An Alternative Strategy in Rapid DNA Extraction Protocol for High Throughput RAPD Analysis in Chickpea and Its Wild Related Species

摘要

ThefirststepofRAPDfingerprintingisthepreparationofthetargetDNAtemplate.TheextractionofDNAfromplantshasbeendescribedbynumerousauthors.EachauthordescribedadifferentmethodtoovercometheproblemsforisolationofgenomicDNA.SomespecieslikeCicerhavecertainmetaboliteslikepolysaccharidesandRNAwhichinterferewithDNAisolationandpolymerasechainreaction(PCR)amplification.Theobjectiveofthisprojectwastodevelophigh-throughputDNAextractionprocedurewithouttheneedforgreenhousespaceorgrowingCicerplants.SeedsweregerminatedofpaperfilterinPetridish,and4-dayoldseedlingtissuewasusedtoextractDNAbymodifiedhighsaltCTABprotocol.Approximately50-150ng/μofgenomicDNAwasisolatedfrom2gofleaftissue.DNAderivedusingthismethodwastestedelectrophoreticallyandthenwasexaminedwithaspectrophotometer:A260/280(Proteincontamination).PolymerasechainreactionamplificationwasperformedusingdifferentRAPDprimersandEcoRI,MseIandHindIIIwereusedforrestrictionenzymereaction.TheisolatedDNAprovedamenabletoPCRamplificationandrestrictiondigestion.Thismethoddoesnotrequireexpensivereagentsandmodernlaboratoryequipmentsandthistechniqueallowsonepersontoextractnearly200storage-stableDNAsamplesdaily,whilekeepingcostsataminimum.Thistechniqueisfast,reproducible,andcanbeappliedforRAPDanalysisandmostofgeneticassays.