1、1The world leader in serving scienceModule8： 实时荧光定量 PCR技术应用陈 涛Field Application ScientistTel: 800 820 8982 / 400 820 89822TaqMan Assay Total SolutionSolutions3Genotyping4Diseases:Diabetes 糖尿病Inflammatory bowel:Gallstones 胆结石Cardiovascular conditions and lipid metabolism: Atrial fibrillation 心房颤动chol
2、esterol 胆固醇转运Neuropsychiatric conditions:Amyotrophic Lateral Sclerosis 肌萎缩侧索硬化Schizophrenia 精神分裂症Bipolar disorder 躁郁症Autoimmune and infectious diseases:Rheumatoid arthritis 类风湿性关节炎Childhood asthma 儿童哮喘Cancer：Prostate cancer 前列腺癌Breast cancer 乳腺癌Colorectal cancer 结肠直肠癌Various traits:Height Blue vs. g
3、reen eyes为什么研究 SNPa change in the nucleotide present at a single base in a known DNA sequence.(Single Nucleotide Polymorphisms )5TaqMan Genotyping Assays6TaqMan SNP基因分型分析试剂盒超过 4,500,000个 SNP基因分型分析试剂盒可供选择，其中包含 3,500,000 HapMap SNP， 70,000个 cSNP另提供超过 10,000个小鼠的 SNP基因分型检测试剂盒SNP Browser软件可以帮助选择和订购所需检测的位
4、点和产品另提供代客设计合成服务7TaqMan Genotyping Assays Workflow1. Combine sample with TaqMan SNP Genotyping Assay2. Perform amplification on thermal cycler3. Read results4. Analyze results using TaqMan GenotyperSofftware TaqMan SNP Genotyping Assays TaqMan Universal PCR Master Mix DNA samplePrepare the reaction m
5、ixPerform PCR QS 12K, ViiA7, 7900HT, 7500, 7500 Fast, StepOne,StepOnePlusTaqMan GenotyperSoftware8Application TaqMan genotyping PCR assaysIL28B rs12979860 genotype is a strong predictor of treatment outcome to PegIFN plus Rbv in HCV-4 monoinfected Patients.The incorporation of the IL28B genotype in
6、the treatment algorithm of HCV-4 patients.Highlight the need to consider this factor in the design and randomization of drug discovery studiesS. De Nicola et al., Hepatology 55, 336 (Feb, 2012).9Copy number variation10 Copy Number Variation (CNV)定义 基因组发生 DNA片段缺失、重复、插入、倒位 片段范围： 50bp-1mb. Most in 1-10
7、kb CNV分布于人类基因组 290, 000 identified CNVs CNV的重要性：与多种疾病表型紧密相 关 HIV/AIDS susceptibility (CCL3L1) Rheumatoid Arthritis风湿性关节炎 Neuropathies 神经系统疾病 (PMP22) Type I diabetes Lupus 红斑狼疮 -免疫系统疾病 (FCGR3B & C4) Crohnsdisease节段性回肠炎 (DEFB4) Cancer Drug Metabolism (e.g. CYP2D6)为什么研究 CNV11CalibratorSample ( )TestSam
8、ple ( )Ct (VIC) = 27.0Ct (FAM) = 28.5Ct = 1.5Ct (VIC)= 27.0Ct (FAM) = 27.5Ct = 0.5Ct = 1Copy number2-Ct 2 = 1Test assay(FAM, YIPF6)Chr XReference assay(VIC, RNase P) Chr 14T a qM a n C N V a s s a y2 . 5 ng w e t D M E gD N A - 1 0 uL - 7 9 0 0Xc nt r l _ 5 5 5 9 3 _ 3 + R N a s e P_ 401232.5 - NA10
9、8592.5 - NA144742.5 - NA144762.5 - NA171022.5 - NA171032.5 - NA171042.5 - NA171052.5 - NA171062.5 - NA171072.5 - NA171082.5 - NA171092.5 - NA171102.5 - NA171112.5 - NA171122.5 - NA171132.5 - NA171142.5 - NA171152.5 - NA171162.5 - NA171172.5 - NA171182.5 - NA171212.5 - NA171222.5 - NA17123gD N ACopy
10、NumberXYXXCopy number variation （CNV）by Relative Quantitation (an example)12TaqMan Copy Number AssaysHuman： 1,600,000 assays Mouse：180,000 assaysEasy to use, robust, and accurate13TaqMan Copy Number Assay Workflow2. Combine and load plate3. Run on ABI real-time PCR instrument4. Analyze with CopyCall
11、erTMSoftware TaqMan Copy Number Assay (Test Assay) TaqMan Copy Number Reference Assay TaqMan Master Mix gDNAPrepare the reaction mixAdd the reaction mix to the prepared DNA reaction platePerform PCR QS 12K, ViiA7, 7900HT, 7500, 7500 Fast, StepOne,StepOnePlusCopyCaller Software1. Prepare Sample14Appl
12、ication A. J. Sharp et al., Nat Genet 40, 322 (Mar, 2008).TaqMan Copy Number AssayThis recurrent 1.5 Mb deletion contains six genes, including a candidate gene for epilepsy(CHRNA7) that is likely responsible for the observed seizure phenotype15Rare Mutation Detection16如何在早期发现肿瘤？17castPCR: Competitiv
13、e Allele-Specific TaqMan PCR 只有突变等位基因 （ mutant allele ） 可以进行扩增野生型等位基因扩增被 Blocker抑制18稀有突变检测试剂盒产品组合 ： 46个癌症基因上的 778个关键突变位点 Wild-type assays for only the subset of mutation targets Internal Positive Control Reagents (IPC kit) Mutation Detector Software适用于 FFPE组织，新鲜、冰冻组织和培养细胞等抽提得到的基因组 DNATaqMan Mutation
14、 Detection Assays (TMDA) 19TaqMan Mutation Detection Assays (TMDA)特点高灵敏度 （High Sensitivity） 可以检测 0.1%突变基因 在不同细胞系中，具有相同的灵敏度高特异性 （High Specificity） 在稀有突变检测应用中，与 standard allele specific PCR和 TaqMan SNP assays相比具有更高的特异性简单操作步骤 （Simple Workflow） 整个工作流程 3 hrs20TaqMan Mutation Detection Assay /cast-PCR Wor
15、kflow1. Select and Order Assays2. Prepare gDNAand reaction mix3. Run by real-time PCR4. Detect & Quantify mutations Target assays Gene reference assays Optional IPC reagents TaqManGenotyping Master Mix Prepare genomic DNA Sample Add Mutation Detection Assay Add Genotyping Master mix (optional: IPC)
16、QS 12K, ViiA7, 7900HT, 7500, 7500 Fast, StepOnePlusMutation Detector Software 21Application A. Didelot et al., Experimental and molecular pathology 92, 275 (Jun, 2012).TaqMan genotyping master mixTaqMan Mutation Detection assay“ less laborious and requires less stringent precautions to prevent cross
17、over”“ an important technology to consider inthe field of mutation detection for clinical purpose“23Percentage of Methylation24为什么研究甲基化CpG岛甲基化与癌症 肿瘤中异常的表观遗传学改变肿瘤的特征之一是不平衡的甲基化。肿瘤的 DNA甲基化改变表现为基因组整体甲基化水平降低和 CpG岛局部甲基化水平异常增高。导致基因组的不稳定。25不同甲基化程度产生不同熔解曲线，通过不同 Tm值进行甲基化程度的区分HRM Melt Curve DynamicsReading a H
18、igh Resolution Melt CurveHigh Resolution Melting (HRM)Tm值： DNA的双螺旋结构打开一半时的温 度一种利用熔解曲线对 DNA 进行分析的方法，利用不同 PCR产物， Tm值不同的原理进行分析 .26High Resolution Melting (HRM) ReagentsSpecially formulated for superior performance and full integration into the AB HRM Workflow MeltDoctor HRM Master Mix MeltDoctor HRM Re
19、agent Kit MeltDoctor HRM Dye MeltDoctor HRM Calibration Plates 384-well 96-well MeltDoctor HRM Positive Control Kit27HRM Experiments Workflow1. Instrument Calibration2. PCR Design & Optimization 3. PCR 4. Sample Melt 5. Data Analyze28ApplilcationCancer Genetics and Cytogenetics 196 (2010)High-resolution DNA melting (HRM) analysisABI 7500 FastReal-Time PCR SystemABI high-resolution melting v 2.0 software29Gene Expression30从单基因到网络调控From single to network31TaqMan Gene Expression Assay
