一种用于PCR的丝状真菌DNA快速粗提方法-豆柴文库

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一种用于PCR的丝状真菌DNA快速粗提方法
Title:ARapidCoarseExtractionMethodforFilamentousFungalDNAforPCR
Abstract:
PolymeraseChainReaction(PCR)isawidelyusedtechniqueinmolecularbiologytoamplifyspecificregionsofDNA.However,obtaininghigh-qualityandpureDNAfromfilamentousfungicanbechallengingduetotheircomplexcellwallstructureandDNAassociationwithproteinsandpolysaccharides.Thispaperpresentsarapidandcost-effectivemethodforcoarseextractionoffilamentousfungalDNA,specificallydesignedforPCRapplications.Themethoddescribedinvolvesaninitialcellwalldisruptionusingacombinationofmechanicalandenzymatictreatments,followedbyDNAisolationandpurification.TheextractedDNAisofsufficientqualityandpurityfordirectuseindownstreamPCRreactions,enablingefficientdetectionandanalysisoffungalgeneticmaterial.
1.Introduction:
PCRhasrevolutionizedmolecularbiologyresearchwithitsabilitytoamplifyspecificDNAregionsfromsmallamountsofstartingmaterial.However,obtaininghigh-qualityDNAfromfilamentousfungicanbechallengingduetotheircomplexcellwallstructureandthepresenceofvariouscontaminants.CoarseextractionmethodsareoftenemployedforpreliminaryDNAisolation,buttheymaynotprovideDNAofsufficientpurityandyieldfordownstreamPCRapplications.ThisstudyaimedtodeveloparapidandefficientmethodforcoarseextractionoffilamentousfungalDNAoptimizedforPCR.
2.MaterialsandMethods:
2.1FungalStrainSelection:
FilamentousfungistrainscommonlyusedinresearchwereselectedtoevaluatetheefficiencyoftheproposedDNAextractionmethod.Thestrainsweregrownonsuitableagarmedia.
2.2SamplePreparation:
Fungalmyceliawereharvestedfromagarplatesandtransferredtoasterilemicrocentrifugetube.Anappropriateamountofextractionbufferwasaddedtothetube,andthesamplewassubjectedtomechanicaldisruptionusingbeadbeating.
2.3CellWallDisruption:
Thefungalcellwallwasfurtherdisruptedthroughenzymatictreatmentwithlyticenzymessuchaslysozymeandchitinase.Theenzymecocktailwasaddedtothesampleandincubatedatanoptimaltemperatureandduration.
2.4DNAIsolationandPurification:
Followingcellwalldisruption,thefungalDNAwas