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1、Affymetrix 表达谱芯片在医学基础研究中的应用,复旦大学医学院 郑汉城,目录,Affymetrix公司介绍 基因芯片原理 表达谱芯片实验流程 表达谱芯片仪器平台 表达谱芯片应用,Affymetrix公司介绍,The Genomics Time Line,Full impact of genomic knowledge yet to be realized Current era is one of knowledge- and tool- building Associate genomic sequences with function Understand impact of po
2、lymorphisms Future utilization of genomic knowledge may allow: Fully integrative Systems models for understanding disease Comprehensive evaluation of drug efficacy and safety Patient stratification and clinical management,Pre-Genomics,Current Era (Transition),Post-Genomics,Pre- 1980s Now, next 15 ye
3、ars Future,医学研究进入组学时代，基因芯片技术提供全基因组的视角,Moving GeneChip to the Clinic Affymetrix致力于将技术应用于临床,Research,Clinic,Clinical Applications,Pre-clinical & Clinical Trials,Discovery,准确 稳定 方便 完善,Affymetrix 基因芯片技术在临床研究中的应用情况,Understanding Patient Response,Mechanism of ActionMechanism of ToxicityEfficacy in pre-cli
4、nicalDetermine dosage,Molecular basis of biology/diseaseDiscover and classify disease subtypesPredict disease outcomeIdentify therapeutic targets,Clinical efficacyClinical safetyTherapy modificationPatient stratification,目前已发表21000篇文献,Affymetrix系统及芯片的生产已经获得 ISO国际认证,在欧洲获得进入临床的许可,美国食品和药物监督局（FDA）已经审批通过
5、了Affymetrix GeneChip System 3000Dx (GCS 3000Dx) 一种用于分析体外诊断（IVD）芯片的仪器系统,Introduction: What GeneChip Can Do? Affymetrix芯片技术囊括基因组转录组水平的研究热点,Eukaryotic Cell,DNA,RNA,DNA Analysis,RNA Analysis,Central Annotation,and Visualization,One Platform many applications,One Genechip System,DNA-Genotyping Family bas
6、ed linkage studies_10k Whole-genome disease association_SNP6.0 Pharmacogenetics_DMET Chromosomal copy number_cyto2.7M Population genetics_SNP6.0 Linkage Disequilibrium _SNP6.0 Resequencing_300k RNA-Expression Analysis Biomarker discovery_U133 plus 2.0 Mechanism of Action_U133 plus 2.0 Transcription
7、factor binding_promoter tiling Transcriptome Analysis_gene ST 1.0 DNA methylation_promoter tiling Alternative splicing_exon,Accessing the Genome Multiple applications enabled on a single platform,RNA,DNA,Whole Genome,Focus,500K,U133,Target Genotyping,CustomExpress,10K,100K,CustomSeq,Rat,Mouse,Tiling
8、,All exon,SNP6.0,SNP5.0,microRNA,DMET,Cyto2.7M,Cyto310k,Affymetrix Facts and Figures,Located in Santa Clara, California Spun off from Affymax N.V. in 1992 Launched the GeneChip system in 1996 Initial Public Offering June 1996 Employees 900 Facilities in California, Massachusetts, United Kingdom , Ch
9、ina and Japan.,基因芯片化学原理,Affymetrix提供的是密度最高的芯片技术,5 m,Millions of copies of a specific oligonucleotide probe,Image of Hybridized GeneChip Array,6500,000 different complementary probes,Single stranded, labeled target,Oligonucleotide probe,GeneChip Array,Hybridized Probe Cell,1.28cm,1.28cm,Harnessing th
10、e Power of Photolithography,光蚀刻原位合成生产工艺是高质量芯片的基础,Quantitative Yes Reproducible Yes,Perfect Match PM,MM Mismatch,独具创新的PM-MM探针设计,Oligo 探针,Probe design,Affymetrix荧光标记系统信号级联放大原理,与PE耦联的SA和cRNA上的生物素结合。 生物素标记的抗体和SA结合。 与PE耦联的SA再与抗体上的生物素结合。,表达谱芯片实验流程,表达谱实验流程,Fragment (heat, Mg2+),Hybridize(16 hours),IVTBioti
11、n-rNTP,cDNA,TotalRNA,Scan,B,B,B,B,Wash & Stain,表达谱芯片应用,Surrogate Strategy Most expression arrays to date,Annotation Strategy Exon arrays Splice variants,Tiling strategy Unbiased look at the genome,多层次的转录水平研究,Known Exons,Unknown transcript,Tiling and Exon arrays are possible by high information conte
12、nt on arrays,表达谱基因芯片重要品质,GeneChip Expression Array Design,Gene Sequence,Multiple oligo probes,Perfect Match,Mismatch,5,3,Expression Catalog Products,Big Three Human Mouse RatModels Arabidopsis B. subtilis Barley Bovine C. elegans Canine Chicken Drosophila E. coli Maize Medicago P. aeruginosa Plasmod
13、ium Anopheles Porcine Rhesus Macaque Rice,Models (cont.) S. aureus Soybean Sugar cane Tomato Vitis vinifera Wheat Xenopus laevis Yeast Zebrafish 30个物种的全基因组Reagents Available for all of the major steps of the assay ControlsCustom,What has been done?,Understanding Disease Mechanism of Disease (large n
14、umber of examples; Leukemia, Psoriasis, Diabetes etc.) Using expression profiles to distinguish disease subtypes (ex: ALL, Yeoh et al. Cancer Cell 1:133-143 (2002) Identify clinical outcome (ex: metastasis Ramaswamy et al., Nature Genetics 33:49-54 (2003)Mechanism of Action Identifying molecular pat
15、hways altered by treatment (ex: SLE, Bennett et al., J. Exp. Med., 197: 711-723 (2003) Identifying molecular pathways altered by combinatorial therapeutic approaches (ex: ALL, Cheok et al., Nature Genetics Response to Therapy Predict Disease recurrence (ex: Hapatocellular Carcinoma Iizuka et al., The Lancet 361: 923-929 (2003) Predict resistance to therapy (ex: Bortezamib/Myeloma Mulligan et al., Poster American Society of Hematology annual meeting Dec. 2002),
