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ChemicalSequencingofDNA

ThisisarapidmethodforchemicalDNAsequencingwhichiscommonlyusedasladderforfootprintingreactionsorforsequencingofshortDNAoligonucleotides.

Reference:Bencinietal.(1984)Biotechniques2:4-5.

SteveHahn/HahnLab

Lastmodified1/6/00

ThemethodbelowworkswellforSequencingofDNAofgreaterthan~40bp.Typically,about150basesofsequencecanbereadfromanalysisona6-8%ureaacrylamidegel.Forsequencingofshortoligonucleotides,thereactiontimesshouldbeincreasedasnotedbelowandthereactionsshouldbeloadedto15%-20%acrylamideureagels.

Ifmakingasequencingladderforfootprinting,useseveralhundredthousandcpmperreactionifpossIBLe.ThiswillgiveenoughsequencedDNAformanygels(typically5,000cpmofthesequencingreactionarerunonalaneofasequencinggelforanovernightfilmexposure).Thefinalyieldofthismethodistypically30%-50%ofthestartingcpm.

TheDNAtobesequencedneedstobeinwater.TEorsaltwillinterferewithsomeofthesequencingreactions.However,1microliterorlessofTEina10microliterreactionwillbeO.K.

1.AliquotDNAinH₂0tothree1.5mlmicrofugetubeslabeled:G+A,A+C,andC+T.Addwatersothatthefinalvolumeineachtubeis9microliters.Addonemicroliterof1microgram/microlitercarrierDNA.ThiswillaidintheDNAprecipitationsteps.
2.TotheT+Ctubes,add15microlitersofhydrazine,mixwellandincubateatroomtemperaturefor10min.Fillthetubewithbutanol,vortexverywell(makesurethereisnotaseparatewaterandbutanolphase)andspininthemicrofugefor5min.Removebutanol,brieflyspinagainandremoveanyremainingbutanol.Dryinspeedvac.ResUSPendin150microliters1/10dilutedpiperidine.Storethesereactionsonicewhilecompletingtheothersequencingreactions.Usepiperidineinthefumehood.
3.TotheG+Atubes,add1.0microliter1MNa/HFormatepH2(useNaOHtoadjustthepHof1Mformicacid).Mixwellandincubate30minat37degrees.Add150microliters1/10dilutedpiperidineandstoreonice.
4.TotheA+Ctube,add1.0microliter30%NaOH.Incubate90degreesfor15min.ThisstepworksbestifperformedinaPCRtubeandincubatedinaPCRinstrumentwithaheatedlidtopreventevaporationduringthereaction.Afterthereactioniscomplete,transfertoa1.5mlmicrofugetube.Add150microliters1/10dilutedpiperidineandstoreonice.
5.Afteralltheabovereactionsarecomplete,incubatetubesat90degreesfor30min.Putaweightontopofthetubestopreventopening.ThisstepcleavestheDNAatthepointofmodification.Cooltubesandspinbeforeopening.
6.Add150microliters70%ethanoltotheA+Creactions.Fillalltubeswithbutanolandvortexverywelltomixphases.Spin5minutesinthemicrofugeandremovethesupernatants.Brieflyrespintoremoveallremainingbutanol.
7.Resuspendpelletsin150microlitersof1%SDS.Fillwithbutanolandprecipitateagain.Afterremovingsupernatant,brieflyspinandremoveanyremainingbutanol.DryinspeedvacandquantitaterADIoactivity.Resuspendinaconvenientvolumeofformamidesequencingdye.
ForsequencingofshortDNAs(lessthan50bp)increasethetimeofhydrazinereactionto12-15min,increasetheFormatereactionto50min,andincreasetheNaOHreactionto25min.

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