫度均一性.熒光定量PCR原理及應(yīng)用熒光定量PCR實驗引物/探針設(shè)計及實驗條件優(yōu)化StratageneMx3000P熒光定量PCR儀QPCRAssayOptimizationDesignSynthesisTestoligosOptimizationPrimersProbeEnzyme,

dNTP,Mg++Serial

dilutionRunandAnalysisTarget/AmpliconChoiceTargetsize:50-150bpfordual-labeledprobes(Taqman)200-400bpforSYBRIfusingacDNAtemplate,spanintronstoavoidgenomicDNAcontamination.Trytoavoidrepetitiveorhighlystructuredregions(CpGislandsandcontrolregionsatthe5’endtendtoformcomplexstructures).QPCRPrimerPropertiesUsethesametargetannealingtemperatureforallyourprimerstofavormultiplexinginthefutureandrunsinglethermalprofileTypically55-60°C(with1°Ctempdifference)AvoidG/Cclampsatthe3’end.Ifrequestedbythesoftware,use100mMmonovalentcationand5mMMg++forprobes,2.5mMforSYBR.AlwaysBLASTyourprimers.Dual-labeledProbeDesignTm10°Chigherthanprimers.35-65%G/C;moreCsthanGs.Avoidrunsof3+ofthesamenucleotide,especiallyGs.5’baseG.3’endofprimerand5’endofprobeonsamestrandbetween1-15bpdistance.Testthatprimersandprobearenotcomplementarytoeachother.Reporter-QuencherPairs

Dye QuencherFAM BHQ-1/TAMRAHEX/JOE BHQ-2TexasRed/ROX BHQ-2Cy5/Quasar670 BHQ-2(or-3)OligoSynthesisPrimers:Usedesaltedprimersforsequence-specificdetection.SYBRGreenprimersshouldbeHPLC-purified.Aliquotstocksolutionof20x(asdeterminedbytheoptimizationstep),typically6-18μMeacholigo.Testprimersbeforeorderingprobe.Probe:Ensurematchingfluorophore/quencherset.Double-HPLCpurified(purificationalsoOK).Aliquotworkingstockat2-4μM(20x).InitialPrimerTestingPrimers:PCRreactionandelectrophoresis.SYBRGreenrunanddissociationanalysis.MeltingCurveAnalysisGoodProbePerformancedRn=0.1to0.9PoorProbePerformancedRn=0.1to0.4

QPCRAssayOptimization

Firstoptimizeprimers(applicationspecific):performprimermatrixassayconfirmoptimalprimerpairwithstandardcurvesensitivityordynamicrangechallengesmultiplexingSecondoptimizeprobeconcentrations:performprobematrixassaywithoptimalprimerpairconfirmoptimalprobeconcentrationwithstandardcurveLastlyoptimizeMg++concentration:

usuallylaststeptoattemptifassayisstillnotperformingoptimallyifmultiplexing,chooseasetconcentrationfortheassay

QPCRAssayOptimizationPrimerconcentrations:100-600nM(startwith300nM).50-300nM(150nM)forSYBRGreen.Probeconcentrations:50-300nM(startwith200nM).Mg++concentration:3.5-5.5mM(startwith5mM).1.5-3.5mM(2.5mM)forSYBRGreen.WhyOptimizePrimers?Goalistogetprimersperformingoptimallysoforwardandreverseprimersannealtosequenceatequalefficiency.Primerannealingisasequencedependentevent.StandardPCRoptimizationusedthermalgradienttofindanannealingtemperatureforwhichbothprimersworkedwelltogether.QPCRhashighersensitivityallowingmorepreciseprimeroptimization.PrimerOptimizationMatrixTwoSchemes:Optimizeconcentrationofforwardandreverseprimersandtotalprimerconcentration.(100-600)or(100,300,600)etc…SYBR:50,150,300Optimizeconcentrationofforwardandreverseprimerswithsametotalprimerconcentration

(Taqman600nMtotal)(SYBRGreen300nMtotal)PrimerConcentrationOptimizationLowestCtIfusingSYBRGreen,cleanmeltcurvesMinimalreplicatevariabilit