羅氏非放射性核酸標記及檢測技術

Non-RadioactiveNucleic

AcidLabelingandDetection非放射性的核酸標記及檢測技術羅氏診斷產(chǎn)品（上海）有限公司應用科學部主要內(nèi)容背景知識非放射性核酸標記方法非放射性標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)特征比較放射性標記地高辛標記安全：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：地高辛標記探針至少可以存放一年，可以預先準備大量的探針，需要的時候取用。無需專用設備：節(jié)省了實驗室用于放置放射性工作平臺的空間?？芍貜褪褂玫碾s交混合物－地高辛標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使用多次。多種檢測方法：化學發(fā)光法，底物顯色法化學發(fā)光法－極短的暴光時間，只需要5－30分鐘，靈敏度可以達到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?類固醇半抗原物質可標記核酸或者蛋白可選擇多種檢測方法:顯色，熒光或者化學發(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標記技術非放射法標記核酸酶法標記:

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

LabeledProbeDIGHighPrime和傳統(tǒng)隨機引物標記法的對比DIGHighPrimeDIGStandardR.P.LabeledDNA(ng)2500200015001000500002004006008001000minPCR法標記探針隨機引物法標記探針模板：完整質粒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寡聚核苷酸標記法－3‘端標記和加尾+DIG-ddUTP+Terminaltransferase+dATP+DIG-dUTP+TerminaltransferasetemplateindependentsynthesisofDNA-tailof40-50ntlengthwithseveralDIG-moleculesincorporated3′-OH5′OligonucleotidewithspecificsequenceadditionofasingleDIG-ddUTPnucleotide非放射性－RNA標記REpSPT18/19insertpromoter轉錄載體，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進行體外轉錄definedlengthina2hincubationat37°CRNAsynthesisbyinvitrotranscriptionfromphagepromoter每ug的DNA模板最多能生成20ugRNA地高辛(DIG)標記試劑盒DIGDNALabelingKitDIG-HighPrimeDIGOligonucleotide3’-EndLabelingKit.(2ndGen)DIGOligonucleotide5’-EndLabelingSetDIGOligonucleotideTailingKit.(2ndGen)DIGRNALabelingKit(SP6/T7)PCRDIGProbeSynthesisKitHybridization雜交標記效率檢測雜交原理有關雜交的產(chǎn)品DIG-QuantificationTeststrip(定量測試條)應用DIG-QuantificationTeststrips檢測

DNAandRNA探針的標記效率DIGRNA標記1hDIGDNAControl-StripDIGDNA標記1h備注:次方法不能對DIG標記的寡核苷酸或PCR探針定量31030100300pg不同的方法標記探針用于雜交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-雜交作為質控檢測在室溫下穩(wěn)定即用試劑可用于所有的雜交反應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顯色檢測化學發(fā)光檢測熒光檢測檢測DIG標記的核酸

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

A-C-U-A-TPPChemiluminescentDetectionCSPD/CDP-StarAntibody-ConjugateLabeled-ProbeTargetDNAMembraneDigoxigeninAlkalinePhosphataseSubstrateSubstrate標記和檢測試劑盒DIG-HighPrimeDNALabelingandDetectionStarterKitI(顯色法)DIG-HighPrimeDNALabelingandDetectionStarterKitII(化學發(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(化學發(fā)光檢測)同源Southern雜交化學發(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