生物芯片技術(shù)生物芯片原理和應(yīng)用.ppt

1、生物芯片技術(shù),生物芯片技術(shù),生物芯片原理和應(yīng)用,何為生物芯片?,生物芯片主要指通過平面微細(xì)加工技術(shù)在固體芯片 表面構(gòu)建的微流體分析單元和系統(tǒng)，以實現(xiàn)對細(xì)胞 、蛋白質(zhì)、核酸以及其他生物組分的準(zhǔn)確、快速、 大信息量的檢測。 他是繼大規(guī)模集成電路之后的又一次具有深遠(yuǎn)意義 的科學(xué)技術(shù)革命。,生物芯片分類,蛋白芯片(Protein Chips),a microarray-based high-throughput protein assay method,Chemiluminescence or Fluorescence based detection methods can be used to vi

2、sualize bound antibodies.,蛋白芯片應(yīng)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 al

3、dehyde 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 will reduce cost by decreasing reagent consumption and improve performance by

4、 greatly increasing the number of assays that can be performed with a single serum sample. Protein-Protein Interaction. DNA- Protein Interaction,基因芯片(Gene chip)技術(shù)是指通過微陣列(Microarray)技術(shù)將高密度DNA片段陣列通過高速機(jī)器人或原位合成方式以一定的順序或排列方式使其附著在如玻璃片等固相表面，以熒光標(biāo)記的DNA探針，借助堿基互補雜交原理，進(jìn)行大量的基因表達(dá)及監(jiān)測等方面研究的最新革命性技術(shù),基因芯片（Gene chip）,基

5、因芯片發(fā)展歷史,Southern color composite of red, blue and green image,This biochip contains all genomic regions that have been reported to be amplified in cancers.,1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,X,Y,Oncogene Targets On the AmpliOnc I Biochip,PDGFB,EGFR1,PDGFRA,MET,FGFR2,WNT1,MYB,HE

6、R2,YES1,HRAS1,CND1,RAF1,GLI,MYC,MDM2,20q13,REL,MYCL1,FGR,FES,ABL,INT2,PIK3CA,NMYC,AKT2,FGFR1,JUNB,AKT1,KRAS2,CDK4,AR,RDA Protocol,RNA extraction and cDNA preparation from archived tissue specimens(tester and driver) Generation of amplified cDNA fragments (amplicons) Subtractive hybridization of ampl

7、icons Enrichment of cDNA fragments from differentially expressed genes,Microarray 用于驗證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,Refer

8、ences 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.,Microarray 還可用于驗證,SSH Differential Display PCR,應(yīng)用之三SNPs & STRs analysis,Single Nucleotide Polymorphisms Short Tandem R

9、epeats Polymerase Ligase,SNPs Typing by Ligase,SNPs Typing by Polymerase（1）,SNPs Typing by Polymerase（2）,STRs Typing（1）,STRs Typing（2）,STRs Typing（3）,應(yīng)用之四 LCM俘獲細(xì)胞的基因表達(dá)分析,LCM: Laser Capture Microdissection,原理,Gene expression profiles of laser-captured adjacent neuronal subtypes,Differential Gene Expr

10、ession 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,Gene expression profiles of laser-captured ad

11、jacent neuronal subtypes,Dorsal Root Ganglion (DRG) Rat Neurons, Nissl stained,cDNA microarray expression patterns of small (S) and large (L) neurons,mRNA enriched in large DRG neurons,mRNA enriched in small DRG neurons,放射性原位雜交驗證結(jié)果,應(yīng)用之五Development of therapeutic Drugs,drug target discovery evaluatio

12、n of animal models of human disease test for drug efficacy test for drug specificity test for drug toxicity,在預(yù)防醫(yī)學(xué)方面，可以使人們盡早地認(rèn)識自身潛在的疾病，并實施有效的防治措施 法醫(yī)學(xué)中，進(jìn)行基因指紋快速識別，以及親子鑒定,其它應(yīng)用,監(jiān)測流行病和傳染病擴(kuò)散 監(jiān)測有害微生物的發(fā)生和傳播 農(nóng)、林、牧、魚等產(chǎn)業(yè)的品種改良和病蟲防治,其它應(yīng)用,生物芯片制作技術(shù) 及相關(guān)產(chǎn)品介紹,生物芯片制作方法分類,原位合成(In Situ Synthesis),Light directed oligonuc

13、leotide 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 di

14、fferent sets of sites and coupling different bases allowing arbitrary DNA probes to be constructed at each site.,預(yù)先合成后點樣(off-chip synthesis),接觸式點樣 非接觸式點樣,接觸式點樣,Best!,接觸式點樣：Chipmaker Pin （Telechem專利）,ArrayIt ChipMaker Pins,Developed in conjunction with Stanford University Single dip in sample multipl

15、e spots Fine (EDM) slot in tip of stainless steel pin Take-up volume 250 nL (saves sample) Spot volume of 100 to 500 pL 100 m to 250 m spot size (high density arrays) Requires 10 to 30 pre-prints to “prime” pin,非接觸式點樣：nQUAD Technology,Characteristics of nQUAD Technology,Wide dispense range Low nanol

16、iter to high microliter Excellent linearity Precise and accurate CVs typically less than 10% Precision less than 5% Non-contact dispense mechanism Easier mechanical alignment (384, 1536,) “On-the-fly” printing possible Capable of dispensing onto membranes or slides Multiple liquid handling modes Asp

17、irate/Dispense Continuous Reagent Dispensing,生物芯片點樣系統(tǒng)必備性能,多針頭自動同時取樣，取樣板為96孔或384孔板 單針取樣量為250到500nL，每次點樣量為100到150pL 點樣直徑為100到150um，系統(tǒng)點陣分辨率為10um 可程控進(jìn)行連續(xù)點樣操作 固相介質(zhì)如玻璃片須通過適當(dāng)機(jī)制進(jìn)行位置固定 點樣針尖必須具備單次取樣連續(xù)點樣的性能,Cartesian - PixSys Series,合成后點樣板 - Cartesian Tech.,PixSys Series 特點：,Combines with TWO dispensing techno

18、logies Contact and Non-contact printing mode ChipMaker quill pins and nQUAD dispensers Pin array Spot Size: 75 to 200um nQUAD quantity: 10nL 10 m positioning resolution 50 slide capacity,Methods in Microarray Detection,CCD Confocal Scanning Microscopy,General Scanning Inc.,權(quán)威的Scanner 專業(yè)供應(yīng)商,ScanArray5000/4000/3000,SCANARRAY3000/4000/5000特點：,全自動檢測及分析系統(tǒng) 適用于D