1、實時熒光定量PCR原理及應用郝 近 技術支持fa229上海吉泰生物科技有限公司內 容 提 綱熒光定量PCR原理及應用熒光定量PCR實驗引物/探針設計及實驗條件優化Stratagene Mx3000P熒光定量PCR儀常規核酸定量方法Southern 雜交Northern 雜交原位雜交傳統 RT-PCR半定量 PCR存在的問題 靈敏度準確性特異性時間Real time PCR定義利用熒光信號對PCR反應的過程進行實時監控目的對起始模板進行定量 優點 靈敏, 重復性, 動態范圍寬, 高通量原理 C(t)值與起始模板濃度的對數成比例應用 腫瘤，微生物檢測，SNP等熒光化學原

2、理SYBR Green ITMTaqMan / TaqMan-MGBMolecular Beacons MGB EclipseTM Probes Lux primersScorpionsTM AmplifluorQ-ZymeOthers.染料法：探針法：TaqMan ProbesUnbound probe free in solutionProbe and primer bind target, FRET occursLight Emission or HeatDonor dye (Reporter)Acceptor dye (Quencher)LightEnergy transferTaqT

3、aq extends and hydrolyzes probe, donor dye free to emit fluorescence - accumulation of signalTaqLight EmissionLight什么是C(t)值C(t)Threshold（域值）傳統的定量方法11,500 counts / 5200 counts = 2.2 fold differenceReal-time 定量Ct 18 and Ct 22, 2(4 Ct shift) = 16 fold difference定量PCR是如何定量的？標 準 曲 線 未 知 樣 品 的 C(t) 值 定量應用

4、之一：病原體檢測與定量絕對定量（特異基因的拷貝數病原體的多少） 標準品（如質粒）：梯度稀釋 未知樣品 陰性對照（NTC）如何確定標準品的量標準品是通過某種方法制備得到的已知拷貝數的含有目標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準品序列信息估算出標準品的分子量B這樣拷貝數A(copy/uL)為： A=C/B*6.02E+14應用之一：病原體檢測與定量HCV (FAM) RNA virus65bp ampliconHIV-1 (HEX) RNA Virus74bp ampliconHBV (CY5) DNA virus 81bp ampliconT

5、aqman Triplex AssayDaniel Candotti - Cambridge, UK應用之二：基因表達差異分析相對定量：基因表達量上升或者表達被抑制的倍數兩種樣品：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用之二：基因表達差異分析RNA抽提RT定量PCRcDNA產物系列稀釋兩個基因的擴增效率Normalizer看家基因應用之二：基因表達差異分析CalibratorSampleGene of Interest目的基因CtCtCtCt Ct1 Ct2應用之二：基因表達差異分析基因表達差異2 /2 Ct1 Ct22 Ct1 -

6、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1XnX0(1+E)n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備和處理腫瘤和正常標準品的準備選擇某種已知表達較高的組織或細胞作為標準品來源提取總RNA，樣本處理方法同前經梯度稀釋：1：10；1：100；1：1000；1：10000；1：100000并自己給稀釋的標準品定義數值：10000，1000，100，10，1等應用之二：基因表達差異分析GAPDHMal表達倍數差異（腫瘤/正常）copy數相對值Xcopy數相對值Y校正后Y/X正常樣品

7、5482130560.5570.5570.129腫瘤樣品87251.599970.1140.072結果計算方法說明在腫瘤樣本中Mal的基因表達量有顯著下調，只相當于正常樣本的0.129倍兩條探針分別針對不同等位基因ACGTACGT應用之三：SNP分析應用之三：SNP分析FAMTETFAMTET熒光定量PCR儀的硬件條件激發熱循環加熱、制冷 樣品檢測熒光定量PCR儀應滿足的要求光源的光譜范圍寬廣，能激發不同的熒光染料能分開不同熒光素的激發光與發射光波長能檢測的靈敏度與動態檢測范圍準確的溫度控制和良好的孔間溫度均一性.熒光定量PCR原理及應用熒光定量PCR實驗引物/探針設計及實驗條件優化Strat

8、agene Mx3000P熒光定量PCR儀QPCR Assay OptimizationDesignSynthesisTest oligosOptimizationPrimersProbeEnzyme,dNTP, Mg+Serial dilutionRun and AnalysisTarget/Amplicon ChoiceTarget size: 50-150 bp for dual-labeled probes (Taqman)200-400 bp for SYBRIf using a cDNA template, span introns to avoid genomic DNA con

9、tamination.Try to avoid repetitive or highly structured regions (CpG islands and control regions at the 5end tend to form complex structures).QPCR Primer PropertiesUse the same target annealing temperature for all your primersto favor multiplexing in the future and run single thermal profileTypicall

10、y 55-60C (with 1C temp difference)Avoid G/C clamps at the 3end.If requested by the software, use 100 mM monovalent cation and 5 mM Mg+ for probes, 2.5 mM for SYBR.Always BLAST your primers.Dual-labeled Probe DesignTm 10C higher than primers.35-65% G/C;more Cs than Gs.Avoid runs of 3+ of the same nuc

11、leotide, especially Gs.5 base G.3 end of primer and 5 end of probe on same strand between 1-15 bp distance.Test that primers and probe are not complementary to each other.Reporter-Quencher Pairs DyeQuencherFAMBHQ-1/TAMRAHEX/JOEBHQ-2Texas Red/ROXBHQ-2Cy5/Quasar670BHQ-2 ( or -3)Oligo SynthesisPrimers:

12、Use desalted primers for sequence-specific detection. SYBR Green primers should be HPLC-purified.Aliquot stock solution of 20 x (as determined by the optimization step), typically 6 - 18M each oligo.Test primers before ordering probe.Probe:Ensure matching fluorophore/quencher set.Double-HPLC purifie

13、d (purification also OK).Aliquot working stock at 2 - 4M (20 x).Initial Primer TestingPrimers:PCR reaction and electrophoresis.SYBR Green run and dissociation analysis.Melting Curve AnalysisGood Probe PerformancedRn = 0.1 to 0.9Poor Probe PerformancedRn = 0.1 to 0.4 QPCR Assay Optimization First opt

14、imize primers (application specific):perform primer matrix assayconfirm optimal primer pair with standard curvesensitivity or dynamic range challenges multiplexingSecond optimize probe concentrations:perform probe matrix assay with optimal primer pairconfirm optimal probe concentration with standard

15、 curveLastly optimize Mg+ concentration: usually last step to attempt if assay is still not performing optimallyif multiplexing, choose a set concentration for the assay QPCR Assay OptimizationPrimer concentrations:100-600 nM (start with 300 nM).50-300 nM (150 nM) for SYBR Green.Probe concentrations

16、:50-300 nM (start with 200 nM).Mg+ concentration: 3.5-5.5 mM (start with 5 mM).1.5-3.5 mM (2.5 mM) for SYBR Green.Why Optimize Primers?Goal is to get primers performing optimally so forward and reverse primers anneal to sequence at equal efficiency. Primer annealing is a sequence dependent event.Sta

17、ndard PCR optimization used thermal gradient to find an annealing temperature for which both primers worked well together.QPCR has higher sensitivity allowing more precise primer optimization.Primer Optimization MatrixTwo Schemes:Optimize concentration of forward and reverse primers and total primer

18、 concentration. (100-600) or (100,300,600) etc SYBR: 50, 150, 300Optimize concentration of forward and reverse primers with same total primer concentration (Taqman 600 nM total) (SYBR Green 300 nM total)Primer Concentration OptimizationLowest CtIf using SYBR Green, clean melt curvesMinimal replicate

19、 variabilityHighest endpoint fluorescenceLower concentrations of total primer multiplexing熒光定量PCR原理及應用熒光定量PCR實驗引物/探針設計及實驗條件優化Stratagene Mx3000P熒光定量PCR儀Stratagenes New Mx3000P!High Performance Personal Price Stratagene 公司成立于1984年,致力于發展生命科學領域的創新產品與科技. 總部位于美國 La Jolla, California .在歐洲與日本設有分公司.共有員工480名.

20、Stratagene 公司簡介一體化設計，徹底摒棄傳統的分離式結構，將擴增系統與檢測系統完美結合，帶來超強的穩定性。Emission Filter WheelExcitation Filter WheelTungsten/Halogen LampHot TopThermal systemFiber Optic Cable and Scanning Arm熱循環系統整板溫度均一性 +/- 0.25C半導體加熱制冷 resistive and convective heat exchange熱蓋最佳反應體積25ul均一性實驗 96 well uniformity run. SYBR Green I

21、 detection96 wellsMean Ct = 18.1St.Dev. = 0.05C.V . = 0.3%Ct Range = 0.26 cyclesMx3000P 光路系統PMT96 well plate石英鹵鎢燈激發光濾鏡輪發射光濾鏡輪掃描式檢測發射光濾鏡發射光濾鏡熒光濾鏡系統ACBEx. Filter350 nm750 nm10 nm InterferenceABCMx3000P激發光源Halogen LampLED石英鹵鎢燈: 中等光強, 波長范圍寬 LED: 光強低, 波長受限制。Alx350FAMTETHEXJOEROXCy5Cy3TAMTxRdMx3000P 可用熒光染料四通道設計，避免光譜重疊Mx3000P 發射光濾鏡輪熒光檢測系統PMT光電倍增管冷CCDCCDPhotodiodes光電二極管CCD與PMT檢測區別無邊緣效應邊緣效應CCDMirror超高的分辨率與靈敏度。2-fold serial dilution (20,000 to 1