QuickandreliablemethodtoanalyzemeioticsegregationpatternsinCoprinuscinereususingthepolymerasechainreactionT.FreedmanandP.J.Pukkila-DepartmentofBIOLOGy,UniversityofNorthCarolina,ChapelHill,NC27599-3280.ItiswellknownthatmultipleauxotrophicMarkersimpedefruitinginCoprinuscinereus.Restrictionfragmentlengthpolymorphismshavebeenusedtoadvantage(Cassidyetal.1984Curr.Genet.8:607-613;Zolanetal.1992Nucl.AcidsRes.20:3993-3999)buttherequiredSouthernanalysisisbothtimeconsumingandlaborious.Herewedemonstrateaquickandreliablemethodusingthepolymerasechainreaction(PCR)(Saikietal.1988Science239:487-491)toscorethepresenceorabsenceofauniqueinsertionamongmeioticsegregants.Todeveloptheoptimumprocedure,wedissectedtetradsfromacrossbetweenastrainthatharboredauniqueinsertionatthetrp1locusandastrainwithouttheinsertion.WelookedforPCRconditionsthatgaveneitherfalsepositivesnorfalsenegatives.

AlthoughitispossIBLetoperformPCRreactionsonfreshmyceliumfromC.cinereuswithoutfirstextractingtheDNA,thismethodissometimesunreliable.FurThermore,onehastorelyonwild-typefluffystrainsforanadequatesupplyofmycelium.Ifthemyceliumisfirstlyophilized,orlyophilizedandthenboiled,falsenegativesarestillobtained.Accordingly,wesimplifiedthestandardmini-prepprocedure(ZolanandPukkila1986Mol.Cell.Biol.6:195-200)andthemethodthatgaveconsistentresultsisreportedhere.Withthismethod,14tetradswereanalyzed,with100%accuracyasjudgedby2:2segregationofthePCR-amplifiedproduct.

SterileEppendorftubescontaining1mlYMDT(0.4%yeastextract,1%maltextract,0.4%dextroseand0.01%L-tryptophan)mediumwereinoculatedwithasmallplugofmyceliumandincubatedat37oC.After5days,theEppendorftubeswerevortexedandspunfor20mininamicrocentrifugeattopspeed.Thesupernatantwasremoved,andthetubeswerefrozeninadryice/ethanolbathfor10minbeforebeingplacedinaspeedvacforatleast5hforlyophilization.Typically,5mgoftissuewasrecovered,andwaseasilypulverizedusingasteelprobe.Threehundredmicrolitersofextractionbufferwasaddedtothedrytissueandthoroughlymixedusingasteriletoothpick.Theextractionbuffer(finalconcentration)was:700mMNaCl,1%CTAB(hexadecyltrimethylammoniumbromide(Sigma)),50mMTris-HCl(pH8),10mMEDTAand1%2-mercaptoethanol(MurrayandThompson1980Nucl.AcidsRes.8:4321-4325).After10minatroomtemperature,300microlitersofSEVAG(1partisoamylalcohol:24partschloroform)wasaddedandmixedbygentlevortexing.After10minatroomtemperature,thetubeswerespunattopspeedfor20min.Thesupernatantwascollectedandstoredat-20oC.Typically,about4microgramsofDNAcanberecoveredbythisprocedureataconcentrationofabout20ng/l,butonly1microliterofthesupernatantisnecessaryforPCR-amplification.

TheDNAsample(1microliterofsupernatant)in10microlitersofwaterwasincubatedinthePCRmachineat94oCinthefirststepofthePCRcycletoavoidproteolyticdegradationofTaqpolymerase.ThefollowingcomponentswereaddedtotheindicatedconcentrationsafterthefirstdenaturationstepforPCR-amplification(finalvolume100microliters):67mMTris-HCl(pH8.8),16mM(NH4)2SO4,10mM2-mercaptoethanol,3mMMgCl2,2%glycerol,1micromoleofeachprimer(seebelow),dATP,dCTP,dGTP(each200uM),dUTP(500uM),and2.5unitsofAmplitaqDNAPolymerase(PerkinElmerCetus).ThePCRbufferisamodificationofthatusedbyJeffreysetal.(1988Nucl.AcidsRes16:10953-10971).ThereactionwasincubatedinaPerkinElmerCetusDNAThermalCycler480machineat94oC,1min,58oC,2minand72oC,3minfor30cycles.Theprimerswereasfollows:HP(5"AGCTCCATGGAATGTGCAGAC3")recognizedthepBluescript(Stratagene)plasmidintegratedinthetrp1locus(Skrzyniaetal.1989Gene81:73-82).Theotherprimer,A934(5"GGACGTACTTGGCA3"),recognizedaninternalregionofthetrp1gene.Thisreactionamplifieda1.6kbfragmentonlyfromDNAofsegregantscontainingtheplasmidinsertion.TwentymicrolitersofthePCRreactionwasrunon0.7%agarosegelsandbandswerevisualizedbyethidiumbromidestaining.

ThisprotocolisfarmoreefficientthanSouthernanalyses.ItshouldallowthesegregationpatternsofRAPDmarkerstobemonitored,ifsuitablemodificationsofMgCl2concentrationandtheannealingtemperatureareused.WeroutinelyusedUTPtopermitthedestructionofcontaminatingamplifiedDNAs.Ifcontaminationisdetectedinanegativecontrol,uracilN-glycosylasecanbeusedsincethisenzymecleavessequenceswhichcontaindUTP.

Theadvantagesofourmethodinclude:1.Thetissueisgrownandlyophilizedinthesametube,whichfacilitatesthesimultaneousanalysisofmanysegregants.2.Onlyoneextractionstepisnecessary.3.Themarkersarescoredbygelelectrophoresis,therebybypassingSouthernanalysis.

SupportedbyNSFgrantDMB-9004176