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From staining methods to DNA sequencing

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MethodsforDNAsequencing

BstDNApolymerase-catalyzedrADIolabeledtwo-stepsequencingreactions(26)aremodifiedfromthosepresentedearlier(25)byalteringtheabsoluteamountsandtherelativedeoxy/dideoxynucleotideratiosintheterminationmixes.Twoseparateterminationmixesprovidedoptimaloverlapforsequencedatastartinginthepolylinkerandextendingtoapproximately600basesfromtheprimingsite.Thistwo-stepformateliminatedtheneedforthechaserequiredintheBstone-stepreaction(25).

Eachextensionreactioncontained500-750ngofBiomekisolatedsingle-strandedDNA,reactionbuffer,nucleotideextensionmix,oligonucleotideprimer(typicallyM13(-40)universalsequencingprimer,seeAppendixD),either[a-32-P]dATPor[a-35-S]dATPandBstpolymerase.Afterthereactionsareextendedfor2minutesat67degCandbrieflycentrifuged,fouraliquotsareremovedandaddedtotheappropriatebase-specificterminationmix.Allnucleotidemixescontainedtheguanosinenucleotideanalog,7-deaza-dGTP,butdifferedintheirdeoxy/dideoxynucleotideratiostoyieldfragmentsranginginsizefromthebeginningofthepolylinkertogreaterthan300basesfromtheprimer,orfragmentsfromabout150togreaterthan600basesfromtheprimerfor"short"or"long"mixes,respectively.Followinganincubationat67degCfor10minandabriefcentrifugation,thereactionsarestoppedbytheadditionofdye/formamide/EDTA,andincubatedat100degC.Whendesired,sequencingreactionsarestoredat-70degCpriortotheadditionofloadingdye.

Whendouble-strandedpUC-basedsubclonesareusedastemplates,theamountofprimerisdoubledandadenaturing/annealingstepisadded.Here,3ugofplasmidDNA,isolatedbyeitherthemini-ormidi-prepdiatomaceousearthmethod,ismixedwithprimer,placedinaboiling-waterbath,andrapidlycooledbyplungingintoanethanol/dry-icebath(28).Followinganincubationonice,theremainingsequencingextensionreagents(reactionbuffer,nucleotideextensionmix,either[a-32-P]dATPor[a-35-S]dATP,andBstpolymerase)areadded.Reactionsareperformedasdescribedaboveforsingle-strandedsequencing.

Protocol

Forsingle-strandedDNAsequencing:

1.Preparethefollowingextensionreactioninamicrocentrifugetube:

750ngM13templateDNA2ulBstreactionbuffer2ulBstnucleotideextensionmix1uloligonucleotideprimer(2.5ng/ul)0.5-1ul[alpha]-32-P-dATPor[alpha]-35-S-dATP1uldilutedBstpolymerase(0.1U/ul)q.s.sterileddH2O12ul

[alpha]-32-P-dATP(PB10384)and[alpha]-35-S-dATP(SJ1304)fromAmersham.
DilutetheBstpolymerase(BioRad170-3406)inBstdilutionbuffer.
2.Incubatethereactionsfor2minutesat67degC,andbrieflycentrifugetoreclaimcondensation.
3.Remove2.5ulaliquotsforeachreactionintothefourbase-specificterminationmixes(eithershortorlong),alreadyPipettedintoaV-bottomedmicrotiterplate(Dynatech).
4.Incubatethereactionsfor10minutesat67degC,andbrieflycentrifugetoreclaimcondensation.ItispossIBLetostorethereactionsat-70degCatthisstage.
5.Stopthereactionsbytheadditionof4ulofagarosegelloadingdyeandincubatefor5-7minutesat100degC.
Fordouble-strandedDNAsequencing:
1.TodenaturetheDNAandannealtheprimer,incubatethefollowingreagentsinaboilingwaterbathfor4-5minutesandrapidlycoolthereactionbyplungingintoanethanol/dryicebath.
3ugplasmidDNA5ngoligonucleotideprimerq.s.sterileddH2O9ul
2.Incubatethereactioninanice-waterbathfor5minutes,andthenaddthefollowingreagents:
2ulBstreactionbuffer2ulBstnucleotideextensionmix0.5-1ul[alpha]-32-P-dATPor[alpha]-35-S-dATP1uldilutedBstpolymerase(0.1U/ul)15ul
[alpha]-32-P-dATP(PB10384)and[alpha]-35-S-dATP(SJ1304)fromAmersham.
DilutetheBstpolymerase(BioRad170-3406)inBstdilutionbuffer.
3.Proceedwiththesequencingreactionasdescribedaboveinsteps2-5forsingle-strandedtemplates
TopreparepolyacrylamidegelsforDNAsequencing,theappropriateamountofureaisdissolvedbyheatinginwaterandelectrophoresisbuffer,therespectiveamountofdeionizedacrylamide-bisacrylamidesolutionisadded,andammoniumpersulfateandTEMEDareaddedtoinitiatepolymerization.Immediatelyaftertheadditionofthepolymerizingagents,thegelsolutionispouredbetweentwoglassplates,tapedtogetherandseparatedbythinspacerscorrespondingtothedesiredthicknessofthegel,takingcaretoavoidandeliminateairbubbles.Priortotaping,theseglassplatesarecleanedwithAlconoxdetergentandhotwater,arerinsedwithdoubledistilledwater,anddriedwithaKimwipe.Typically,thenotchedglassplateistreatedwithasilanizingreagentandthenrinsedwithdoubledistilledwater.Afterpouring,thegelimmediatelyislaidhorizontallyandawellformingcombisinsertedintothegelandheldinplacebymetalclamps.Thepolyacrylamidegelsareallowedtopolymerizeforatleast30minutespriortouse.Afterpolymerization,thecombandthetapeatthebottomofthegelareremoved.Theverticalelectrophoresisapparatusisassembledbyclampingthetopandbottombufferwellsontothegel,andaddingrunningbuffertothebufferchambers.Thewellsarecleanedbycirculatingbufferintothewellswithasyringeand,immediatelypriortotheloadingofeachsample,theureaineachwellissuctionedoutwithamouthpipette.
Eachbase-specificsequencingreactionterminatedwiththeshortterminationmixisloadedusingamouthpipetteontoa0.15mmX50cmX20cm,denaturing5%polyacrylamidegelandelectrophoresedfor2.25hoursat22mA.Thereactionsterminatedwiththelongterminationmixtypicallyaredividedinhalfandloadedontotwo0.15mmX70cmX20cmdenaturing4%polyacrylamidegels.Onegeliselectrophoresedat15mAfor8-9hoursandtheotheriselectrophoresedfor20-24hoursat15mA.Afterelectrophoresis,theglassplatesareseparatedandthegelisblottedtoWhatmanpaper,coveredwithplasticwrap,driedbyheatingonaHoefervacuumgeldrier,andexposedtoX-rayfilm.Dependingontheintensityofthesignalandwhethertheradiolabelis32-Por35-S,exposuretimesvariedfrom4hourstoseveraldays.Afterexposure,thefilmsaredevelopedbyprocessingindeveloperandfixersolutions,rinsedwithwater,andairdried.Theautoradiogramthenisplacedonalight-boxandthesequenceismanuallyreadandthedatatypedintoacomputer.
Protocol
1.Prepare8Murea,polyacrylamidegelsaccordingtothefollowingrecipe(100ml),dependinginthedesiredpercentage:
4%5%6%urea48g48g48g40%A&B10ml12.5ml15ml10XMTBE10ml10ml10mlddH2O42ml39.5ml37ml15%APS500ul500ul500ulTEMED50ul50ul50ul
Urea(5505UA)isfromGibco/BRL.
2.Combinetheurea,MTBEbuffer,andwaterandincubatefor5minutesat55degCandthenstirtodissolvetheurea.
3.Coolbriefly,addtheA&B,mix,anddegasundervacuumfor5minutes.
4.Whilestirring,addtheAPSandTEMEDpolymerizationagentsandthenimmediatelypourinbetweentwotapedglassplateswith0.15mmspacers.(Priortotaping,thenotched,frontglassplateshouldbetreatedwithasmallamountofsilanizingreagentandthenrinsedwithddH2O).
5.Insertthewellformingcomb,clamp,andallowthegeltopolymerizeforatleast30minutes.
6.Priortoloading,removethetapearoundthebottomofthegelandthewell-formingcomb.Assembletheverticalelectrophoresisapparatusbyclampingtheupperandlowerbufferchamberstothegelplates,andadd1XMTBEelectrophoresisbuffertothechambers.
7.Flushthesamplewellswithasyringecontainingrunningbuffer,andimmediatelypriortoloadingeachsample,flushthewellwithrunningbufferusinggelloadingtips.
9.Load1-2ulofsampleintoeachwellusingaPipettemanwithgel-loadingtips,andthenelectrophoreseaccordingthefollowingguidelines(duringelectrophoresis,coolthegelwithafan):
terminationelectrophoresisreactionpolyacrylamidegelconditionsshort5%,0.15mmX50cmX20cm2.25hoursat22mAlong4%,0.15mmX70cmX20cm8-9hoursat15mAlong4%,0.15mmX70cmX20cm20-24hoursat15mA
10.Afterelectrophoresis,removethebufferwells,thetape,andprythegelplatesapart.Thegelshouldadheretobackplate.Blotthegeltoa40cmX20cmsheetof3MMWhatmanpaper,coverwithplasticwrap,anddryonaHoefergeldryerfor25minutesat80degC
11.PlacethedriedgelinacassetteandexposetoKodakXRP-1film.
12.Developthefilmfor1-5minutesinKodakGBXdeveloper,rinseindistilledwaterfor30seconds,fixinKodakGBXfixerfor5minutes,andthenrinseagainindistilledwaterfor30seconds.Allowthefilmtoairdry.
Eachbase-specificfluorescent-labeledcyclesequencingreactionroutinelyincludedapproximately100or200ngBiomekisolatedsingle-strandedDNAforAandCorGandTreactions,respectively.Double-strandedcyclesequencingreactionssimilarlycontainedapproximately200or400ngofplasmidDNA,isolatedusingeitherthestandardalkalinelysisorthediatomaceousearthmodifiedalkalinelysisprocedures.AllreagentsexcepttemplateDNAareaddedinonepipettingstepfromapremixofpreviouslyaliquottedstocksolutionsstoredat-20degC(seeAppendixB).Topreparethereactionpremixes,reactionbufferiscombinedwiththebase-specificnucleotidemixes.Priortouse,thebase-specificreactionpremixesarethawedandcombinedwithdilutedTaqDNApolymeraseandtheindividualfluorescentend-labeleduniversalprimers(seeAppendixC)toyieldthefinalreactionmixes,thataresufficientfor24templatesamples.
Oncetheabovemixesareprepared,fouraliquotsofsingleordouble-strandedDNAarepipettedintothebottomofeach0.2mlthin-walledreactiontube,correspondingtotheA,C,G,andTreactions,andthenanaliquotoftherespectivereactionmixesisaddedtothesideofeachtube.Thesetubesarepartofa96-tube/retainersettrayinamicrotiterplateformat,whichfitsintoaPerkinElmerCetusCycler9600.Stripcapsaresealedontothetube/retainersetandtheplateiscentrifugedbriefly.Theplatethenisplacedinthecyclerwhoseheatblockhadbeenpreheatedto95degC,andthecyclingprogramimmediatelystarted.Thecyclingprotocolconsistedof15-30cyclesofseven-temperatures:
95degCdenaturation
55degCannealing
72degCextension
95degCdenaturation
72degCextension
95degCdenaturation,and
72degCextension,linkedtoa4degCfinalsoakfile.
Atthisstage,thereactionsfrequentlyarefrozenandstoredat-20degCforuptoseveraldays.Priortopoolingandprecipitation,theplateiscentrifugedbrieflytoreclaimcondensation.Theprimerandbase-specificreactionsarepooledintoethanol,andtheDNAisprecipitatedanddried.Thesesequencingreactionscouldbestoredforseveraldaysat-20degC.
Protocol
1.Pipette1or2ulofeachDNAsample(100ng/ulforM13templatesand200ng/ulforpUCtemplates)intothebottomofthe0.2mlthin-walledreactiontubes(RobbinsScientific).Usethe1ulsampleforAandCreactions,andthe2ulsampleforGandTreactions.Meanwhile,preheatthePECetusThermocycler9600to95degC(Program#2).
2.PreparetheTaqpolymerasedilution.AmpliTaqpolymerase(N801-0060)isfromPerkin-ElmerCetus.
30ulAmpliTaq(5U/ul)30ul5XTaqreactionbuffer130ulddH20190uldilutedTaqfor24clones
3.PreparetheA,C,G,andTbasespecificmixesbyaddingbase-specificprimeranddilutedTaqtoeachofthebasespecificnucleotide/bufferpremixes:
A,C/G,T60/120ul5XTaqcyclesequencingmix30/60uldilutedTaqpolymerase30/60ulrespectivefluorescentend-labeledprimer120/240ul
4.Sealthereactiontubescarefullywiththestripcaps,andcentrifugebrieflyat2500rpm.Placethetube/retainersetinthe9600Cycler,abortthesoakfileprogram,andrunprogram#11.Thisprogramwillcyclethesequencingreactionsfor30cyclesofseventemperatures(30cyclesof95degCdenaturationfor4seconds;55degCannealingfor10seconds;72degCextensionfor1minute;95degCdenaturationfor4seconds;72degCextensionfor1minute;95degCdenaturationfor4seconds;and72degCextensionfor1minute),andthenwilllinktoa4degCsoakfileuntilthatprogramisaborted.(Itispossibletofreezethereactionsat-20degCaftercycling,priortothepoolingstep).
5.Brieflycentrifugetheplatetoreclaimcondensation.Poolthefourbasespecificreactionsinto250ulof95%ethanol.
6.Precipitatethesequencingreactions,andstorethedriedsamplesat-20degC.
OneofthemajorproblemsinDNAcyclesequencingisthatwhenfluorescentprimers(1)areusedthereactionconditionsaresuchthatthenestedfragmentsetdistributionishighlydependentuponthetemplateconcentrationinthereactionmix.WehaverecentlyobservedthatthenestedfragmentsetdistributionfortheDNAcyclesequencingreactionsusingthefluorescentlabelledterminators(8)ismuchlesssensitivetoDNAconcentrationthanthatobtainedwiththefluorescentlabelledprimerreactionsasdescribedabove.Inaddition,thefluorescentterminatorreactionsrequireonlyonereactiontubepertemplatewhilethefluorescentlabelledprimerreactionsrequireonereactiontubeforeachofthefourterminators.Thislatterpointallowsthefluorescentlabelledterminatorreactionstobepipettedeasilyina96wellformat.Theprotocolused,asdescribedbelow,iseasilyinterfacedwiththe96welltemplateisolationand96wellreactionclean-upproceduresalsodescribedherein.Byperformingallthreeofthesestepsina96wellformat,theoverallprocedureishighlyreproducableandthereforelesserrorprone.
Protocol
1.Place0.5ugofsingle-strandedor1ugofdouble-strandedDNAin0.2mlRobbinsPCRtubes.
2.Add1ul(forsinglestrandedtemplates)or4ul(fordouble-strandedtemplates)of0.8uMprimerand9.5ulofABIsuppliedpremixtoeachtube,andbringthefinalvolumeto20ulwithddH2O.
3.Centrifugebrieflyandcycleasusualusingtheterminatorprogramasdescribedbythemanufacturer(i.e.preheatat96oCfollowedby25cyclesof96oCfor15seconds,50oCfor1second,60oCfor4minutes,andthenlinktoa4oChold).
4.ProceedwiththespincolumnpurificationusingeithertheCentri-SepcolumnsorG-50microtiterplateproceduresgivenbelow.
TerminatorReactionClean-UpviaCentri-SepColumns
1.Gentlytapthecolumntocausethegelmaterialtosettletothebottomofthecolumn.
2.Removethecolumnstopperandadd0.75mldH2O.
3.Stopperthecolumnandinvertitseveraltimestomix.Allowthegeltohydrateforatleast30minutesatroomtemperature.Columnscanbestoredforafewdaysat4鸆;longerstorageinwaterisnotrecommended.Allowcolumnsthathavebeenstoredat4鸆towarmtoroomtemperaturebeforeuse.Removeanyairbubblesbyinvertingthecolumnandallowingthegeltosettle.Removetheupper-endcapfirstandthenremovethelower-endcap.Allowthecolumntodraincompletely,bygravity.(Note:Ifflowdoesnotbeginimmediatelyapplygentlepressuretothecolumnwithapipetbulb.)
4.Insertthecolumnintothewashtubeprovided.
5.Spininavariable-speedmicrocentrifugeat1300gfor2minutestoremovethefluid.
6.RemovethecolumnfromthewashtubeandinsertitintoaSampleCollectionTube.
7.Carefullyremovethereactionmixture(20ml)andloaditontopofthegelmaterial.Ifthesampleswereincubatedinacyclinginstrumentthatrequiredoverlayingwithoil,carefullyremovethereactionfrombeneaththeoil.Avoidpickingupoilwiththesample,althoughsmallamountsofoil(<1ml)inthesamplewillnotaffectresults.Oilattheendofthepipettipcontainingthesamplecanberemovedbytouchingthetipcarefullyonacleansurface(e.g.,thereactiontube).Useeachcolumnonlyonce.
8.Spininavariable-speedmicrocentrifugewithafixedanglerotor,placethecolumninthesameorientationasitwasinforthefirstspin--thisisimportantbecausethesurfaceofthegelwillbeatanangleinthecolumnafterthespin.
9.Drythesampleinavacuumcentrifuge.Donotapplyheat.Donotoverdry.Ifdesired,reactionscanbeethanolprecipitated.
TerminatorReactionClean-UpviaSephadexG-50FilledMicrotiterFormatFilterPlates
ThefollowingprotocolwasdevelopedattheC.ElegansGenomeSequencingCenteratWashingtonUniversity,St.Louis,Missouri,wasconveyedtousbyDr.RichardWilson,andnowhasbeenmodifiedtotakeadvantageoftheMillipore45ulColumnLoader(cat.#MACL09645).AdditionalinformationaboutthisprocedurealsoisavailableattheMilliporewebsite.
PreparationofSephadexG-50containingMicrotiterFilterPlates:
1.AdddrySephadexG-50totheMillipore(cat.#MACL09645)45ulColumnLoader.
2.RemovetheexcessofresinfromthetopoftheColumnLoaderwiththescrapersupplied.
3.PlaceMultiScreenHVPlate(MilliporeMAHVN4550)upside-downontopoftheColumnLoader.
4.InvertbothMultiScreenHVPlateandColumnLoader.
5.TapontopoftheMilliporeColumnLoadertoreleasetheresin.
6.Usingamulti-channelpipettor,add300ulofddH2Otoeachwelltoswelltheresin.andletstandatroomtemperaturefor3hours.
7.OncetheminicolumnsareswolleninMultiScreenplates,theycanbesealedwithsaranwrapandstoredintherefrigeratorat4degCforseveralweeks.Abatchofplatesalsocanbestoredintherefrigeratorat4degCforseveralweeksinasealedplasticcontainerwithadamptoweltoassuretheplatesarekeptmoist.
8.Whenneeded,thematrixcontainingfilterplateistapedoveramicrotiterplateandcentrifugedfor2minutesat1500RPMinaBeckmanGS-6Rtopackthecolumnsandtoremoveanyaccessbuffer.
**Olderprocedurenolongerused,replacedbysteps1-8above**1.Weighout8-10gofSephadexG-50Beaddiameter:50-150micron(medium)andtransfertoasterilebottle.2.Add100mlofsterilewater.3.Letitstandatroomtemperaturefor3hoursorplaceat4oCovernight.Packingandusingthecolumns:1.Sephadexsettlesout;therefore,youmustresUSPendbeforeaddingtotheplateandalsoafterfillingevery8to10wells.2.Add400ulofmixedSephadexG-50toeachwellofmicrotiterfilterplate(MilliporeMAHVN4550).3.Placemicrotiterfilterplateontopofanothermicrotiterplatetocollectwaterandtapesidessotheydonotflyapartduringcentrifugation.4.Spinat1500rpmfor2minutes.5.Discardwaterthathasbeencollectedinthemicrotiterplate.6.Againplacethemicrotiterfilterplateontopofthemicrotiterplatetocollectwaterandtapesidessotheydonotflyapartduringcentrifugation.7.Addanadditional100-200ulofSephadexG-50tofillthemicrotiterfilterplatewells.8.Spinat1500rpmfor2minutes.**endofolderprocedurenolongerused**
Usingthe96wellformatmicrotiterplatefiltersfordye-terminatorclean-up
9.Discardwaterthathasbeencollectedinthelowermicrotiterplate.
10.Placethecollectionmicrotiterfilterplateontopofamicrotiterplatetocollectsampleandtapesidessotheydonotflyapartduringcentrifugation.
11.Add20ulterminatorreactiontoeachSephadexG-50containingwells.Ifasmallerreactionvolumewasused(typically5-7ul),thenadd10ulofwatertodilutetothelargervolumeandimproverecovery.
12.Spinat1500rpmfor2minutes.
13.DrythecollectedeffluentinaSpeed-Vacforapproximately1-2hours.
Single-strandeddye-terminatorreactionsrequiredapproximately2ugofphenolextractedM13-basedtemplateDNA.TheDNAisdenaturedandtheprimerannealedbyincubatingDNA,primer,andbufferat65degC.Afterthereactioncooledtoroomtemperature,alpha-thio-deoxynucleotides,fluorescent-labeleddye-terminators,anddilutedSequenase[TM]DNApolymeraseareaddedandthemixtureisincubatedat37degC.Thereactionisstoppedbyaddingammoniumacetateandethanol,andtheDNAfragmentsareprecipitatedanddried.Toaidintheremovalofunincorporateddye-terminators,theDNApelletisrinsedtwicewithethanol.Thedriedsequencingreactionscouldbestoreduptoseveraldaysat-20degC.
Double-strandeddye-terminatorreactionsrequiredapproximately5ugofdiatomaceousearthmodified-alkalinelysismidi-preppurifiedplasmidDNA.Thedouble-strandedDNAisdenaturedbyincubatingtheDNAinsodiumhydroxideat65degC,andafterincubation,primerisaddedandthereactionisneutralizedbyaddinganacid-buffer.Reactionbuffer,alpha-thio-deoxynucleotides,fluorescent-labeleddye-terminators,anddilutedSequenase[TM]DNApolymerasethenareaddedandthereactionisincubatedat37degC.AmmoniumacetateisaddedtostopthereactionandtheDNAfragmentssimilarlyareprecipitated,rinsed,dried,andstored.
Protocol
ForSingle-strandedreactions:
1.Addthefollowingtoa1.5mlmicrocentrifugetube:
4ulssDNA(2ug)4ul0.8uMprimer2ul10xMOPSbuffer2ul10xMn[2+]/isocitratebuffer12ul
2.TodenaturetheDNAandannealtheprimer,incubatethereactionat65-70degCfor5minutes.Allowthereactiontocoolatroomtemperaturefor15minutes,andthenbrieflycentrifugetoreclaimcondensation.
3.Toeachreaction,addthefollowingreagentsandincubatefor10minutesat37degC.(Formorethanonereaction,apotofthereagentsshouldbemade).
7ulABIterminatormix(401489)2uldilutedSequenase[TM](3.25U/ul)1ul2mMa-SdNTPs22ul
TheundilutedSequenase[TM](70775)fromUnitedStatesBiochemicalsis13U/ulandshouldbediluted1:4withUSBdilutionbufferpriortouseresultinginaworkingdilutionof3.25U/ul.
4.Add20ul9.5Mammoniumacetateand100ul95%ethanoltostopthereactionandvortex.
5.PrecipitatetheDNAinanice-waterbathfor10minutes.Centrifugefor15minutesat12,000rpminamicrocentrifugeat4degC.Carefullydecantthesupernatant,andrinsethepelletbyadding300ulof70-80%ethanol.Vortexandcentrifugeagainfor15minutes,andcarefullydecantthesupernatant.
6.Repeattherinsesteptoinsureefficientremovaloftheunincorporatedterminators.(Alternatively,afterthefirstrinsestep,dropletsofsupernatantcanberemovedbycarefullyabsorbingthemwithaQ-tipcottonswaborarolledupKimwipe).
7.DrytheDNAfor5-10minutes(oruntildry)intheSpeedy-Vac,andstorethedriedreactionsat-20degC.
Fordouble-strandedreactions:
1.Addthefollowingtoa1.5mlmicrocentrifugetube:
5uldsDNA(5ug)4ul1NNaOH3ulddH2O12ul
2.Incubatethereactionat65-70degCfor5minutes,andthenbrieflycentrifugetoreclaimcondensation.
3.Addthefollowingreagentstoeachreaction,vortex,andbrieflycentrifuge:
3ul8uMprimer9ulddH2O4ulMOPS-Acidbuffer28ul
4.Toeachreaction,addthefollowingreagentsandincubatefor10minutesat37degC.(Formorethanonereaction,apotofthereagentsshouldbemade).
4ul10XMn[2+]/isocitratebuffer6ulABIterminatormix2uldilutedSequenase[TM](3.25U/ul)1ul2mM[alpha]-S-dNTPs22ul
TheundilutedSequenase[TM]fromUnitedStatesBiochemicalsis13U/ulandshouldbediluted1:4withUSBdilutionbufferpriortouseresultinginaworkingdilutionof3.25U/ul.
5.Add60ul8Mammoniumacetateand300ul95%ethanoltostopthereactionandvortex.
6.PrecipitatetheDNAinanice-waterbathfor10minutes.Centrifugefor15minutesat12,000rpminamicrocentrifugeat4degC.Carefullydecantthesupernatant,andrinsethepelletbyadding300ulof80%ethanol.Vortexandcentrifugeagainfor15minutes,andcarefullydecantthesupernatant.
7.Repeattherinsesteptoinsureefficientremovaloftheunincorporatedterminators.(Alternatively,afterthefirstrinsestep,dropletsofsupernatantcanberemovedbycarefullyabsorbingthemwithaQ-tipcottonswaborarolledupKim-wipe).
8.DrytheDNAfor5-10minutes(oruntildry)intheSpeedy-Vac.
PolyacrylamidegelsforfluorescentDNAsequencingarepreparedasdescribedaboveexceptthatthegelmixisfilteredpriortopolymerization.Optically-ground,lowfluorescenceglassplatesarecarefullycleanedwithhotwater,distilledwater,andethanoltoremovepotentialfluorescentcontaminantspriortotaping.Denaturing6%polyacrylamidegelsarepouredinto0.3mmX89cmX52cmtapedplatesandfittedwith36wellformingcombs.Afterpolymerization,thetapeandthecombareremovedfromthegelandtheoutersurfacesoftheglassplatesarecleanedwithhotwater,andrinsedwithdistilledwaterandethanol.ThegelisassembledintoanABIsequencer,andthecheckedbylaser-scanning.IfbaselinealterationsareobservedontheABI-associatedMacintoshcomputerdisplay,theplatesarerecleaned.Subsequently,thebufferwellsareattached,electrophoresisbufferisadded,andthegelispre-electrophoresedfor10-30minutesat30W.
Priortosampleloading,thepooledanddriedreactionproductsareresuspendedinformamide/EDTAloadingbufferbyvortexingandthenheatedat90degC.AsamplesheetiscreatedwithintheABIdatacollectionsoftwareontheMacintoshcomputerwhichindicatedthenumberofsamplesloadedandthefluorescent-labeledmobilityfiletouseforsequencedataprocessing.Aftercleaningthesamplewellswithasyringe,theodd-numberedsequencingreactionsareloadedintotherespectivewellsusingamicropipettorequippedwithaflat-tippedgel-loadingtip.Thegeltheniselectrophoresedfor5minutesbeforethewellsarecleanedagainandtheevennumberedsamplesareloaded.Thefilterwheelusedfordye-primersanddye-terminatorsisspecifiedontheABI373ACPU,alsowhereelectrophoresisconditionsareadjusted.Typicallyelectrophoresisanddatacollectionarefor10hoursat30WontheABI373Athatisfittedwithaheat-distributingaluminumplateincontactwiththeouterglassgelplateintheregionbetweenthelaserstopandthesampleloadingwells(26).
Afterdatacollection,animagefileiscreatedbytheABIsoftwarewhichrelatedthefluorescentsignaldetectedtothecorrespondingscannumber.Thesoftwarethendeterminedthesamplelanepositionsbasedonthesignalintensities.Afterthelanesaretracked,thecross-sectionofdataforeachlaneareextractedandprocessedbybaselinesubtraction,mobilitycalculation,spectraldeconvolution,andtimecorrection.OntheMacintoshcomputer,thecollecteddatacanbeviewedinseveralformats.Theoverallgraphicsimageofthegelcanbedisplayedtoassesstheaccuracyoflanetracking,andthedatafromeachsamplelanecanbeviewedaseitherafour-colorrawfluorescentsignalversusscannumber,asachromatogramofprocessedsequencedata,orasastringofnucleotides.Afterprocessing,thesequencedatafilesaretransferredtoaSPARCSTation2usingNFSShare.
Protocol
1.Prepare8Murea,4.75%polyacrylamidegels,asdescribedabove,usinga36-wellformingcomb.Alternatively,therecipecanbescaleduptooneliter.
2.Priortoloading,removethetapefromaroundtheentiregelandcarefullycleantheoutersurfaceofthegelplateswithhotwater.Rinsetheglasswithdistilledwaterandthenwithethanol,andallowtheethanoltoevaporate.
3.AssemblethegelplatesintoanABI373ADNASequencerbyplacingtheplatesontheledgeinthebottombufferwellandclampingthegelintoplacewiththeblackclampsattachedtothelaserstop.
4.ChecktheglassplatesbyclosingtheABIlidandselecting"StartPre-run"andthen"PlateCheck"fromtheABIdisplay.AdjustthePMTontheABIdisplay("Calibration","PMT")sothatthelowerscan(usuallytheblue)linecorrespondstoanintensityvalueof800-1000asdisplayedontheMacintoshcomputerdatacollectionwindow.Ifthebaselineoffour-colorscanlinesisnotflat,recleantheglassplates.
4.Attachthetopbufferandthealignmentbrace,andfillbothbufferwellswith1XMTBEelectrophoresisbuffer.Affixthealuminumheatdistributionplatebysettingitonthelaserstopagainsttheglassplates.
5.Pre-electrophoresethegelfor10-30minutesbychoosing"StartPre-run"and"Pre-runGel".
6.UseMakeSampleSheetOUtocreateasamplesheetordothisfromwithintheABIdatacollectionsoftwarebyenteringthenamesandthefluorescentmobilityfile("b920_21.mob"forfluorescent-labeledM13-21universalforwardprimer,"DyePrimer{M13RP1}"forfluorescent-labeledM13universalreverseprimer,"DyeTerm{anyprimer}"forAmpliTaqTerminators,and"DyeTerm{T7}-SetB"forSequenase[TM]fluorescent-labeleddyeterminators)touseforanalysis.ThisMacintoshprogramandtherelatedfilesareavailablefromourftpsiteatftp://ftp.genome.ou.edu/asastuffit1.5.1,binhexedfile.
7.Preparethesamplesforloading.Add3ulofFEtothebottomofeachtube,vortex,heatat90degCfor3minutes,andcentrifugetoreclaimcondensation.
8.Abortthepre-electrophoresis,andflushthesamplewellswithelectrophoresisbufferwithasyringe.Usingflat-tippedgelloadingpipettetips,loadeachodd-numberedsample.Pre-electrophoresethegelforatleast5minutes,flushthewellsagain,andthenloadeacheven-numberedsample.
9.Begintheelectrophoresis(30Wfor10hours)runbyselecting"StartRun"ontheABIdisplayandbychoosing"BeginDataCollection"fromthecontrollerboxwithintheABIdatacollectionsoftwareontheMacintosh.
10.Afterdatacollection,theABIsoftwarewillautomaticallyopenthedataanalysissoftware,whichwillcreatetheimagedgelfile,extractthedataforeachsamplelane,andprocessthedata.Checktheimagedgelfileforsampletracking,andthentransfertheresultsfoldercontainingthesequencetracefilestoaSPARCstation2wheretheharddiskismountedontheether-nettedMacintoshcomputerviaNFSShare.
SequencingdoublestrandedDNAtemplateshasbecomeacommonandefficientprocedure(10)forrapidlyobtainingsequencedatawhileavoidingpreparationofsinglestrandedDNA.DoublestrandedtemplatesofCDNAscontaininglongpoly(A)tractsaredifficulttosequencewithvectorprimerswhichannealdownstreamofthepoly(A)tail.Sequencingwiththeseprimersresultsinalongpoly(T)ladderfollowedbyasequencewhichisdifficulttoread.Inanattempttosolvethisproblemwesynthesizedthreeprimerswhichcontain(dT)17andeither(dA)or(dC)or(dG)atthe3"end.Wereasonedthatthepresenceofthesethreebasesatthe3"endwould"anchor"theprimersattheupstreamendofthepoly(A)tailandallowsequencingoftheregionimmediatelyupstreamofthepoly(A)region.
Usingthisprotocol,over300bpofreadablesequencecouldbeobtained.Wehaveappliedthisapproachtoseveralotherpoly(A)-containingcDNAcloneswithsimilarresults.SequencingoftheoppositestrandofthesecDNAsusinginsert-specificprimersoccurreddirectlyupstreamofthepoly(A)region.Theabilitytodirectlyobtainsequenceimmediatelyupstreamfromthepoly(A)tailofcDNAsshouldbeofparticularimportancetolargescaleeffortstogeneratesequence-taggedsites(STSs)(11)fromcDNAs(12,13).
Protocol1.Synthesizeanchoredpoly(dT)17withanchorsof(dA)or(dC)or(dG)atthe3"endonaDNAsynthesizeranduseafterpurificationonOligonucleotidePurificationCartridges.
2.Forsequencingwithanchoredprimers,denature5-10mgofplasmidDNAinatotalvolumeof50mlcontaining0.2Msodiumhydroxideand0.16mMEDTAbyincubationat65oCfor10minutes.
3.Addthethreepoly(dT)anchoredprimers(2pmolofeach)andimmediatelyplacethemixtureonice.Neutralizethesolutionbyadding5mlof5MammoniumacetatepH7.0.
4.PrecipitatetheDNAbyadding150mlofcold95%ethanolandwashthepellettwicewithcold70%ethanol.
5.Drythepelletfor5minutesandthenresuspendinMOPS-Acidbuffer.
6.Annealtheprimersbyheatingthesolutionfor2minutesat65oCfollowedbyslowcoolingtoroomtemperaturefor15-30minutes.
7.Performsequencingreactions,usingmodifiedT7DNApolymeraseanda-[32P]dATP(>1000Ci/mmole)usingtheprotocoldescribedearlier.
ThefollowingisarapidandefficientmethodforsequencingclonedcDNAsbasedonPCRamplification(14),randomshotguncloning(1,3,15),andautomatedfluorescentsequencing(16).ThismethodwasdevelopedinourlaboratorybecauseoncethesequenceofagenomicDNAcontainingcosmidisobtainedandputativeexonsarepredicted,thecorrespondingcDNAsshouldbesequencedinatimelymanner.However,thepresentlyimplementeddirectedcDNAsequencingstrategies,i.e.primerwalking(17)andExonucleaseIIIdeletion(18),arebothtimeconsumingandlaborintensive,whilethealternative,i.e.randomlyshearingtheintactplasmidfollowedbyshotgunsequencing(1,3,15),leadstoasignificantnumberofclonescontainingtheoriginalcDNAcloningvectorratherthanthedesiredcDNAinsert.
ThisisaPCR-basedapproachwherethe"universal"forwardand/orreverseprimingsiteswereexcludedfromtheresultingPCRproductbychoosingaprimerpairthatlaybetweentheusual"universal"forwardandreverseprimingsitesandthemultiplecloningsitesoftheStratageneBluescriptvector.ThesetwoPCRprimers,withthesequence5"-TCGAGGTCGACGGTATCG-3"fortheforwardor-16bsprimerand5"-GCCGCTCTAGAACTAGTG-3"forthereverseor+19bsprimer,nowhavebeenusedtoamplifysufficientquantitiesofcDNAinsertsinthe1.2to3.4kbsizerangesothattherandomshotgunsequencingapproachdescribedbelowcouldbeimplemented.
Protocol
1.Incubatefour100ulPCRreactions,eachcontainingapproximately100ngofplasmidDNA,100pmolesofeachprimer,50mMKCl(dilutefrom1Mstock),10mMTris-HClpH8.5(dilutefrom1Mstock),1.5mMMgCl2(dilutefrom1Mstock),0.2mMofeachdNTP(dilutefrom100mMstock),and5unitsofPE-CetusAmplitaqin0.5mlsnapcaptubesfor25cyclesof95oCfor1min.,55oCfor1min.and72oCfor2min.inaPE-Cetus48tubeDNAThermalCycler.
2.AfterpoolingthefourreactionstoobtainsufficientquantitiesofPCRproductforthesubsequentstepstheaqueoussolutioncontainingthePCRproductisplacedinanAeroMistNEBulizer,broughtto2.0mlbyaddingapproximately0.5to1.0mlofglycerol,andequilibratedat-20oCbyplacingitineitheranisopropylalcohol/dryiceorsaturatedaqueousNaCl/dryicebathfor10min.
3.Thesampleisnebulizedat-20oCbyapplying25-30psinitrogenpressurefor2.5min.FollowingethanolprecipitationtoconcentratetheshearedPCRproduct,thefragmentswerebluntendedandphosphorylatedbyincubationwiththeKlenowfragmentofE.coliDNApolymeraseandT4polynucleotidekinaseasdescribedpreviously.Fragmentsinthe0.4to0.7kbrangewereobtainedbyelutionfromalowmeltingagarosegel.

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发布于： 2018-12-26 阅读（144）

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