slide1 l.
Download
Skip this Video
Loading SlideShow in 5 Seconds..
生物芯片技术 PowerPoint Presentation
Download Presentation
生物芯片技术

Loading in 2 Seconds...

  share
play fullscreen
1 / 67
sani

生物芯片技术 - PowerPoint PPT Presentation

163 Views
Download Presentation
生物芯片技术
An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.

- - - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - - -
Presentation Transcript

  1. 生物芯片技术 生物芯片技术

  2. 生物芯片原理和应用

  3. 何为生物芯片? 生物芯片主要指通过平面微细加工技术在固体芯片 表面构建的微流体分析单元和系统,以实现对细胞 、蛋白质、核酸以及其他生物组分的准确、快速、 大信息量的检测。 他是继大规模集成电路之后的又一次具有深远意义 的科学技术革命。

  4. 生物芯片分类

  5. 蛋白芯片(Protein Chips) a microarray-based high-throughput protein assay method Chemiluminescence or Fluorescence based detection methods can be used to visualize bound antibodies.

  6. 蛋白芯片应用 • Diagnostic immunoassay that allows the simultaneous high-throughput analysis of known autoantigens. In order to quantify antibodies in the sera of patients with autoimmune diseases, Recombinant antigens and control proteins were immobilized on slides with reactive aldehyde groups as replicas in serial dilutions of the various antigens thereby allowing accurate determination of autoantibody titer using minimal amounts of serum. Miniaturized and highly paralleled immunoassays like these willreduce costbydecreasing reagent consumptionand improve performance by greatly increasing the number of assays that can be performed with a single serum sample. • Protein-Protein Interaction. • DNA- Protein Interaction

  7. 基因芯片(Gene chip) • 基因芯片(Gene chip)技术是指通过微阵列(Microarray)技术将高密度DNA片段阵列通过高速机器人或原位合成方式以一定的顺序或排列方式使其附着在如玻璃片等固相表面,以荧光标记的DNA探针,借助碱基互补杂交原理,进行大量的基因表达及监测等方面研究的最新革命性技术

  8. 基因芯片发展历史 Southern & Northern Blot Dot Blot Macroarray Microarray

  9. Types of DNA Chips

  10. Comparison of DNA Chip Technologies Oligo-Chip cDNA-Chip Genomic Chip 8 n or 20 n < 2,000 n > 50,000 n Sensitivity of DNA chip based assays is a function of: • Probe and target DNA/RNA (Complexity) • Chip surface (autofluorescence & non-spec. bkg) • Attachment chemistry/methodology (hyb. efficiency & crosshyb.) • Hybridization efficiency (lots of factors) • Detection technology (signal type, efficiency, noise) genomic analysis sequencing expression expression

  11. Why Genomic Biochips? • Screen specimens to determine gene copy number changes • Establish correlations between gene copy number changes and disease biology • Determine the interaction of multiple genes on the initiation and progression of disease • Accelerate development of products for genomic disease management to guide therapeutic intervention • Combined with expression chips, gives full understanding of disease process

  12. DNA Chip Technology • Solid support (glass, plastic, metal, silicon) • Miniaturized array of DNA (genetic material) • Work on the biochemical principle of DNA/DNA hybridization • Hybridized probes (DNA molecules) are fluorescently labeled

  13. Tumor Sample Genomic Expression Assay Formats 1. Extract genomic DNA from tissue 1. Extract mRNA from tissue Normal Sample 2. Produce cDNA by RT & Label 2. Label 3. Mix with labeled reference DNA 3. Mix with labeled reference cDNA 4. Hybridize to Chip 4. Hybridize to Chip 5. Wash and Image 5. Wash and Image

  14. Areas of Biochip Applications • Academic research of genetic diseases • Cancer • Prenatal genetics • General genetic diseases • Infectious diseases • Drug discovery • Animal farming/veterinary • Industrial (fermentation, corrosion) • Environmental

  15. 应用之一基因表达谱(gene expression pattern) • Research Use. • Clinical Diagnostic Use. Biological Sample Functional Information One Disease——One Gene Expression Pattern

  16. Research Use——From Sequence to function • 计算Ratio 值 (= Cy3/Cy5) • 在 0.5-2.0 之外的定义为在两样本中有明显差异表达。进而获取初步功能信息。 • Clustering

  17. Research Use——From Sequence to function Challenges • 庞大的数据库 • 数学模型

  18. Research Use ——From Sequence to function 可以大大推进包括人类(后)基因组计划在内的各类基因组研究,通过比较不同个体或物种之间以及同一个体在不同生长发育阶段、正常和疾病状态下基因及其表达的差异,寻找和发现新的基因,研究基因的功能以及生物体在进化、发育、遗传等过程中的规律。

  19. Research Use ——From Sequence to function 基因芯片可为研究不同层次多基因协同作用的生命过程提供手段。 将在研究人类重大疾病如癌症、心血管疾病等相关基因及作用机理方面发挥巨大的作用。

  20. Clinical Diagnostic Use One Disease=One Expression Pattern • Academic research of genetic diseases • Cancer • Prenatal genetics • General genetic diseases • Infectious diseases • HAV、HBV、HCV….. • HIV

  21. Clinical Diagnostic Use 生物芯片能为现代医学科学及医学诊断学的发展提供强有力的手段,促进医学从“系统、血管、组织和细胞层次”(通常称之为“第二阶段医学”)向“DNA、RNA、蛋白质及其相互作用层次”(第三阶段医学)过渡,使之快速进入实际应用。

  22. Prototype AmpliOnc™ I Biochip This biochip contains all genomic regions that have been reported to be amplified in cancers. AmpliOncTM I Biochip after hybridization; color composite of red, blue and green image

  23. Oncogene Targets On the AmpliOnc™ I Biochip FGR NMYC MYCL1 RAF1 HRAS1 EGFR1 KRAS2 FGFR1 REL PDGFRA WNT1 CND1 GLI INT2 MDM2 CDK4 MYB MET PIK3CA MYC ABL FGFR2 11 9 10 8 12 7 3 6 1 5 4 2 JUNB AR PDGFB HER2 AKT2 20q13 YES1 20 21 22 19 Y 16 FES 17 18 AKT1 15 14 13 X

  24. RDA Protocol • RNA extraction and cDNA preparation from archived tissue specimens(tester and driver) • Generation of amplified cDNA fragments (‘amplicons’) • Subtractive hybridization of amplicons • Enrichment of cDNA fragments from differentially expressed genes

  25. Microarray 用于验证RDA • Shotgun subcloning of RDA fragments • Picking transformed libraries for long-term propagation • Amplification of RDA inserts in 96-well plate format for arraying • Hybridization of cDNA amplicons to microarrays

  26. References aboutCoupling of RDA & Microarray • Schena, M. et al. (1995)Science, 270, 467 • Lockhart,et al.(1996).Nature Biotechnology, 14, 1675 • DeRisi, et al. (1996). Nature Genet., 14, 457.

  27. Microarray 还可用于验证 • SSH • Differential Display PCR

  28. 应用之三——SNPs & STRs analysis • Single Nucleotide Polymorphisms • Short Tandem Repeats • Polymerase • Ligase

  29. SNPs Typing ——by Ligase

  30. SNPs Typing ——by Polymerase(1)

  31. SNPs Typing ——by Polymerase(2)

  32. STRs Typing(1)

  33. STRs Typing(2)

  34. STRs Typing(3)

  35. 应用之四 ——LCM俘获细胞的基因表达分析 LCM: Laser Capture Microdissection

  36. 原理

  37. Gene expression profiles of laser-captured adjacent neuronal subtypes • Differential Gene Expression • between Large- and Small-sized • Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained • Large DRG Neurons: • myelinated • fast-conducting • transmit mechanosensory information • Small DRG Neurons: • unmyelinated • slow-conducting • transmit nociceptive information

  38. Gene expression profiles of laser-captured adjacent neuronal subtypes Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained

  39. cDNA microarray expression patterns of small (S) and large (L) neurons

  40. mRNA enriched in large DRG neurons

  41. mRNA enriched in small DRG neurons

  42. 放射性原位杂交验证结果

  43. 应用之五——Development of therapeutic Drugs • drug target discovery • evaluation of animal models of human disease • test for drug efficacy • test for drug specificity • test for drug toxicity

  44. 其它应用 在预防医学方面,可以使人们尽早地认识自身潜在的疾病,并实施有效的防治措施 法医学中,进行基因指纹快速识别,以及亲子鉴定

  45. 其它应用 监测流行病和传染病扩散 监测有害微生物的发生和传播 农、林、牧、鱼等产业的品种改良和病虫防治

  46. 生物芯片制作技术 及相关产品介绍

  47. 生物芯片制作方法分类

  48. 原位合成(In Situ Synthesis) Light directed oligonucleotide synthesis. A solid support is derivatized with a covalent linker molecule terminated with a photolabile protecting group. Light is directed through a mask to deprotect and activate selected sites, and protected nucleotides couple to the activated sites. The process is repeated, activating different sets of sites and coupling different bases allowing arbitrary DNA probes to be constructed at each site.