A practical method for the preparation of total DNA from filamentous fungi

ApracticalmethodforthepreparationoftotalDNAfromfilamentousfungi

M.I.Borges,M.O.Azevedo,R.Bonatelli,Jr.,M.S.S.FelipeandS.Astolfi-FilhoDepartamentodeBIOLOGiaCelular,UniversidadedeBrasilia,70910Brasilia,andDepartamentodeGenetica,UniversidadedeCampinas,13100Campinas,Brazil

MostmethodsofDNApreparationfromfungiaretime-consumingduetotheneedtofirstmakeprotoplasts,expensiveforchemicalssuchascesiumchloride,orsuitableonlyforsmallscalepreparations.WehavedevelopedasimplemethodfortotalDNApreparation,yieldingaproductofqualitysuitableforrestrictiondigestionandlibraryconstruction.

ToextractDNA,0.5to2.0goffreshorfrozenmyceliumisgroundinliquidnitrogen.ThemycelialpowderisdividedbetweentwoSorvalltubeseachcontaining15mlofcoldspermidine-SDSbuffer(4mMspermidine,10mMEDTA,0.1MNaCl,0.5%SDS,10mMß-mercaptoethanol,40mMTris-HClpH8.0),andthoroughlyshaken.Themixtureisthenimmediatelyextractedwith1voldoubledistilledphenol.Afterasecondphenolextraction,theaqueousphaseisextractedwith1volchloroform.Totheresultingaqueousphaseisadded10%ofthatvolumeof3MsodiumacetatepH5.5.DNAisthenprecipitatedbyadditionof2volsofcoldabsoluteethanol,andrecoveredeitherbyspoolingitoutorbycentrifugationat10,000xgfor10minutes.Afterwashingwith70%coldethanol,theDNAisair-driedatroomtemperature,redissolvedinTEbuffer(20mMTris-HClpH7.5,0.1mMEDTA)toaDNAconcentrationcirca0.5mg/mlandstoredat-20°C.Purityandaveragefragmentsizearecheckedbyelectrophoresis.

ThemethodishighlyreproducIBLe,andresultsover70%recoveryofgoodqualityDNA(A260/A280circa1.85).Withoptimalgrindingandnucleaseinhibition,theDNAisofhighmolecularweight(>23kb),withminimalsmearonelectrophoresis.ThemethodhasbeenusedforNeurosporacrassa,Aspergillusnidulans,DactyliumdendroidesandHumicolagrisea.Forallfourspecies,theDNApreparedhasbeensuccessfullyrestrictedandreligated.DNAfromthismethodhasbeenusedintheconstructionofgenomiclibrariesfromHumicolagriseaandDactyliumdendroides.