Loss of Heterozygosity

Thismethodwassuccessfulinourlabusingprostatetissueandforourspecificobjectives.Investigatorsmustbeawarethattheywillneedtotailorthefollowingprotocolfortheirownresearchobjectivesandtissueunderstudy.

ThismethodisusedtodetectgenomicDNAdeletionsintumorcells.Foramoredetaileddiscussionofapplyingthisapproachtomicrodissectedsamples,seeAllelicLossStudiesinProstateMPatNCI.

1.Reagents

1. DNAsample(seeProcessingofMicrodissectedTissue-DNA-basedAnalysis)
2. ProteinaseK(Sigma)
3. ProteinaseKbuffer(0.05Mtris-HCL,0.001MEDTA,1%Tween20,0.1mg/mlproteinaseK,pH8.0)
4. AmpliTaqGoldBuffer(PerkinElmer)
5. dNTPmixture(PerkinElmer)
6. Primers
7. DEPC-treatedH₂O
8. AmpliTaqGoldPolymerase(PerkinElmer)
9. a-³²PdCTP,6000Ci/mmol(NENDupont)
10. Formamide,99%(Fluka)
11. Bromophenolblue-Xylenecyanole(Sigma),reconstitutedasdirected
12. GelMix-6sequencinggelsolution(LifeTechnologies)
13. Ammoniumpersulfate(Biorad)
14. 10XTBEbuffer(0.89MTrisBase,0.89MBoricAcid0.02MDisodiumEDTA)(AdvancedBiotechnologies)
15. Acrylease(Stratagene)
16. Glasscleaner(e.g.,Windex,GlassPlus)
17. 95%ethanol

2.Equipment

1. Thermalcycler(MJResearch)
2. Sequencinggelelectrophoresisapparatus(GibcoBRL)
3. Highvoltagepowersupply
4. Geldryer(LifeTechnologies)
5. Glassplates,31.0x38.5cm(LifeTechnologies)
6. 0.4mmspacers(LifeTechnologies)
7. Castingboot(LifeTechnologies)
8. Sharktoothcomb(LifeTechnologies)
9. Smallclamps
10. Whatmanblottingpaper,3mmthickness
11. KodakBiomaxMRorARfilm
12. Filmcassette(AmershamLifeScience)
13. Filmprocessor

3.TimeRequirements

1. Gelpreparation:1.5-2hours.Polymerizationrequires1hour,butmaystandovernight.
2. LOHreactions:2.5hours(approximately1hourforset-up,1.5forPCR)
3. High-resolutiondenaturingpolyacrylamidegelelectrophoresis:1-3hours.Twentyminutesforset-up.Electrophoresistimevariesaccordingtoproductsize.
4. Geldrying:1hour
5. AutorADIography:1hour-2days

4.Methods

TIP:Investigatorsmustbeespeciallycarefulwhenusingthismethodologytoanalyzearchivaltissuespecimens.FormalinfixationinparticularresultsinDNAthatisdifficulttoamplifyandoftenproducesinconsistentPCRresults,includingartifactualalleliclossandpooramplificationoflargeproducts.Therefore,whenthistechniqueisusedtoanalyzearchivalsamples,itishighlyrecommendedthatreplicateexperiments(multipleindependentdissections,triplicatePCRreactions,etc.)beusedtoverifyresults.

A:LCMandProteinaseKTreatment

1. ObtainmicrodissectedcellsusingtheLCMprocedure.

   TIP:Thenumberofcellsneededtosuccessfullyperformtheassayvariesdependingonthequalityandprocessingconditionsofthetissuesamples.Onethousandcellsisrecommendedasagoodstartingpoint.

2. SUSPendapproximately1000microdissectedcellsin20µlproteinaseKbuffer.
3. Incubateovernightat37°C.

B:PreparetheGlassPlates

TIP:UseAccuwipesforcleaningpurposes,astheywillnotleavelintbehindandarenon-abrasive.

1. Cleanglassplatestwicewithglasscleaner.
2. Repeatusing95%EtOH.
3. SpraysmallplatewithAcrylease.
4. SpreadAcryleaseevenlyusingacircularmotion.
5. Buffdry.
6. Quicklyassembletheplateswithouttouchingthecleansurface.
7. Place0.4mmspacersontheedgesofthelargerplate.
8. Placethesmallerglassplateontopofthelargerplateandspacers.
9. Securetheplateswithacastingboot(tapeorclampsmaybesubstitutedforthecastingboot).

C:PolymerizetheGel

TIP:Acrylamideisaneurotoxin.Besuretowearglovesandalabcoatwhenworkingwiththissubstance.

1. Add480µlof10%ammoniumpersulfateto75mlofGel-mix-6.
2. Mixbyinversion.
3. Holdthenozzleofthebottleatthecornerofthegelcast.
4. Holdthegelcastata45^oangletothebenchandpourthegelbetweentheplates.Ifbubblesgettrappedbetweentheplates,removethembytappingtheoutsideoftheplatesorbytippingtheplatesupright.
5. Insertthestraightsideofthecombapproximately1cmintothegel.Ifbubblesareintroducedatthispoint,removethecombandusetheteethofthecombtosweepoutsmallbubbles.
6. Clampthetopoftheplatestogether.
7. Allowthegeltopolymerizeforatleastonehour.

TIP:Thegelcanbelefttopolymerizeovernight.However,ifbubblesappear,thegelhasbeguntoseparatefromtheplates.Tominimizeseparation,wrapthegelinplasticfilmandstoreat4°Cuntiluse.

D:PCRReaction

TIP:Investigatorsmustbeespeciallycarefulwhenusingthismethodologytoanalyzearchivaltissuespecimens.FormalinfixationinparticularresultsinDNAthatisdifficulttoamplifyandoftenproducesinconsistentPCRresults,includingartifactualalleliclossandpooramplificationoflargeproducts.Ifthistechniqueistobeutilizedforanalysisofarchivalsamples,wehighlyrecommendthatreplicateexperiments(multipleindependentdissections,triplicatePCRreactions,etc.)beusedtoverifyresults.

1. Removereagentsfromthefreezerbeforebeginningtheprocedure.
   • Thawthoroughlybeforeuse.
   • Prepareallreactionsonice.
   • PreparethereducedcytosinemixturepriortobeginningtheLOHreactionsetup.
   • Vortexallreagents,withtheexceptionofTaqGoldPolymerasebeforebeginningthePCRreactionsetup.
2. Prepare320µlreducednucleotidemixture:

   Aliquot1µlofeachDNAsampleintoaseparatePCRtubeandsetaside.

   TIP:DNAthatisrecoveredfrommicrodissectedsamplesand"semi-purified"usingaone-stepproteinaseKbufferwillsometimesproduce"non-specific"PCRproductsinadditiontothetruealleles.Moreover,largeralleleswillsometimesamplifymuchlesswellthansmalleralleles.Thus,normal-cellDNArecoveredfromthesametissuesectionasthetumorDNAservesasthebestcontrolfordeterminingthepresenceorabsenceofallelicloss.

   Preparesufficientvolumeofthereactionmixtureinaseparatetubeforallreactiontubes:

   ReducedNucleotideMix 10µl dATP,10mM 10µl dGTP,10mM 10µl dTTP,10mM 2.0µl dCTP,10mM 288µl DEPC-treatedH2O

   Thoroughlymixthereactionmixturebypipettinganddispense9µlofthereactionmixtureintoeachtubecontainingDNAsample.

   TIP:BesuretomixtheLOHreactionmixturewiththeDNAsamplebypipetting.ThisisespeciallycriticalforDNAfrommicrodissectedsamplesthathasbeenprocessedthroughaone-stepproteinaseK-based"purification."

   Capthereactiontubesandplacetheminathermalcycler.

   CyclethereactionsaccordingtoTmofthespecificprimerset.

   AfterPCR,removethesamplesfromthethermalcycleranddispense2µlofformamide/dyesolution(95%formamide,20mMEDTA,0.05%bromophenolblue,0.05%xylenecyanole)intoeachreactiontube.

   Storereactionsat4°Cuntilthegelisreadyforloading.

   TIP:Investigatorsmaywanttoconsiderthe"touchdown"procedureforPCRbyDonRH,CoxPT,WainwrightBJ,BakerK,Mattickjs:"Touchdown"PCRtocircumventspuriousprimingduringgeneamplification.NuclAcidsRes19:4008,1991.Advantagesinclude:

   • Muchcleanerbands,sincebystartingwithahighannealingtemperatureof66degreesandlowering1degreeeverycycle,thefirstPCRproductsarethemostspecificones.
   • Theexactsameprotocolcanbeusedforallprimers.

   E:FinalizeGelPreparation

   1. Removethegelfromthecastingboot.
   2. Pushthespacersintothegeluntiltheyareflushwiththesmallerglassplatetopreventthebufferfromleakingduringelectrophoresis(spacerstendtogetpushedoutofthegelduringpolymerization).
   3. Placethegelinthesequencingapparatusandclosethebufferreleasevalve.
   4. Pour500mlof0.5XTBEbufferintheupperchamberand500mlof1XTBEbufferinthelowerchamber.
   5. RemovethecombandclearbubblesfromtheloadingareawithaPipette.
   6. Inserttheteethofthecombapproximately1mmintothegel.
   7. Pre-heatthegelat1700voltsfor15-20minutes.

   F:GelLoading

   1. Removethesamplesfromthefreezer.
   2. Denaturethesamplesinathermalcyclerat95°Cfor5mins.
   3. Removesamplesfromthethermalcyclerandimmediatelyplaceonice,withanicepackontopofthesamples,for1min.
   4. Turnoffthepowersupply.
   5. Adjustcombifithasbeenpushedoutofthegelduringpre-heating.
   6. Load4µlofeachsampleperwell.

      TIP:Itisbesttoskiplanestoavoidcontaminationcausedbyleakingbetweenthewells.

   7. Runthegelat1700voltsfor1-2hours(runningtimebasedonPCRproductsize).

   G:SeparatetheGel

   1. Turnoffthepowersupply.
   2. Drainbufferchambers(buffermustbedisposedofinaliquidradioactivewastecarboy).
   3. Removethegelfromthesequencingapparatus.
   4. Separatetheplatesbyremovingthespacersandinsertingthetipsoftwothinspatulasintheirplace.
   5. Gentlyliftthespatulasuntilthetopplateseparatesfromthelowerplateandgel.
   6. PlaceWhatmanpaperonthegel.
   7. SlowlypeeltheWhatmanpaperandgelofftheglassplate.
   8. Coverthegelwithplasticwrapanddryonageldryerfor1hour.

   H:Autoradiography

   1. Removetheplasticwrapfromthegel.
   2. Placethegelinanautoradiographycassette.
   3. Exposefilm1hour-2daysusingKodakBioMaxMRorARfilm.

      TIP:UseMRfilmformaximumresolutionofbands.AnintensifyingscreenisusefulwhenanalyzingPCRproductsfromsmallnumbersofmicrodissectedcells.

   ReferencesDebelenkoLV,BrambillaE,AgarwalSK,SwalwellJI,KesterMB,LubenskyIA,ZhuangZ,GuruSC,ManickamP,OlufemiSE,ChandrasekharappaSC,CrabtreeJS,KimYS,HeppnerC,BurnsAL,SpiegelAM,MarxSJ,LiottaLA,CollinsFS,TravisWD,Emmert-BuckMR.IdentificationofMEN1genemutationsinsporadiccarcinoidtumorsofthelung.HumMolGenet6(13):2285-90,1997.

   Emmert-Buck,MR,Lubensky,IA,Dong,Q,Chandrasekharappa,C,Guru,SC,Manickam,P,Keseter,M,Olufemi,S-E,Agarwal,S,Burns,AL,Spiegel,AM,Collins,FS,Marx,SJ,Zhuang,Z,Liotta,LA,Debelenko,LV.LocalizationofthemultipleendocrineneoplasiaTypeI(MEN1)genebasedontumordeletionmapping.CancerRes57:1855-8,1997.

   Emmert-BuckMR,VockeCD,PozzattiRO,DurayPH,JenningsSB,FlorenceCD,ZhengpingZ,BostwickDG,LiottaL,andLinehanWM.Alleliclossonchromosome8p12-21inmicrodissectedprostaticintraepithelialneoplasia.CancerRes55:2959-62,1995.

   
   
   您的称呼：
   单位名称：
   您的邮箱：
   您的电话：
   品牌与型号：
   其他要求：
   新闻动态
   行业前沿
   技术文章
   最新产品
   • Gendepot公司 GenDEPOT细胞afp免疫荧光
     2017-12-04
   • Gendepot抗体品牌 GenDEPOT生物检测试剂盒
     2020-10-05
   • 代理 报价
     2018-09-04
   • 普瑞麦迪(北京)实验室技术有限公司_Premedical Laboratories Co...
     2021-03-24
   • GenDEPOT代理创亚化工(上海)有限公司-创亚化工(上海)有限公司
     2021-03-24
   • 全部产品分类_普瑞麦迪(北京)实验室技术有限公司_丁香通
     2021-03-25
   • 普瑞麦迪-首页
     2021-03-24
   • 普瑞麦迪-首页
     2021-03-24
   • [原创]石家庄凯达生物工程有限公司商业计划书doc下载_爱问共享资料
     2020-03-27
   • 三日疟原虫荧光PCR检测试剂盒优惠促销品牌:百奥莱博北京盖德...
     2019-09-04
   • GenDEPOT中国特约代理商(2021年度)
     2020-06-29
   • 暗黑破坏神2_暗黑破坏神2下载_修改器_存档_补丁_攻略_专题_游侠...
     2017-05-01
   • GenDEPOT 品牌介绍及代理商/经销商名单品牌专区
     2019-03-09
   • 厂家直供10ml玻璃瓶 透明棕色 留样瓶 试剂瓶 样品瓶 10ml ...
     2020-09-24
   • feldan_产品目录
     2021-04-03
   • GenDEPOT 品牌介绍及代理商/经销商名单品牌专区
     2019-03-17
   • ...different methods for generation of singlestranded DNA...
     2021-09-11
   • 敌克松杀菌剂价格_敌克松杀菌剂价格商家/厂家/公司/
     2021-08-13
   • TissueHarvestProtocol资讯
     2021-08-11
   • ...different methods for generation of singlestranded DNA...
     2021-09-13
   • 【进口代理清关】进口代理清关价格_进口代理清关报价
     2021-08-08
   • Tissue Harvest Protocol General Surgery and …
     2021-08-24
   • Tissue Harvest Protocol General Surgery and …
     2021-08-17
   • gendepot
     2021-08-31
   • Gendepot/ABTS显色剂（高对比度），A3510/A3510-050-500ml
     2026-07-11
   • Gendepot/Celecoxib，>99%，BC016/300 mg/BC016-300
     2026-07-11
   • Gendepot/His-Tag Mouse Monoclonal Antibody, 1mg/ml, Ab1005/1mg/Ab1005-101
     2026-07-11
   • Gendepot/Distilled Water, W0325/500 ml/W0325-050
     2026-07-11
   • Gendepot/X-Gal，5-溴-4-氯-3-吲哚基β-D-半乳吡喃糖苷，X0320/X0320-101-10 g
     2026-07-11
   • Gendepot/牛血清白蛋白，细胞培养试验，CA068/100 g/CA068-010
     2026-07-11
   • Gendepot/HRV 3C蛋白酶，T9800/1000单位/T9800-010
     2026-07-11
   • Gendepot/离心管，50 ml，PP，无菌，塞式密封盖，自立式，袋装，LC8006/500/CS/LC8006-500
     2026-07-11
   Copyright@苏州蚂蚁淘生物科技有限公司 2003-2021  188进口试剂采购网版权所有     苏ICP备17049038号-9

   友情链接：  其他

   188进口试剂采购网 www.188bio.cn -中国试剂网,试剂网,化学试剂网,国药试剂,抗体公司,试剂公司,试剂盒公司,苏州试剂公司,北京化学试剂公司,天津化学试剂,试剂商城,试剂代理,流式抗体 细胞库查询 sitemap

   Reactionmixture/reactiontube 1.0µl TaqBuffer 0.8µl Reducednucleotidemixture 0.2µl Forwardprimer,20µM 0.2µl Reverseprimer,20µM 6.6µl DEPCtreatedH20 0.1µl a-32PdCTP 0.1µl AmpliTaqGoldPolymerase Totalvolume=9µl