原理应用探针设计和应用实例课件

1、实时荧光定量PCR原理及应用郝 近 技术支持fa229上海吉泰生物科技有限公司内 容 提 纲荧光定量PCR原理及应用荧光定量PCR实验引物/探针设计及实验条件优化Stratagene Mx3000P荧光定量PCR仪常规核酸定量方法Southern 杂交Northern 杂交原位杂交传统 RT-PCR半定量 PCR存在的问题 灵敏度准确性特异性时间Real time PCR定义利用荧光信号对PCR反应的过程进行实时监控目的对起始模板进行定量 优点 灵敏, 重复性, 动态范围宽, 高通量原理 C(t)值与起始模板浓度的对数成比例应用 肿瘤，微生物检测，SNP等荧光化学原

2、理SYBR Green ITMTaqMan / TaqMan-MGBMolecular Beacons MGB EclipseTM Probes Lux primersScorpionsTM AmplifluorQ-ZymeOthers.染料法：探针法：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）传统的定量方法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？标 准 曲 线 未 知 样 品 的 C(t) 值 定量应用

4、之一：病原体检测与定量绝对定量（特异基因的拷贝数病原体的多少） 标准品（如质粒）：梯度稀释 未知样品 阴性对照（NTC）如何确定标准品的量标准品是通过某种方法制备得到的已知拷贝数的含有目标片段的核苷酸序列通常通过紫外定量等方法测得浓度C(ng/uL)通过核苷酸的相对分子量和标准品序列信息估算出标准品的分子量B这样拷贝数A(copy/uL)为： A=C/B*6.02E+14应用之一：病原体检测与定量HCV (FAM) RNA virus65bp ampliconHIV-1 (HEX) RNA Virus74bp ampliconHBV (CY5) DNA virus 81bp ampliconT

5、aqman Triplex AssayDaniel Candotti - Cambridge, UK应用之二：基因表达差异分析相对定量：基因表达量上升或者表达被抑制的倍数两种样品：Calibrator（对照，如正常组织） 与Sample（待测样品）两个基因：目的基因与看家基因应用之二：基因表达差异分析RNA抽提RT定量PCRcDNA产物系列稀释两个基因的扩增效率Normalizer看家基因应用之二：基因表达差异分析CalibratorSampleGene of Interest目的基因CtCtCtCt Ct1 Ct2应用之二：基因表达差异分析基因表达差异2 /2 Ct1 Ct22 Ct1 -

6、Ct2这个公式的前提是看家基因和待测基因的扩增效率相同且都为1XnX0(1+E)n总RNADNaseIcDNA总RNART无RTRNaseHcDNARNaseH无检查是否样品中有基因组DNA污染真正用于检测的样品样品的准备和处理肿瘤和正常标准品的准备选择某种已知表达较高的组织或细胞作为标准品来源提取总RNA，样本处理方法同前经梯度稀释：1：10；1：100；1：1000；1：10000；1：100000并自己给稀释的标准品定义数值：10000，1000，100，10，1等应用之二：基因表达差异分析GAPDHMal表达倍数差异（肿瘤/正常）copy数相对值Xcopy数相对值Y校正后Y/X正常样品

7、5482130560.5570.5570.129肿瘤样品87251.599970.1140.072结果计算方法说明在肿瘤样本中Mal的基因表达量有显著下调，只相当于正常样本的0.129倍两条探针分别针对不同等位基因ACGTACGT应用之三：SNP分析应用之三：SNP分析FAMTETFAMTET荧光定量PCR仪的硬件条件激发热循环加热、制冷 样品检测荧光定量PCR仪应满足的要求光源的光谱范围宽广，能激发不同的荧光染料能分开不同荧光素的激发光与发射光波长能检测的灵敏度与动态检测范围准确的温度控制和良好的孔间温度均一性.荧光定量PCR原理及应用荧光定量PCR实验引物/探针设计及实验条件优化Strat

8、agene Mx3000P荧光定量PCR仪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荧光定量PCR原理及应用荧光定量PCR实验引物/探针设计及实验条件优化Stratagene Mx3000P荧光定量PCR仪Stratagenes New Mx3000P!High Performance Personal Price Stratagene 公司成立于1984年,致力于发展生命科学领域的创新产品与科技. 总部位于美国 La Jolla, California .在欧洲与日本设有分公司.共有员工480名.

20、Stratagene 公司简介一体化设计，彻底摒弃传统的分离式结构，将扩增系统与检测系统完美结合，带来超强的稳定性。Emission Filter WheelExcitation Filter WheelTungsten/Halogen LampHot TopThermal systemFiber Optic Cable and Scanning Arm热循环系统整板温度均一性 +/- 0.25C半导体加热制冷 resistive and convective heat exchange热盖最佳反应体积25ul均一性实验 96 well uniformity run. SYBR Green I

21、 detection96 wellsMean Ct = 18.1St.Dev. = 0.05C.V . = 0.3%Ct Range = 0.26 cyclesMx3000P 光路系统PMT96 well plate石英卤钨灯激发光滤镜轮发射光滤镜轮扫描式检测发射光滤镜发射光滤镜荧光滤镜系统ACBEx. Filter350 nm750 nm10 nm InterferenceABCMx3000P激发光源Halogen LampLED石英卤钨灯: 中等光强, 波长范围宽 LED: 光强低, 波长受限制。Alx350FAMTETHEXJOEROXCy5Cy3TAMTxRdMx3000P 可用荧光染料四通道设计，避免光谱重叠Mx3000P 发射光滤镜轮荧光检测系统PMT光电倍增管冷CCDCCDPhotodiodes光电二极管CCD与PMT检测区别无边缘效应边缘效应CCDMirror超高的分辨率与灵敏度。2-fold serial dilution (20,000 to 1