分子生物學(xué)研究進(jìn)展05Biochip1029課件

1、Microarray Analysis鄭 芳Fang Zheng Ph.D.Prof. Tianjin Medical University1Microarray Analysis鄭 芳1Presentation outline Introduction DNA Microarray Tissue Aarray Analysis Flowarray Analysis2Presentation outline IntroducPresentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analy

2、sis3Presentation outline IntroducDefinition of MicroarrayMicroarray Analysis: Introduction 生物芯片：能對(duì)生物成分或生物分子進(jìn)行快速并行處理和分析的技術(shù)。檢測(cè)原理：利用特異性的分子間的相互作用，如核酸雜交、抗原-抗體特異性結(jié)合、蛋白-蛋白間特異性結(jié)合等。4Definition of MicroarrayMicro1. General IntroductionIf you get the gene, you will get the protein.If you want a protein, get it

3、s gene!51. General IntroductionIf you Abnormality of Gene structureAbnormality of Gene expressionCentral dogmaTechnologyPCRRE analysis Hybridization DNA sequencingDNA fingerprintingDNA microarrayRT-PCR FQ-PCRNorthern blotDNA microarray(Gene chip)Western-blotIHC stainELIZAProtein chipTissue ArrayDNAm

4、RNAProteintranscriptiontransplantationRT6Abnormality of Abnormality of History of Microarray developmentSouthern & Northern BlotDot BlotMacroarrayMicroarrayMicroarray Analysis: Introduction 7History of Microarray developmMicroarray ：Mini-laboratoryMicroarrayComputer-chipHistory of Microarray develop

5、mentMicroarray Analysis: Introduction 1950s: Computer-chip2000s: Bio-Microarray8Microarray ：Mini-laboratoryMicMicroarray Analysis: Introduction Microarray development Motivation Low-automaticity of the analytical apparatus Difficult to integrate all the steps together, including sample preparation,

6、biochemical event, consequence detection & analysis.9Microarray Analysis: IntroductMicroarray 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 10Micro

7、array Analysis: IntroductMicroarray Analysis: Introduction Classification of Microarray DNA/gene microarray Protein chip Tissue Array Flowarray11Microarray Analysis: IntroductDNA Microarray AnalysisPrinciple of Microarrays DNA芯片技術(shù)是指在固相支持物上原位合成（in situ synthesis）寡核苷酸探針，或者直接將大量的DNA探針以顯微打印的方式有序的固化于支持物表

8、面，然后與標(biāo)記的樣品雜交，通過對(duì)雜交信號(hào)的檢測(cè)分析即可得出樣品的遺傳信息（基因序列及表達(dá)的信息）。12DNA Microarray AnalysisPrincipDNA Microarray: miniaturized array having up to tens of thousands of single-stranded DNA attached to it.DNA Microarray AnalysisDefinition of Microarrays13DNA Microarray: miniaturized aMicroarray Analysis: Introduction M

9、icroarray development 生物芯片主要特征:高通量: 極高的樣品并行處理能力 High throughput: high level of parallel processing capability 微量化：所需樣品量減少，靈敏度高 High level of sensitivity自動(dòng)化：全過程自動(dòng)化、標(biāo)準(zhǔn)化程度高 Automatization、standardization14Microarray Analysis: Introduct1. Disease diagnosisApplications of biochip technologyMicroarray Ana

10、lysisMolecular mechanisms of the diseases Treatment strategyof the diseases 應(yīng)用DNA芯片可在DNA水平檢測(cè)與疾病相關(guān)的內(nèi)源性或外源性基因；應(yīng)用表達(dá)芯片可以在表達(dá)水平分析同一組織在不同的發(fā)育階段，正常及病理狀態(tài)基因表達(dá)的差異，也可分析不同組織同一基因在正常及病理狀態(tài)下表達(dá)的差異，從而為疾病診斷與分類，病原微生物分型提供科學(xué)依據(jù)。151. Disease diagnosisApplicatio2. DNA sequencingHuman genome project; HGP：To identify and determ

11、ine DNA sequence of human genomeSequencing by hybridization, SBH (雜交測(cè)序)： 建立在特定序列的DNA與特定長(zhǎng)度的寡核苷酸集合的雜交的基礎(chǔ)之上。未知DNA序列可以通過鑒定與靶DNA序列形成完整的雙鏈體寡核苷酸的重疊區(qū)域來確定. 65536個(gè)八核苷酸點(diǎn)陣可測(cè)定約200個(gè)核苷酸的序列；一百萬個(gè)十二核苷酸點(diǎn)陣可測(cè)定約1000個(gè)堿基對(duì).Microarray AnalysisApplications of biochip technologies162. DNA sequencingMicroarray An3. Search new g

12、enes 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 AnalysisApplications of biochip technology173. Search new genesMicroarray 5.Drug screening 在基因水平上尋找藥物靶標(biāo)，毒性對(duì)基因的影響。 臨床

13、許多細(xì)菌對(duì)藥物具有抗藥性，但是不同的亞型對(duì)同一藥物的敏感性不同。DNA芯片技術(shù)已被用于鑒定結(jié)核菌及非典型分支桿菌的基因型與耐利福平的相關(guān)性以及HIV產(chǎn)生抗藥性與其逆轉(zhuǎn)錄酶及蛋白酶基因突變的相關(guān)性。這為臨床選擇敏感藥物提供了有效手段。Applications of biochip technologyMicroarray Analysis185.Drug screening Applications 6. Proteinogram analysis 基因組計(jì)劃后的另一項(xiàng)重要計(jì)劃-蛋白組(Proteomics)計(jì)劃. Contrast analysis of proteinogram on nor

14、mal and cancer cells by Protein chipsApplications of biochip technologyMicroarray Analysis7. Miniaturization of analytical apparatus196. Proteinogram analysisApplicQuantitative monitoring of gene expression patterns with a complementary DNA microarray. Schena, M., et al., Science 1995;270:467470Para

15、llel 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;14:457460References:Microarray AnalysisApplications of bioc

16、hip technology20Quantitative monitoring of g212122222323Presentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis24Presentation outline IntroducDNA Microarray AnalysisPrinciple of Microarrays DNA芯片技術(shù)是指在固相支持物上原位合成（in situ synthesis）寡核苷酸探針，或者直接將大量的DNA探針以顯微打印的方式有序的固化于支持物

17、表面，然后與標(biāo)記的樣品雜交，通過對(duì)雜交信號(hào)的檢測(cè)分析即可得出樣品的遺傳信息（基因序列及表達(dá)的信息）。又被稱為基因芯片（gene chips）、DNA陣列（DNA array）、cDNA芯片（cDNA chips）、寡核苷酸陣列（oligonucleotide array）等。25DNA Microarray AnalysisPrincipDNA Microarray AnalysisPrinciple of Microarrays Microarray assays are based on hybridization of a single-stranded DNA labeled with

18、 a fluorescent tag to a complementary molecule attached to the chip26DNA Microarray AnalysisPrincipMicroarrays 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 probe

19、s which bind to a specific geneDNA Microarray AnalysisPrinciple of Microarrays27Microarrays enable simultaneouConstructionPlace 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)Hy

20、bridized probes (DNA molecules) are fluorescently labeledDNA Microarray AnalysisPrinciple of Microarrays28ConstructionDNA Microarray AnaWhen 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 molecu

21、le in test tube.DNA Microarray AnalysisPrinciple of Microarrays29When each spot in a microarray Chip preparation 芯片制備 Sample preparation 樣品制備 Work on the biochemical principle of DNA/DNA hybridization 雜交反應(yīng)Analyze hybridization pattern 結(jié)果分析DNA Microarray AnalysisBasic idea of Microarrays30 Chip prepa

22、ration 芯片制備DNA MicDNA Microarray AnalysisBasic idea of Microarrays31DNA Microarray AnalysisBasic iGene expressionGene disease relationGene-gene interactionFinding Co-Regulated GenesUnderstanding Gene Regulatory NetworksDNA Microarray AnalysisOne gene at a time before, now tens of thousands simultane

23、ously32Gene expressionDNA Microarray DNA 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-s

24、cale, high-throughput analysis33DNA Microarray AnalysisMicroar DNA 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 expre

25、ssion studies34 DNA Microarray AnalysiscDNA MDNA Microarray AnalysisSample preparation and hybridization to cDNA microarrays35DNA Microarray AnalysisSample DNA Microarray AnalysisComparison of Gene Expression PatternsBetween Normal Lung and Lung Carcinomafor Identification of Genes Associatedwith Ne

26、oplastic TransformationNormalTumorNormalTumorPatient 1Patient 236DNA Microarray AnalysisCompari用于基因轉(zhuǎn)錄水平的檢測(cè)、基因組分析和后基因組研究 美國(guó)斯坦福大學(xué)Davis等用PCR法從外周血淋巴細(xì)胞cDNA文庫中得到了1046種cDNA片段，制成芯片。然后與熱處理的T淋巴細(xì)胞和未處理的T細(xì)胞種提取的mRNA反轉(zhuǎn)錄出的單鏈cDNA片段雜交。經(jīng)激光共聚焦掃描系統(tǒng)分析，共發(fā)現(xiàn)17個(gè)差異表達(dá)的基因，其中11個(gè)是被熱誘導(dǎo)的，6個(gè)是被熱抑制的。經(jīng)序列分析證實(shí)，其中3個(gè)是未報(bào)導(dǎo)的新基因。DNA Microarray

27、 AnalysisDNA microarrays application37用于基因轉(zhuǎn)錄水平的檢測(cè)、基因組分析和后基因組研究DNA MiMicroarray Analysis: Development Microarray designs and applicationsMicroarray Probes cDNAs (from libraries or PCR products) Oligonucleotides Genomic DNA fragments38Microarray Analysis: DevelopmeMicroarray Analysis: Development Micr

28、oarray designs and applicationsApplications Gene expression studies Oligo-mediated sequencing Genetic mapping studies Mutation analysis Polymorphism analysis39Microarray Analysis: DevelopmeThe tools provided to us by our understanding of molecular biology, are what allow us to interrogate DNA/RNA fo

29、r the purpose of identifying disease causing by genetic alterations.DNA Microarray AnalysisDNA microarrays application40The tools provided to us by ouPresentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis41Presentation outline IntroducTissue Array AnalysisTissue mi

30、croarrays 1998 Kononen, Nature Medicineallow one to identify and evaluate highly specialized gene expression patterns and thus are a high-throughput tool (for diagnosis).Tissue MicroarrayDNA MicroarrayTissue microarrays 42Tissue Array AnalysisTissue miComplementary uses of DNA and tissue arraysDNA m

31、icroarrays 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 Analysis43Complementary uses of DNA and TMAs often

32、used for validating tumor genes in cancer researchThe 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

33、 in the clinic. Tissue Array Analysis44TMAs often used for validatingdonor block array block slide Tissue Microarray (TMA) TechnologyKononen et al. Nature Medicine 1998Tissue Array Analysis45donor block array block Tissue Microarray (TMA) TechnologyHundreds of tiny (typically 0.6 mm diameter) cylind

34、rical 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 Anal

35、ysis46Tissue Microarray (TMA) TechTissue Array Section0.6 mm 0.2mm 700 TissueSamplesTissue Array Analysis47Tissue Array Section0.6 mm Real picture: brown stainingby D. SeligsonTissue Array Analysis48Real picture: brown stainingbyKi-67 Expression in Kidney CancerHigh GradeLow GradeMessage: brown stai

36、ning related to tumor gradeTissue Array Analysis49Ki-67 Expression in Kidney CanMultiple 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 h

37、ave 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 Analysis50Multiple measurements per patiProperties of TMA DataHighly skewed, non-normal, semi-continuous.Often a good idea to model

38、 as ordinal variables with many levels.Staining scores of the same markers are highly correlated Tissue Array Analysis51Properties of TMA DataHighly sPresentation outline Introduction DNA Microarray Tissue Array Analysis Flowarray Analysis52Presentation outline IntroducFlowarray AnalysisBasic knowledge of flow cytometry53Flowarray AnalysisBasic knowle+流式分析Flow cytometric analysis捕獲標(biāo)記待測(cè)配體檢測(cè)探針包被探針Coating probe熒光微載體microcarrierFlowarray AnalysisWorking principle of flowarray analysis54+流式分析捕獲標(biāo)記待測(cè)配體檢測(cè)探針包被探針熒光微載體Flo P