分子生物学研究进展05Bioch.ppt

Microarray Analysis,郑 芳Fang Zheng Ph.D.Prof.Tianjin Medical University,Presentation outline,Introduction DNA Microarray Tissue Aarray Analysis Flowarray Analysis,Presentation outline,Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis,Definition of Microarray,Microarray Analysis:Introduction,生物芯片：能对生物成分或生物分子进行快速并行处理和分析的技术。检测原理：利用特异性的分子间的相互作用，如核酸杂交、抗原-抗体特异性结合、蛋白-蛋白间特异性结合等。,1.General Introduction,If you get the gene,you will get the protein.If you want a protein,get its gene!,Abnormality of Gene structure,Abnormality of Gene expression,PCRRE analysis Hybridization DNA sequencingDNA fingerprintingDNA microarray,RT-PCR FQ-PCRNorthern blotDNA microarray(Gene chip),Western-blotIHC stainELIZAProtein chipTissue Array,History of Microarray development,Microarray Analysis:Introduction,Microarray：Mini-laboratory,Microarray,Computer-chip,History of Microarray development,Microarray Analysis:Introduction,1950s:Computer-chip2000s:Bio-Microarray,Microarray Analysis:Introduction,Microarray development,Motivation Low-automaticity of the analytical apparatus Difficult to integrate all the steps together,including sample preparation,biochemical event,consequence detection&analysis.,Microarray Analysis:Introduction,Microarray development,Aim of microarray application:To integrate those noncontinuous analytic processes into a chip or a microarray,and made them continuous and miniaturized,Microarray Analysis:Introduction,Classification of Microarray,DNA/gene microarray Protein chip Tissue Array Flowarray,DNA Microarray Analysis,Principle of Microarrays,DNA芯片技术是指在固相支持物上原位合成（in situ synthesis）寡核苷酸探针，或者直接将大量的DNA探针以显微打印的方式有序的固化于支持物表面，然后与标记的样品杂交，通过对杂交信号的检测分析即可得出样品的遗传信息（基因序列及表达的信息）。,DNA Microarray:miniaturized array having up to tens of thousands of single-stranded DNA attached to it.,DNA Microarray Analysis,Definition of Microarrays,Microarray Analysis:Introduction,Microarray development,生物芯片主要特征:高通量:极高的样品并行处理能力 High throughput:high level of parallel processing capability 微量化：所需样品量减少，灵敏度高 High level of sensitivity自动化：全过程自动化、标准化程度高 Automatization、standardization,1.Disease diagnosis,Applications of biochip technology,Microarray Analysis,Molecular mechanisms of the diseases,Treatment strategyof the diseases,应用DNA芯片可在DNA水平检测与疾病相关的内源性或外源性基因；应用表达芯片可以在表达水平分析同一组织在不同的发育阶段，正常及病理状态基因表达的差异，也可分析不同组织同一基因在正常及病理状态下表达的差异，从而为疾病诊断与分类，病原微生物分型提供科学依据。,2.DNA sequencingHuman genome project;HGP：To identify and determine DNA sequence of human genomeSequencing by hybridization,SBH(杂交测序)：建立在特定序列的DNA与特定长度的寡核苷酸集合的杂交的基础之上。未知DNA序列可以通过鉴定与靶DNA序列形成完整的双链体寡核苷酸的重叠区域来确定.65536个八核苷酸点阵可测定约200个核苷酸的序列；一百万个十二核苷酸点阵可测定约1000个碱基对.,Microarray Analysis,Applications of biochip technologies,3.Search new genes 4.Mutation and polymorphism analysis To detect and analyze DNA variation and polymorphism on a large scale Detection of genetic mutation Inherited diseases,and tumor,Microarray Analysis,Applications of biochip technology,5.Drug screening 在基因水平上寻找药物靶标，毒性对基因的影响。临床许多细菌对药物具有抗药性，但是不同的亚型对同一药物的敏感性不同。DNA芯片技术已被用于鉴定结核菌及非典型分支杆菌的基因型与耐利福平的相关性以及HIV产生抗药性与其逆转录酶及蛋白酶基因突变的相关性。这为临床选择敏感药物提供了有效手段。,Applications of biochip technology,Microarray Analysis,6.Proteinogram analysis 基因组计划后的另一项重要计划-蛋白组(Proteomics)计划.Contrast analysis of proteinogram on normal and cancer cells by Protein chips,Applications of biochip technology,Microarray Analysis,7.Miniaturization of analytical apparatus,Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Schena,M.,et al.,Science 1995;270:467470Parallel human genome analysis:Microarray-based expression monitoring of 1000 genes.Schena,M.,et al.,PNAS 1993:1061410619Use of a cDNA microarray to analyse gene expression patterns in human cancer.DeRisi,J.,et al.,Nature Genetics 1996;14:457460,References:,Microarray Analysis,Applications of biochip technology,Presentation outline,Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis,DNA Microarray Analysis,Principle of Microarrays,DNA芯片技术是指在固相支持物上原位合成（in situ synthesis）寡核苷酸探针，或者直接将大量的DNA探针以显微打印的方式有序的固化于支持物表面，然后与标记的样品杂交，通过对杂交信号的检测分析即可得出样品的遗传信息（基因序列及表达的信息）。又被称为基因芯片（gene chips）、DNA阵列（DNA array）、cDNA芯片（cDNA chips）、寡核苷酸阵列（oligonucleotide array）等。,DNA Microarray Analysis,Principle of Microarrays,Microarray assays are based on hybridization of a single-stranded DNA labeled with a fluorescent tag to a complementary molecule attached to the chip,Microarrays enable simultaneous measurement of the expression levels of thousands of genes in a sampleMicroarray:Glass slide with a matrix of thousands of spots printed on to itEach spot contains probes which bind to a specific gene,DNA Microarray Analysis,Principle of Microarrays,ConstructionPlace array of probes on microchipProbe is oligonucleotide 25 bases long that characterizes gene or genomeChip is about 2cm by 2cmSolid support(glass,plastic,metal,silicon)Hybridized probes(DNA molecules)are fluorescently labeled,DNA Microarray Analysis,Principle of Microarrays,When each spot in a microarray is attached a unique DNA molecule,it can be used to detect presence/absence or even concentration of a particular type of DNA molecule in test tube.,DNA Microarray Analysis,Principle of Microarrays,Chip preparation 芯片制备 Sample preparation 样品制备 Work on the biochemical principle of DNA/DNA hybridization 杂交反应Analyze hybridization pattern 结果分析,DNA Microarray Analysis,Basic idea of Microarrays,DNA Microarray Analysis,Basic idea of Microarrays,Gene expressionGene disease relationGene-gene interactionFinding Co-Regulated GenesUnderstanding Gene Regulatory Networks,DNA Microarray Analysis,One gene at a time before,now tens of thousands simultaneously,DNA Microarray Analysis,Microarrays designs and applications:cDNA microarrays in gene expression studies,cDNA Microarray cDNA clone inserts are printed onto glass slides at high density As many as 25,000 cDNAs can be applied to a single glass slide Enables large-scale,high-throughput analysis,DNA Microarray Analysis,cDNA Microarrays Thousands of known genes are available Requires special instrumentation for generation of microarrays and for analysis of results,Microarrays designs and applications:cDNA microarrays in gene expression studies,DNA Microarray Analysis,Sample preparation and hybridization to cDNA microarrays,DNA Microarray Analysis,Comparison of Gene Expression PatternsBetween Normal Lung and Lung Carcinomafor Identification of Genes Associatedwith Neoplastic Transformation,用于基因转录水平的检测、基因组分析和后基因组研究 美国斯坦福大学Davis等用PCR法从外周血淋巴细胞cDNA文库中得到了1046种cDNA片段，制成芯片。然后与热处理的T淋巴细胞和未处理的T细胞种提取的mRNA反转录出的单链cDNA片段杂交。经激光共聚焦扫描系统分析，共发现17个差异表达的基因，其中11个是被热诱导的，6个是被热抑制的。经序列分析证实，其中3个是未报导的新基因。,DNA Microarray Analysis,DNA microarrays application,Microarray Analysis:Development,Microarray designs and applications,Microarray Probes cDNAs(from libraries or PCR products)Oligonucleotides Genomic DNA fragments,Microarray Analysis:Development,Microarray designs and applications,Applications Gene expression studies Oligo-mediated sequencing Genetic mapping studies Mutation analysis Polymorphism analysis,The tools provided to us by our understanding of molecular biology,are what allow us to interrogate DNA/RNA for the purpose of identifying disease causing by genetic alterations.,DNA Microarray Analysis,DNA microarrays application,Presentation outline,Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis,Tissue Array Analysis,Tissue microarrays 1998 Kononen,Nature Medicineallow one to identify and evaluate highly specialized gene expression patterns and thus are a high-throughput tool(for diagnosis).,Tissue microarrays,Complementary uses of DNA and tissue arrays,DNA microarrays for gene expression Used to find biomarkersRelatively few tumor samples but thousands of markers(genes)TMAs for protein expression patternsUsed to validate biomarkerslarge number(hundreds)of tumors,relatively few markers,Tissue Array Analysis,TMAs often used for validating tumor genes in cancer research,The current classification systems of tumors are partially predictive of outcomes,and are based primarily upon morphologic criteria(grade,stage)The hope is that tumor markers may lead to improved diagnostic,prognostic and therapeutic applications in the clinic.,Tissue Array Analysis,donor block array block slide,Tissue Microarray(TMA)Technology,Kononen et al.Nature Medicine 1998,Tissue Array Analysis,Tissue Microarray(TMA)Technology,Hundreds of tiny(typically 0.6 mm diameter)cylindrical tissue cores-densely and precisely arrayed into a single histologic paraffin block.From this new array block,up to 300 serial 4-8 m thick sections may be produced.Targets for protein expression by immuno-histochemical studies and fluorescence in situ hybridization(FISH).,Tissue Array Analysis,Tissue Array Section,Tissue Array Analysis,Real picture:brown staining,by D.Seligson,Tissue Array Analysis,Ki-67 Expression in Kidney Cancer,High Grade,Low Grade,Message:brown staining related to tumor grade,Tissue Array Analysis,Multiple measurements per patient:Several spots per tumor sample and several“scores”per spot,Maximum intensity=Max Percent of cells staining=Pos Percent of cells staining with the maximum intensity=PosMax Spots have a spot grade:NL,1,2,.Indicator of missingness,Each patient(tumor sample)is usually represented by multiple spots 3 tumor spots 1 matched normal spot,Tissue Array Analysis,Properties of TMA Data,Highly skewed,non-normal,semi-continuous.Often a good idea to model as ordinal variables with many levels.Staining scores of the same markers are highly correlated,Tissue Array Analysis,Presentation outline,Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis,Flowarray Analysis,Basic knowledge of flow cytometry,+,+,流式分析Flow cytometric analysis,捕获,标记,待测配体,检测探针,包被探针Coating probe,荧光,微载体microcarrier,Flowarray Analysis,Working principle of flowarray analysis,Principle：用高聚分子微粒为载体,在微载体表面进行探针-配体杂交,用流式细胞仪检测标记抗体的荧光强度.,Flowarray Analysis,Working principle of flowarray analysis,Technique characteristic：微量化、数字化、图像化和高通量化 microamount,digitalization,topography and high throughput Application：Research,clinical diagnosis,drug development,Flowarray Analysis,Working principle of flowarray analysis,Principle of high throughput analysis,Flowarray Analysis,(From Prof.Y LIN),BD CBATM液态芯片（科研）试剂盒（美国 2001）Luminex 100液态芯片（科研）试剂盒（美国2004）FlowChipTM流式芯片试剂盒（西安微通2007）临床诊断 免疫监测 药物研发,current situation of Flowarray technology,Flowarray Analysis,(From Prof.Y LIN),RF-IgA,RF-IgM,Normal,RA,Semi-works production 1:Rheumatoid factor,Flowarray Analysis,肾移植无排斥,肾移植排斥,IL-5,IFN-,IL-6,IL-2,Semi-works production 2:organ-graft refection,Flowarray Analysis,(From Prof.Y LIN),Thank you for your attention!,