分子生物学研究进展05Biochip1029课件.ppt

1、Microarray Analysis1 Introduction DNA Microarray Tissue Aarray Analysis Flowarray Analysis2 Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis3Definition of MicroarrayMicroarray Analysis:Introduction 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

2、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 structureAbnormality of Gene expressionCentral dogmaTechnologyPCRRE analysis Hybridization DNA seq

3、uencingDNA fingerprintingDNA microarrayRT-PCR FQ-PCRNorthern blotDNA microarray(Gene chip)Western-blotIHC stainELIZAProtein chipTissue ArrayDNAmRNAProteintranscriptiontransplantationRT6History of Microarray developmentMicroarray Analysis:Introduction 7Microarray：Mini-laboratoryMicroarrayComputer-chi

4、pHistory of Microarray developmentMicroarray Analysis:Introduction 1950s:Computer-chip2000s:Bio-Microarray8Microarray Analysis:Introduction Microarray development Motivationu Low-automaticity of the analytical apparatusu Difficult to integrate all the steps together,including sample preparation,bioc

5、hemical event,consequence detection&analysis.9Microarray 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 10Microarray Analysis:Introduction Classificat

6、ion of Microarray DNA/gene microarray Protein chip Tissue Array Flowarray11DNA 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

7、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 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

8、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 mechanisms 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

9、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，病原微生物分型提供科学依据。152.2.DNA sequencingHuman genome project;HGP：To identify and determine DNA sequence of

10、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.65536 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

11、.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 genetic mutationl Inherited diseases,and tumor Microarray AnalysisApplications of biochip technology1

12、75.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。Applicat

13、ions 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 chipsApplications of biochip technologyMicroarray Analysis7.Miniaturization of analytical apparatus19 Qua

14、ntitative 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 genes.Schena,M.,et al.,PNAS 1993:1061410619 Use of a cDNA microarray to analyse gene expression

15、patterns in human cancer.DeRisi,J.,et al.,Nature Genetics 1996;14:457460References:Microarray AnalysisApplications of biochip technology20212223 Introduction DNA Microarray Tissue Array 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

16、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）。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

17、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 hybridization of a single-stranded DNA labeled with a fluorescent tag to a complementary molecule attached to the chip26DNA Micro

18、array AnalysisPrinciple of Microarrays27DNA Microarray AnalysisPrinciple of Microarrays28DNA Microarray AnalysisPrinciple of Microarrays29DNA Microarray Analysisof Microarrays30DNA Microarray Analysisof Microarrays31DNA Microarray AnalysisOne gene at a time before,now tens of thousands simultaneousl

19、y32DNA Microarray AnalysisMicroarrays designs 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

20、 Microarray AnalysiscDNA Microarrays Thousands 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 hybrid

21、ization to cDNA microarraysRNA IsolationReverseTranscriptionFluorescentTagFluorescentTagDNA“Chip”Mix and Applyto ArrayControlPopulationTreatedPopulation35DNA Microarray AnalysisComparison of Gene Expression PatternsBetween Normal Lung and Lung Carcinomafor Identification of Genes Associatedwith Neop

22、lastic TransformationNormalTumorNormalTumorPatient 1Patient 236DNA Microarray AnalysisDNA microarrays application37Microarray Analysis:Development Microarray designs and applicationsMicroarray Probes cDNAs(from libraries or PCR products)Oligonucleotides Genomic DNA fragments38Microarray Analysis:Dev

23、elopment Microarray designs and applicationsApplications Gene expression 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 i

24、dentifying disease causing by genetic alterations.DNA Microarray AnalysisDNA microarrays application40 Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis41Tissue Array AnalysisTissue microarrays 1998 Kononen,Nature Medicineallow one to identify and evaluate highly specialized gene

25、expression patterns and thus are a high-throughput tool(for diagnosis).42Tissue Array Analysis43Tissue Array Analysis44donor block array block slide Tissue Microarray(TMA)TechnologyKononen et al.Nature Medicine 1998Tissue Array Analysis45Tissue Microarray(TMA)TechnologyHundreds of tiny(typically 0.6

26、 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).Tiss

27、ue Array Analysis460.6 mm 0.2mm 700 TissueSamplesTissue Array Analysis47by D.SeligsonTissue Array Analysis48High GradeLow GradeMessage:brown staining related to tumor gradeTissue Array Analysis49 Maximum intensity=Max Percent of cells staining=Pos Percent of cells staining with the maximum intensity

28、=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 Analysis50Tissue Array Analysis51 Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis52Flowarray Analys

29、is53+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 flowarray 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 analy

30、sis55l Technique characteristic：微量化、数字化、图像化和高通量化 microamount,digitalization,topography and high throughputl Application：Research,clinical diagnosis,drug developmentFlowarray AnalysisWorking principle of flowarray analysis56Principle of high throughput analysis Flowarray Analysis(From Prof.Y LIN)57cu

31、rrent situation of Flowarray technologyFlowarray Analysis(From Prof.Y LIN)58100101102103104FL1-HF061128IgA.001100101102103104FL1-HF061128.001100101102103104FL1-HF061128.017100101102103104FL1-HF061129IgA.011RF-IgARF-IgMNormalRASemi-works production 1:Rheumatoid factorFlowarray Analysis59Y052405.00710

32、0101102103104FL2-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)606162