罗氏非放射性核酸标记及检测技术

Non-RadioactiveNucleic

AcidLabelingandDetection非放射性的核酸标记及检测技术罗氏诊断产品（上海）有限公司应用科学部主要内容背景知识非放射性核酸标记方法非放射性标记的核酸杂交非放射性核酸检测方法IsotopeLabBenefits对使用者健康无影响更高的灵敏度

comparedtoradioactivity更快获得结果

comparedtoisotopicprocedures探针可以至少保存一年多种用途

bywideproductrange方便操作

ofhandlingAdvantagesofNon-radioactiveLabelinganddetection:

DetectionofFragileXHybridizationwith32P-labeledprobeTarget:10µghumangenomicDNAProbe:RPL-labeledHybr.:HomeBrewExp.:o.n.12345678910111213DetectionofFragileXHybridizationwithDIG-labeledprobeTarget:10µghumangenomicDNAProbe:PCRlabeledHybr.:RocheprotocolExp.:20min12345678Non-Radioactivevs.Radioactive

(放射性与非放射性比较)特征比较放射性标记地高辛标记安全：放射性同位素会引起的健康危害使得备受争议，迫切需要采用一种更安全的方法作为替代。操作：即使是使用很小量的的放射性同位素都需要获得许可证，并在特定的放射性同位素实验室进行，所有的污染废弃物都必须小心的收集和弃置，所有这些都需要耗费人力财力。半衰期：由于放射性的快速衰减，用高特异活性标记的特定探针只能使用几天。检测方法：放射性探针通常是采用昂贵的X射线胶片来检测，这种方法比较费时，因为需要较长的暴光时间，并需要特殊的显影仪器。磷影像具有更高的灵敏度，但是更贵，使得很多实验室不能承受。安全：没有健康和环境的危害，没有不合适的安全限制。稳定：地高辛标记探针至少可以存放一年，可以预先准备大量的探针，需要的时候取用。无需专用设备：节省了实验室用于放置放射性工作平台的空间。可重复使用的杂交混合物－地高辛标记的探针可以存放于杂交缓冲液中，新鲜变性以后可以重复使用多次。多种检测方法：化学发光法，底物显色法化学发光法－极短的暴光时间，只需要5－30分钟，灵敏度可以达到0.03pgDNA或者0.1pgRNA。SouthernBlot可以在0.3ug的人胎盘DNA中检测出单拷贝的基因。底物显色法－检测的时间长一点，灵敏度低一点（16小时后可以检测出0.1pg的DNA）。地高辛DigoxigeninDigitalispurpureaDigoxigeninoccursexclusivelyinD.purpureaandD.lanata地高辛唯一来源于毛地黄和毛花毛地黄中WhatisDIG?类固醇半抗原物质可标记核酸或者蛋白可选择多种检测方法:显色，荧光或者化学发光快速安全灵敏的同位素代替品FeaturesofDiGoxigeninStructureofDIG-ModifiedNucleotidesCoupledvia碱稳定EtherBondAvailable:DIG-UTPDIG-dUTPDIG-ddUTPUTPSpacerODIGEtherbondStructureofDIG-dUTPCoupledvia碱不稳定OUTPSpacerDIGEsterbondO标记技术非放射法标记核酸酶法标记:

Klenow进行随机引物标记DNAPolI/DNaseI进行缺口平移标记PCR方法进行标记（TaqDNAPolymerase,/PwoDNAPolymeraseorExpand)*寡核苷酸3´-标记或用末端转移酶加尾标记DNA*用SP6-,T3-orT7RNAPolymerases*进行体外转录标记RNA用AMV/M-MuLV合成cDNAC.therm/Taq,C.therm/PwoorC.therm/Expand进行RT-PCR*LabelingmixesorkitsavailablefromRocheAppliedScience非放射性随机引物标记法3´OHdNTPsDIG-dUTPSingle-StrandedTemplate++Klenow-Enzymeoptimalspacingbetweenhaptensof20-30nucleotidesforoptimalrecognitionbyantibody

LabeledProbeDIGHighPrime和传统随机引物标记法的对比DIGHighPrimeDIGStandardR.P.LabeledDNA(ng)2500200015001000500002004006008001000minPCR法标记探针随机引物法标记探针模板：完整质粒DNATaqPolymeraseSpecificPrimers,(e.g.formultiplecloningsite)模板：纯化DNA插入片断KlenowPolymeraseRandomHexamerPrimersUnlabeledTemplateLabeledProbeFragments(sizerange:300-800-1500bpLabeledProbe“Full-Size”PrimaryExtensionProductsStandardDirectDetectionAssay

LabelingoftwodifferentprobesbyRPLSpottingofdilutionseriesanddirectdetectionwithchemiluminescenceCalculationofdilutionseriesaccordingto1µgoftemplateDNAResult:Foruseinhybridizationitisimportant,thatthe0.3pgspot(Probe1)ideallythe0.1pgspot(Probe2)isvisibleAmountofDIG-labeledDNAperspot10310.30.10pgControlProbe1Probe2探针纯化PurificationoflabeledprobepriortohybridizationItisnotnecessarytopurifylabeledprobesfromunincorporatedDIG-dUTPIfaprobecausesbackgroundproblems,e.g.ifthetemplateDNAwasisolatedwithahomebrewmethodapplythe

HighPurePCRProductPurificationKit寡聚核苷酸标记法－3‘端标记和加尾+DIG-ddUTP+Terminaltransferase+dATP+DIG-dUTP+TerminaltransferasetemplateindependentsynthesisofDNA-tailof40-50ntlengthwithseveralDIG-moleculesincorporated3´-OH5´OligonucleotidewithspecificsequenceadditionofasingleDIG-ddUTPnucleotide非放射性－RNA标记REpSPT18/19insertpromoter转录载体，含有噬菌体启动子和插入DNA片断用某一限制性内切酶将载体线性化+ATP,CTP,GTP,UTP,+DIG-UTP+SP6,T3orT7RNApolymerase进行体外转录definedlengthina2hincubationat37°CRNAsynthesisbyinvitrotranscriptionfromphagepromoter每ug的DNA模板最多能生成20ugRNA地高辛(DIG)标记试剂盒DIGDNALabelingKitDIG-HighPrimeDIGOligonucleotide3’-EndLabelingKit.(2ndGen)DIGOligonucleotide5’-EndLabelingSetDIGOligonucleotideTailingKit.(2ndGen)DIGRNALabelingKit(SP6/T7)PCRDIGProbeSynthesisKitHybridization杂交标记效率检测杂交原理有关杂交的产品DIG-QuantificationTeststrip(定量测试条)应用DIG-QuantificationTeststrips检测

DNAandRNA探针的标记效率DIGRNA标记1hDIGDNAControl-StripDIGDNA标记1h备注:次方法不能对DIG标记的寡核苷酸或PCR探针定量31030100300pg不同的方法标记探针用于杂交PositivelyChargedNylonMembrances (正电荷尼龙膜)DIGEasyHybBuffer优点:无毒性(尿素取代甲酰胺)无DNase和RNase,无菌

Southern和Northern-杂交作为质控检测在室温下稳定即用试剂可用于所有的杂交反应HybridizationBagswithSpout －带管口的杂交袋去除气泡的步骤：尽可能去袋中的除气泡挤压小部分的液体到管口内盖上盖子检测方法Detection地高辛(DIG)检测方法NBT/BCIP显色检测化学发光检测荧光检测检测DIG标记的核酸

Youneed:Anti-DIG-AntibodycoupledtoAlkalinePhosphataseAsubstrateforAlkalinePhosphatase,Colorsubstrate:NBT/BCIP(membranesandISH)Chemiluminescentsubstrates:CSPDorCDP-Star(nylonmembranesonly)原位杂交:Anti-DIG-FluoresceinAnti-DIG-Rhodamine检测DIG标记的核酸化学发光底物X-RayFilm-C-G-T-G-A-T-A-G-C-

A-C-U-A-TPPChemiluminescentDetectionCSPD/CDP-StarAntibody-ConjugateLabeled-ProbeTargetDNAMembraneDigoxigeninAlkalinePhosphataseSubstrateSubstrate标记和检测试剂盒DIG-HighPrimeDNALabelingandDetectionStarterKitI(显色法)DIG-HighPrimeDNALabelingandDetectionStarterKitII(化学发光法)DIGNorthernStarterKitDIGDNALabelingandDetectionKitDIGNucleicAcidDetectionKitNBT/BCIPColorSubstratesandReactionProducts(显色检测)fragmentsize(bp)49072176176612301033653517453394298,298234,234220194,194同源Southern杂交中显色法检测DNA

醋酸纤维膜

尼龙膜(unchargedmembrane)fragmentsize(bp)49072176176612301033653517453394298,298234,234220194,194300100301031300100301031DNAloadedperlane(pg)DNAloadedperlane(pg)TargetDNA:Dilutionbuffer:Probe:Detection:pBR328digestedseparatelywithEcoRl,Bg/landHinfl;50µg/mlHerringspermDNA10mMTris-HCL,1mMEDTA,pH8Digoxigenin-labeledpBR328DNANBT/BCIP16hoursChemiluminescenceSubstratesandReactionProducts(化学发光检测)同源Southern杂交化学发光检测的灵敏度pBR328-fragments100Target:pBR328digestedwithBamHl,BgllandHinfl,100;10;1pgloadedperlaneProbe:20ng/mlDIG-labeledpBR328DNA(randompriming)Polaroid-film,5min.exp.X-ray-film,3min.exp.fragmentsize(bp)49072176176612301033653517453394298(d)234(d)101100101<70fg不同发光底物CSPD/CDP-STAR比较典型的Southern杂交用Southern-Blot杂交法检测同源单拷贝基因(CourtesyofThomasLahaye,Univ.Aachen,Germany)Target:Probe:Hybridization:Washes:Detection:Exposure.10mgDNAof6differentbarleycultivars,cutwithEcoRI,separatedon0.8%agarosegel,followedbycapillarytransferSingle-copyDNAsequenceofbarleylabeledbyPCR

DIGEASYHYB,38°Co.n.2µlPCRproduct/ml

2x5min.roomtemp.2xSSC,0.1%SDS 2x15min.68°C;0.5xSSC,0.1%SDSCSPD8min.r典型的Northern杂交(CourtesyofC.Kraemer,InstituteforMolecularGenetics,UniversityofMainz,Germany)

DetectionoftheDmXGenefromDrosophilaMelanogasteronaNorthernBlotTarget:Probe:Exposure:1µgpoly(A)+RNAextractedfrom

D.melanogasteradult(lane1)and500ngpoly(A)+RNAfromlarvae(lane2),separatedonaMOPS-formaldehydegelfollowedbycapillarytransferAntisenseRNA5min.(

32P-labeledprobe14days!)11.5kb1