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1、ANALYTICALBIOCHEMISTRYAnalyticalBiochemistry342(2005)93–102www.elsevier.com/locate/yabioEvaluationofthinWlmsofagaroseonglassforhybridizationofDNAtoidentifyplantpathogenswithmicroarraytechnologya,ca,b,¤cC.A.Koch,P.C.H.Li,R.S.UtkhedeaDepartmentofMolecularBiologyandBiochemistry,SimonFras
2、erUniversity,Burnaby,BC,CanadaV5A1S6bDepartmentofChemistry,SimonFraserUniversity,Burnaby,BC,CanadaV5A1S6cAgricultureandAgri-FoodCanada,P.O.Box1000,Agassiz,BC,CanadaV0M1A0Received15February2005Availableonline30April2005AbstractAgarose-coatedglassslides,afteractivation,werespottedwitham
3、ine-modiWedoligonucleotideprobesusingamanualeight-pinarrayingdevice.Twoprobes,designedtoidentifytwocommongreenhousefungalplantpathogens,DidymellabryoniaeandBotrytiscinerea,werehybridizedwithpolymerasechainreaction(PCR)-ampliWedXuorescentlylabeledDNAextractedfrompurecultureandfromdisea
4、sedplanttissue.Theprobeseasilydistinguishedthesepathogensfromeachotherwithoutcrossreaction.ThicknessoftheagaroselayerandlengthofthesampleDNAwereimportantfactorsaVectinghybridizationeYciencyofimmobilizedprobetoPCRproduct.ThesefactorsdidnotaVecthybridizationwithshortcomplementaryoligonu
5、cleotide.ProbesWxedonagarose-coatedslidescoulddiVerentiatesamplesasreadilyasprobesonnylonbutwithpotentiallyhigherspotdensityandgavemuchbettersignalthanprobesonsilylatedslides.Theuseofplainglassslides,agarose,andamanualarrayermakesthistechniqueusefulfordevelop-ingspecializedandinexpens
6、iveDNAmicroarraysonasolidrigidsubstrate.2005ElsevierInc.Allrightsreserved.Keywords:Microarray;AgarosesubstrateArraysofDNAoligonucleotidesimmobilizedonretainingamonolayerofimmobilizedoligonucleotidesolidsubstratescanbeusedtoidentifyunknownsam-probe(2Dimmobilization),higherprobeconcent
7、rationsplesbysimultaneouslytestingformanyorganismsandreducedtheeYciencyandkineticsofhybridizationpre-havebeenusedtosuccessfullyidentifyhuman,otherani-sumablyduetocrowding[7].Thishindrancewasmal,andplantpathogens[1–4].Commonsolidreducedinthegelpadsbecauseofthegreaterreactionsubstratesa
8、regla
