分子生物学研究进展05Bioch.ppt

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1、Microarray Analysis郑郑 芳芳Fang Zheng Ph.D.Prof.Tianjin Medical University1Presentation outline Introduction DNA Microarray Tissue Aarray Analysis Flowarray Analysis2Presentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis3Definition of MicroarrayMicroarray Analysis:Int

2、roduction 生物芯片：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。析的技术。检测原理：检测原理：利用特异性的分子间的相互作用，利用特异性的分子间的相互作用，如核酸杂交、抗原如核酸杂交、抗原-抗体特异性结合、蛋白抗体特异性结合、蛋白-蛋白蛋白间特异性结合等。间特异性结合等。41.General IntroductionIf you get the gene,you will get the protein.If you want a protein,get its gene!5Abnormality of Gene struct

3、ureAbnormality of Gene expressionCentral dogmaTechnologyPCRRE analysis Hybridization DNA sequencingDNA fingerprintingDNA microarrayRT-PCR FQ-PCRNorthern blotDNA microarray(Gene chip)Western-blotIHC stainELIZAProtein chipTissue ArrayDNAmRNAProteintranscriptiontransplantationRT6History of Microarray d

4、evelopmentSouthern&Northern Southern&Northern BlotBlotDot Dot BlotBlotMacroarrayMacroarrayMicroarrayMicroarrayMicroarray Analysis:Introduction 7Microarray：Mini-laboratoryMicroarrayComputer-chipHistory of Microarray developmentMicroarray Analysis:Introduction 1950s:Computer-chip2000s:Bio-Microarray8M

5、icroarray Analysis:Introduction Microarray development Motivationu Low-automaticity of the analytical apparatusu Difficult to integrate all the steps together,including sample preparation,biochemical event,consequence detection&analysis.9Microarray Analysis:Introduction Microarray development Aim of

6、 microarray application:To integrate those noncontinuous analytic processes into a chip or a microarray,and made them continuous and miniaturized 10Microarray Analysis:Introduction Classification of Microarray DNA/gene microarray Protein chip Tissue Array Flowarray11DNA Microarray AnalysisPrinciple

7、of Microarrays DNADNA芯片技术芯片技术是指在固相支持物上原位合成（是指在固相支持物上原位合成（in situ in situ synthesissynthesis）寡核苷酸探针，或者直接将大量的）寡核苷酸探针，或者直接将大量的DNADNA探针以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）。传信息（基因序列及表达的信息）。12DNA MicroarrayDNA Microarray:mi

8、niaturized array having up to:miniaturized array having up to tens of thousands of single-stranded DNA attached tens of thousands of single-stranded DNA attached to it.to it.DNA Microarray AnalysisDefinition of Microarrays13Microarray Analysis:Introduction Microarray development 生物芯片主要特征生物芯片主要特征:高通量

9、高通量:极高的样品并行处理能力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，灵敏度高微量化：所需样品量减少，灵敏度高 High level of sensitivity自动化：自动化：全过程自动化、标准化程度高全过程自动化、标准化程度高 Automatization、standardization141.Disease diagnosisApplications of biochip technologyMicroarray AnalysisMolecular mecha

10、nisms of the diseases Treatment strategyof the diseases 应用应用DNADNA芯片可在芯片可在DNADNA水平检测与疾病相关的内源性或外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。疾病诊断与分类，病原微生

11、物分型提供科学依据。152.2.DNA sequencingHuman genome project;HGP：To identify and determine DNA sequence of human genomeSequencing by hybridization,SBH(杂交测序杂交测序)：建立在特定序列的建立在特定序列的DNADNA与特定长度的寡核苷酸集合的杂与特定长度的寡核苷酸集合的杂交的基础之上。未知交的基础之上。未知DNADNA序列可以通过鉴定与靶序列可以通过鉴定与靶DNADNA序列形序列形成完整的双链体寡核苷酸的重叠区域来确定成完整的双链体寡核苷酸的重叠区域来确定.6553

12、6 65536个八核苷酸点阵可测定约个八核苷酸点阵可测定约200200个核苷酸的序列；个核苷酸的序列；一百万个十二核苷酸点阵可测定约一百万个十二核苷酸点阵可测定约10001000个碱基对个碱基对.Microarray AnalysisApplications of biochip technologies163.Search new genes 4.Mutation and polymorphism analysisl To detect and analyze DNA variation and polymorphism on a large scalel Detection of gene

13、tic mutationl Inherited diseases,and tumor Microarray AnalysisApplications of biochip technology175.Drug screening 在基因水平上寻找药物靶标，毒性对基因的影响。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。同一药物的敏感性不同。DNA芯片技术已被用于鉴定结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核菌及非典型分支杆菌的基因型与耐利福平的相关性以

14、及及HIV产生抗药性与其逆转录酶及蛋白酶基因突变的相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。关性。这为临床选择敏感药物提供了有效手段。Applications of biochip technologyMicroarray Analysis186.Proteinogram analysis 基因组计划后的另一项重要计划基因组计划后的另一项重要计划-蛋白组蛋白组(Proteomics)计划计划.Contrast analysis of proteinogram on normal and cancer cells by Protein chipsApp

15、lications of biochip technologyMicroarray Analysis7.Miniaturization of analytical apparatus19 Quantitative monitoring of gene expression patterns with a complementary DNA microarray.Schena,M.,et al.,Science 1995;270:467470 Parallel human genome analysis:Microarray-based expression monitoring of 1000

16、 genes.Schena,M.,et al.,PNAS 1993:1061410619 Use of a cDNA microarray to analyse gene expression patterns in human cancer.DeRisi,J.,et al.,Nature Genetics 1996;14:457460References:Microarray AnalysisApplications of biochip technology20212223Presentation outline Introduction DNA Microarray Tissue Arr

17、ay Analysis Flowarray Analysis24DNA Microarray AnalysisPrinciple of Microarrays DNADNA芯片技术芯片技术是指在固相支持物上原位合成（是指在固相支持物上原位合成（in situ in situ synthesissynthesis）寡核苷酸探针，或者直接将大量的）寡核苷酸探针，或者直接将大量的DNADNA探针以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样品杂交，通过对杂交信号的检测分析即可得出样品的遗

18、传信息（基因序列及表达的信息）。传信息（基因序列及表达的信息）。又被称为基因芯片（又被称为基因芯片（gene chipsgene chips）、）、DNADNA阵列（阵列（DNA DNA arrayarray）、）、cDNAcDNA芯片（芯片（cDNAcDNA chips chips）、寡核苷酸阵列）、寡核苷酸阵列（oligonucleotideoligonucleotide array array）等。）等。25DNA Microarray AnalysisPrinciple of Microarrays Microarray assays are based on hybridizatio

19、n of a single-stranded DNA labeled with a fluorescent tag to a complementary molecule attached to the chip26Microarrays enable simultaneous measurement of the expression levels of thousands of genes in a sampleMicroarray:n nGlass slide with a matrix of thousands of Glass slide with a matrix of thous

20、ands of spots printed on to itspots printed on to itn nEach spot contains probes which bind to a Each spot contains probes which bind to a specific genespecific geneDNA Microarray AnalysisPrinciple of Microarrays27ConstructionPlace array of probes on microchipPlace array of probes on microchipProbe

21、is Probe is oligonucleotideoligonucleotide 25 bases long that 25 bases long that characterizes gene or genomecharacterizes gene or genomeChip is about 2cm by 2cmChip is about 2cm by 2cmSolid support(glass,plastic,metal,silicon)Solid support(glass,plastic,metal,silicon)Hybridized probes(DNA molecules

22、)are Hybridized probes(DNA molecules)are fluorescently labeledfluorescently labeledDNA Microarray AnalysisPrinciple of Microarrays28When 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

23、 test tube.DNA Microarray AnalysisPrinciple of Microarrays29pp Chip preparation 芯片制备芯片制备pp Sample preparation 样品制备样品制备pp Work on the biochemical principle of DNA/DNA hybridization 杂交反应杂交反应ppAnalyze hybridization pattern 结果分析结果分析DNA Microarray AnalysisBasic idea Basic idea of Microarrays30DNA Microar

24、ray AnalysisBasic idea Basic idea of Microarrays31Gene expressionGene disease relationGene-gene interactionFinding Co-Regulated GenesUnderstanding Gene Regulatory NetworksDNA Microarray AnalysisOne gene at a time before,now tens of thousands simultaneously32DNA Microarray AnalysisMicroarrays designs

25、 and applications:cDNA microarrays in gene expression studiescDNA 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 analysis33 DNA Microarray AnalysiscDNA Microarrays Thousand

26、s of known genes are available Requires special instrumentation for generation of microarrays and for analysis of resultsMicroarrays designs and applications:cDNA microarrays in gene expression studies34DNA Microarray AnalysisSample preparation and hybridization to cDNA microarrays35DNA Microarray A

27、nalysisComparison of Gene Expression PatternsBetween Normal Lung and Lung Carcinomafor Identification of Genes Associatedwith Neoplastic TransformationNormalTumorNormalTumorPatient 1Patient 236用用于于基基因因转转录录水水平平的的检检测测、基基因因组组分分析和后基因组研究析和后基因组研究 美美美美国国国国斯斯斯斯坦坦坦坦福福福福大大大大学学学学DavisDavisDavisDavis等等等等用用用用PCR

28、PCRPCRPCR法法法法从从从从外外外外周周周周血血血血淋淋淋淋巴巴巴巴细细细细胞胞胞胞cDNAcDNAcDNAcDNA文文文文库库库库中中中中得得得得到到到到了了了了1046104610461046种种种种cDNAcDNAcDNAcDNA片片片片段段段段，制制制制成成成成芯芯芯芯片片片片。然然然然后后后后与与与与热热热热处处处处理理理理的的的的T T T T淋淋淋淋巴巴巴巴细细细细胞胞胞胞和和和和未未未未处处处处理理理理的的的的T T T T细细细细胞胞胞胞种种种种提提提提取取取取的的的的mRNAmRNAmRNAmRNA反反反反转转转转录录录录出出出出的的的的单单单单链链链链cDNAcDN

29、AcDNAcDNA片片片片段段段段杂杂杂杂交交交交。经经经经激激激激光光光光共共共共聚聚聚聚焦焦焦焦扫扫扫扫描描描描系系系系统统统统分分分分析析析析，共共共共发发发发现现现现17171717个个个个差差差差异异异异表表表表达达达达的的的的基基基基因因因因，其其其其中中中中11111111个个个个是是是是被被被被热热热热诱诱诱诱导导导导的的的的，6 6 6 6个个个个是是是是被被被被热热热热抑抑抑抑制的。经序列分析证实，其中制的。经序列分析证实，其中制的。经序列分析证实，其中制的。经序列分析证实，其中3 3 3 3个是未报导的新基因。个是未报导的新基因。个是未报导的新基因。个是未报导的新基因。D

30、NA Microarray AnalysisDNA microarrays application37Microarray Analysis:Development Microarray designs and applicationsMicroarray Probes cDNAs(from libraries or PCR products)Oligonucleotides Genomic DNA fragments38Microarray Analysis:Development Microarray designs and applicationsApplications Gene ex

31、pression studies Oligo-mediated sequencing Genetic mapping studies Mutation analysis Polymorphism analysis39The 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

32、 AnalysisDNA microarrays application40Presentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis41Tissue Array AnalysisTissue microarrays 1998 Kononen,Nature Medicineallow one to identify and evaluate highly specialized gene expression patterns and thus are a high-thro

33、ughput tool(for diagnosis).Tissue MicroarrayTissue MicroarrayDNA MicroarrayDNA MicroarrayTissue microarraysTissue microarrays 42Complementary uses of DNA and tissue arraysComplementary uses of DNA and tissue arraysDNA microarrays for gene expression DNA microarrays for gene expression Used to find b

34、iomarkersUsed to find biomarkersRelatively Relatively few few tumor tumor samples samples but but thousands thousands of markers(genesof markers(genes)TMAsTMAs for protein expression patterns for protein expression patternsUsed to validate biomarkersUsed to validate biomarkerslarge large number numb

35、er(hundreds)(hundreds)of of tumors,tumors,relatively relatively few markers few markers Tissue Array Analysis43TMAs often used for validating TMAs often used for validating tumor genes in cancer researchtumor genes in cancer researchThe current classification systems of tumors are The current classi

36、fication systems of tumors are partially predictive of outcomes,and are based partially predictive of outcomes,and are based primarily upon morphologic criteria(grade,primarily upon morphologic criteria(grade,stage)stage)The hope is that tumor markers may lead to The hope is that tumor markers may l

37、ead to improved diagnostic,prognostic and therapeutic improved diagnostic,prognostic and therapeutic applications in the clinic.applications in the clinic.Tissue Array Analysis44donor block array block slide Tissue Microarray(TMA)TechnologyKononen et al.Nature Medicine 1998Tissue Array Analysis45Tis

38、sue Microarray(TMA)TechnologyHundreds 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-histochemica

39、l studies and fluorescence in situ hybridization(FISH).Tissue Array Analysis46Tissue Array SectionTissue Array Section0.6 mm 0.2mm 700 TissueSamplesTissue Array Analysis47Real picture:brown stainingReal picture:brown stainingby D.SeligsonTissue Array Analysis48Ki-67 Expression in Kidney CancerKi-67

40、Expression in Kidney CancerHigh GradeLow GradeMessage:brown staining related to tumor gradeTissue Array Analysis49Multiple measurements per patient:Multiple measurements per patient:Several spots per tumor sample and several“scores”per spotSeveral spots per tumor sample and several“scores”per spot M

41、aximum 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 spotTissue Array Analysis50Propert

42、ies of TMA DataProperties of TMA DataHighly skewed,non-normal,semi-continuous.n nOften a good idea to model as ordinal Often a good idea to model as ordinal variables with many levels.variables with many levels.Staining scores of the same markers are highly correlated Tissue Array Analysis51Presenta

43、tion outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis52Flowarray AnalysisBasic knowledge of flow cytometryBasic knowledge of flow cytometry53+流式分析Flow cytometric analysis捕获标记待测配体待测配体检测探针检测探针包被探针Coating probe荧光荧光微载体microcarrierFlowarray AnalysisWorking principle of flowarr

44、ay analysis54l Principle：用高聚分子微粒为载体用高聚分子微粒为载体,在微载体表面在微载体表面进行探针进行探针-配体杂交配体杂交,用流式细胞仪检测标用流式细胞仪检测标记抗体的荧光强度记抗体的荧光强度.Flowarray AnalysisWorking principle of flowarray analysis55l Technique characteristic：微量化、数字化、图像化和高通量化 microamount,digitalization,topography and high throughputl Application：Research,clinic

45、al diagnosis,drug developmentFlowarray AnalysisWorking principle of flowarray analysis56Principle of high throughput analysis Flowarray Analysis(From Prof.Y LIN)57BD CBABD CBABD CBABD CBATMTMTMTM液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国 2001200120012001）Luminex 100Luminex 100Luminex 1

46、00Luminex 100液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国液态芯片（科研）试剂盒（美国2004200420042004）FlowChipFlowChipFlowChipFlowChipTMTMTMTM流式芯片试剂盒（西安微通流式芯片试剂盒（西安微通流式芯片试剂盒（西安微通流式芯片试剂盒（西安微通2007200720072007）临床诊断临床诊断临床诊断临床诊断 免疫监测免疫监测免疫监测免疫监测 药物研发药物研发药物研发药物研发current situation of Flowarray technologyFlowarray Analysis

47、(From Prof.Y LIN)58RF-IgARF-IgMNormalRASemi-works production 1:Rheumatoid factorFlowarray Analysis59Y052405.007100101102103104FL2-HY052405.008100101102103104FL2-HY052405.020100101102103104FL2-HY051705IL2.004100101102103104FL2-HY051705IL2.005100101102103104FL2-HY051705IL6.004100101102103104FL2-HY051705IL6.005100101102103104FL2-HY052405.019100101102103104FL2-H肾移植无排斥肾移植无排斥肾移植肾移植排斥排斥IL-5IFN-IL-6IL-2Semi-works production 2:organ-graft refectionFlowarray Analysis(From Prof.Y LIN)6061Thank you for your attention!62