A simple, rapid procedure for the isolation of DNA for PCR from Gibberella fujikuroi (Fusarium section Liseola)

Thepolymerasechainreaction(PCR)isamethodforamplifyingspecificsegmentsofDNAdefinedbythesmallprimersusedtostartthereaction.Usingarbitrarilychosen10-baseprimers,onecangenerate"randomamplifiedpolymorphicDNA"(RAPD)Markers(Williamsetal.1991Nucl.AcidsRes.18:6531-6535).TheseDNAfragments,separatedbyelectrophoresisinanagarosegel,canbeusedasmarkersforstudyinggeneticvariationwithinandamongfungalpopulations.ArapidandsimpleprocedureforisolatingfungalDNAfrommultipleisolatesisneededifthistechniqueistobeusefulfordiagnosisandscreeningofnaturalpopulations.WehavedevelopedamethodforisolatingDNAwithoutgrindingfromFusariumculturesgrownonagarslantsusingamodificationoftheSDSminiprepmethodofLeeetal.(1988FungalGeneticsNewsletter35:23-24).

1.Placeacube(0.5cc)ofmycelia-coveredagarfroma5day-oldslantina1.5mlmicrocentrifugetube.2.Filltubewithliquidnitrogenandletitevaporate.Repeat.Nogrindingisnecessary.3.Immediatelyadd500ulof65Clysisbuffer(50mMTrispH8,50mMEDTA,3SDS,1BME,and0.1mg/mlProteinaseK).Vortextubeandplacein65Cwaterbathfor1hr.Vortexafter30minand60min.4.Extractwith500ulphenol.Spintube5minat8000rpminmicrocentrifugetoseparatephases.Remove450uloftheaqueousphase.5.Extractwith450ulbufferedphenol.Spin.Remove400uloftheaqueousphase.6.Extractwith400ulchloroform:isoamylalcohol::24:1.Spin.Remove350uloftheaqueousphase.7.Add50ul7.5Mammoniumacetate.Gentlymix.Add880ul95ethanol.Inverttomix.Placein-20Cfreezer30mintoovernight.8.SpindownDNApelletfor20minat13,000rpm.Rinsepelletin70ethanol.Drypelletandresuspendin20ulTE(10mMTris,1mMEDTA,pH8).

DNAwaspreparedfromisolatesgrownoncompleteandminimalslants(Correlletal.1987Phytopathology77:1640-1646).WeobtainedenoughDNAfromeachisolatefor20reactions.Weused1ulofDNAforPCRwithourprimer,designatedECORI(5""-ATGAATTCGC-3"").Toeachreactiontubeonicewasadded42ulsterile,glass-distilledanddeionizedwater;5ul10XbufferfromPromega(500mMKCl,100mMTris-HClpH9,15mMMgCl,0.1gelatinw/v,1TritonX-100),1ulof50uMprimer,1ulof1mMdNTP""sand1ulDNAsolution.Tubeswereboiledfor2.5min.Tubeswerereturnedtoice,then1unitofTaqpolymerase(Promega)wasadded.Tubeswerespunfor5secina4Cmicrofugeandthen100ulmineraloilwasadded.TubeswereplacedinaPTC-100programmablethermalcontrollerfromM.J.Researchwiththefollowingprogram:Step1.92C30sec.Step2.35C1min.Step3.Slope35to72at1degreeCevery8sec.Step4.72C2min.Step5.cycletoStep1.45times.Step6.72C7min.andStep7.end.20ulofeachreactionmixwasloadedintoa1.3agarosegelwithTBEbuffer.After2.0hoursat56V,thegelwasstainedwithethidiumbromideandphotographed.Theresultingpatterns.ofamplifiedDNAfragmentsarelistedinTable1.

Table1.RAPDmarkerpatternsforGibberellafujikuroiisolatesusingtheECORIprimer

Strain DNA fragments, kb +/- SE A2910 1.661 +/- .044, 0.597 +/- .015 A2911 1.698 +/- .098, 0.561 +/- .031A3957 1.678 +/- .032, 0.639 +/- .015X3974 1.661 +/- .044

Acknowledgements:Contribution91-404-J,KansasAgriculturalExperimentStation,KansasStateUniversity,Manhattan.SupportedbyagrantfromtheKansasStateBoardofAgriculture(CornCommissionandGrainSorghumCommission),bytheKansasAgriculturalExperimentStateHatchProject547,andbytheSorghum/MilletCollaborativeResearchSupportProgram(INTSORMIL)AID/DAN-1254-G-00-0021-00fromtheAgencyforInternationalDevelopment,Washington,DC.