羅氏非放射性核酸標(biāo)記及檢測技術(shù)

Non-RadioactiveNucleic

AcidLabelingandDetection非放射性的核酸標(biāo)記及檢測技術(shù)羅氏診斷產(chǎn)品（上海）有限公司應(yīng)用科學(xué)部主要內(nèi)容背景知識非放射性核酸標(biāo)記方法非放射性標(biāo)記的核酸雜交非放射性核酸檢測方法IsotopeLabBenefits對使用者健康無影響更高的靈敏度

comparedtoradioactivity更快獲得結(jié)果

comparedtoisotopicprocedures探針可以至少保存一年多種用途

bywideproductrange方便操作

ofhandlingAdvantagesofNon-radioactiveLabelinganddetection:

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

(放射性與非放射性比較)特征比較放射性標(biāo)記地高辛標(biāo)記安全：放射性同位素會引起的健康危害使得備受爭議，迫切需要采用一種更安全的方法作為替代。操作：即使是使用很小量的的放射性同位素都需要獲得許可證，并在特定的放射性同位素實驗室進(jìn)行，所有的污染廢棄物都必須小心的收集和棄置，所有這些都需要耗費人力財力。半衰期：由于放射性的快速衰減，用高特異活性標(biāo)記的特定探針只能使用幾天。檢測方法：放射性探針通常是采用昂貴的X射線膠片來檢測，這種方法比較費時，因為需要較長的暴光時間，并需要特殊的顯影儀器。磷影像具有更高的靈敏度，但是更貴，使得很多實驗室不能承受。安全：沒有健康和環(huán)境的危害，沒有不合適的安全限制。穩(wěn)定：地高辛標(biāo)記探針至少可以存放一年，可以預(yù)先準(zhǔn)備大量的探針，需要的時候取用。無需專用設(shè)備：節(jié)省了實驗室用于放置放射性工作平臺的空間?？芍貜?fù)使用的雜交混合物－地高辛標(biāo)記的探針可以存放于雜交緩沖液中，新鮮變性以后可以重復(fù)使用多次。多種檢測方法：化學(xué)發(fā)光法，底物顯色法化學(xué)發(fā)光法－極短的暴光時間，只需要5－30分鐘，靈敏度可以達(dá)到0.03pgDNA或者0.1pgRNA。SouthernBlot可以在0.3ug的人胎盤DNA中檢測出單拷貝的基因。底物顯色法－檢測的時間長一點，靈敏度低一點（16小時后可以檢測出0.1pg的DNA）。地高辛DigoxigeninDigitalispurpureaDigoxigeninoccursexclusivelyinD.purpureaandD.lanata地高辛唯一來源于毛地黃和毛花毛地黃中WhatisDIG?類固醇半抗原物質(zhì)可標(biāo)記核酸或者蛋白可選擇多種檢測方法:顯色，熒光或者化學(xué)發(fā)光快速安全靈敏的同位素代替品FeaturesofDiGoxigeninStructureofDIG-ModifiedNucleotidesCoupledvia堿穩(wěn)定EtherBondAvailable:DIG-UTPDIG-dUTPDIG-ddUTPUTPSpacerODIGEtherbondStructureofDIG-dUTPCoupledvia堿不穩(wěn)定OUTPSpacerDIGEsterbondO標(biāo)記技術(shù)非放射法標(biāo)記核酸酶法標(biāo)記:

Klenow進(jìn)行隨機(jī)引物標(biāo)記DNAPolI/DNaseI進(jìn)行缺口平移標(biāo)記PCR方法進(jìn)行標(biāo)記（TaqDNAPolymerase,/PwoDNAPolymeraseorExpand)*寡核苷酸3′-標(biāo)記或用末端轉(zhuǎn)移酶加尾標(biāo)記DNA*用SP6-,T3-orT7RNAPolymerases*進(jìn)行體外轉(zhuǎn)錄標(biāo)記RNA用AMV/M-MuLV合成cDNAC.therm/Taq,C.therm/PwoorC.therm/Expand進(jìn)行RT-PCR*LabelingmixesorkitsavailablefromRocheAppliedScience非放射性隨機(jī)引物標(biāo)記法3′OHdNTPsDIG-dUTPSingle-StrandedTemplate++Klenow-Enzymeoptimalspacingbetweenhaptensof20-30nucleotidesforoptimalrecognitionbyantibody

LabeledProbeDIGHighPrime和傳統(tǒng)隨機(jī)引物標(biāo)記法的對比DIGHighPrimeDIGStandardR.P.LabeledDNA(ng)2500200015001000500002004006008001000minPCR法標(biāo)記探針隨機(jī)引物法標(biāo)記探針模板：完整質(zhì)粒DNATaqPolymeraseSpecificPrimers,(e.g.formultiplecloningsite)模板：純化DNA插入片斷KlenowPolymeraseRandomHexamerPrimersUnlabeledTemplateLabeledProbeFragments(sizerange:300-800-1500bpLabeledProbe“Full-Size”PrimaryExtensionProductsStandardDirectDetectionAssay

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

HighPurePCRProductPurificationKit寡聚核苷酸標(biāo)記法－3‘端標(biāo)記和加尾+DIG-ddUTP+Terminaltransferase+dATP+DIG-dUTP+TerminaltransferasetemplateindependentsynthesisofDNA-tailof40-50ntlengthwithseveralDIG-moleculesincorporated3′-OH5′OligonucleotidewithspecificsequenceadditionofasingleDIG-ddUTPnucleotide非放射性－RNA標(biāo)記REpSPT18/19insertpromoter轉(zhuǎn)錄載體，含有噬菌體啟動子和插入DNA片斷用某一限制性內(nèi)切酶將載體線性化+ATP,CTP,GTP,UTP,+DIG-UTP+SP6,T3orT7RNApolymerase進(jìn)行體外轉(zhuǎn)錄definedlengthina2hincubationat37°CRNAsynthesisbyinvitrotranscriptionfromphagepromoter每ug的DNA模板最多能生成20ugRNA地高辛(DIG)標(biāo)記試劑盒DIGDNALabelingKitDIG-HighPrimeDIGOligonucleotide3’-EndLabelingKit.(2ndGen)DIGOligonucleotide5’-EndLabelingSetDIGOligonucleotideTailingKit.(2ndGen)DIGRNALabelingKit(SP6/T7)PCRDIGProbeSynthesisKitHybridization雜交標(biāo)記效率檢測雜交原理有關(guān)雜交的產(chǎn)品DIG-QuantificationTeststrip(定量測試條)應(yīng)用DIG-QuantificationTeststrips檢測

DNAandRNA探針的標(biāo)記效率DIGRNA標(biāo)記1hDIGDNAControl-StripDIGDNA標(biāo)記1h備注:次方法不能對DIG標(biāo)記的寡核苷酸或PCR探針定量31030100300pg不同的方法標(biāo)記探針用于雜交PositivelyChargedNylonMembrances (正電荷尼龍膜)DIGEasyHybBuffer優(yōu)點:無毒性(尿素取代甲酰胺)無DNase和RNase,無菌

Southern和Northern-雜交作為質(zhì)控檢測在室溫下穩(wěn)定即用試劑可用于所有的雜交反應(yīng)HybridizationBagswithSpout －帶管口的雜交袋去除氣泡的步驟：盡可能去袋中的除氣泡擠壓小部分的液體到管口內(nèi)蓋上蓋子檢測方法Detection地高辛(DIG)檢測方法NBT/BCIP顯色檢測化學(xué)發(fā)光檢測熒光檢測檢測DIG標(biāo)記的核酸

Youneed:Anti-DIG-AntibodycoupledtoAlkalinePhosphataseAsubstrateforAlkalinePhosphatase,Colorsubstrate:NBT/BCIP(membranesandISH)Chemiluminescentsubstrates:CSPDorCDP-Star(nylonmembranesonly)原位雜交:Anti-DIG-FluoresceinAnti-DIG-Rhodamine檢測DIG標(biāo)記的核酸化學(xué)發(fā)光底物X-RayFilm-C-G-T-G-A-T-A-G-C-

A-C-U-A-TPPChemiluminescentDetectionCSPD/CDP-StarAntibody-ConjugateLabeled-ProbeTargetDNAMembraneDigoxigeninAlkalinePhosphataseSubstrateSubstrate標(biāo)記和檢測試劑盒DIG-HighPrimeDNALabelingandDetectionStarterKitI(顯色法)DIG-HighPrimeDNALabelingandDetectionStarterKitII(化學(xué)發(fā)光法)DIGNorthernStarterKitDIGDNALabelingandDetectionKitDIGNucleicAcidDetectionKitNBT/BCIPColorSubstratesandReactionProducts(顯色檢測)fragmentsize(bp)49072176176612301033653517453394298,298234,234220194,194同源Southern雜交中顯色法檢測DNA

醋酸纖維膜

尼龍膜(unchargedmembrane)fragmentsize(bp)49072176176612301033653517453394298,298234,234220194,194300100301031300100301031DNAloadedperlane(pg)DNAloadedperlane(pg)TargetDNA:Dilutionbuffer:Probe:Detection:pBR328digestedseparatelywithEcoRl,Bg/landHinfl;50μg/mlHerringspermDNA10mMTris-HCL,1mMEDTA,pH8Digoxigenin-labeledpBR328DNANBT/BCIP16hoursChemiluminescenceSubstratesandReactionProducts(化學(xué)發(fā)光檢測)同源Southern雜交化學(xué)發(fā)光檢測的靈敏度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不同發(fā)光底物CSPD/CDP-STAR比較典型的Southern雜交用Southern-Blot雜交法檢測同源單拷貝基因(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雜交(CourtesyofC.Kraemer,InstituteforMolecularGenetics,UniversityofMainz,Germany)

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

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

32P-labeledprobe14days!)11.5kb1