Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases 英文参考文献.docVIP

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Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases 英文参考文献.doc

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Amplification of cox2 (~620 bp) from 2 mg of Up to 129 Years Old Herbarium Specimens, Comparing 19 Extraction Methods and 15 Polymerases 英文参考文献

Amplificationofcox2(,620bp)from2mgofUpto129 YearsOldHerbariumSpecimens,Comparing19 ExtractionMethodsand15Polymerases SabineTelle,MarcoThines* UniversityofHohenheim,InstituteofBotany210,Stuttgart,Germany Abstract During the past years an increasing number of studies have focussed on the useof herbarium specimens for molecular phylogeneticinvestigationsandseveralcomparativestudieshavebeenpublished.However,inthestudiesreportedsofar usuallyratherlargeamountsofmaterial(typicallyaround100mg)weresampledforDNAextraction.Thisequalsanamount roughly equivalent to 8cm2 of a medium thick leaf. For investigating the phylogeny of plant pathogens, such large amountsoftissueareusuallynotavailableorwouldirretrievablydamagethespecimens.Throughsystematiccomparisonof 19 DNA extraction protocols applied to only 2mg of infected leaf tissue and testing 15 different DNA polymerases, we could successfully amplify a mitochondrial DNA region (cox2; ,620bp) from herbarium specimens well over a hundred years old. We conclude that DNA extraction and the choice of DNA polymerase are crucial factors for successful PCR amplification from small samples of historic herbarium specimens. Through a combination of suitable DNA extraction protocolsandDNApolymerases,onlyafractionofthepreservedplantmaterialcommonlyusedisnecessaryforsuccessful PCR amplification. This facilitates the potential use of a far larger number of preserved specimens for molecular phylogeneticinvestigationandprovidesaccesstoawealthofgeneticinformationinpreservedinspecimensdepositedin herbariaaroundtheworldwithoutreducingtheirscientificorhistoricalvalue. Citation:TelleS,ThinesM(2008)Amplificationofcox2(,620bp)from2mgofUpto129YearsOldHerbariumSpecimens,Comparing19ExtractionMethods and15Polymerases.PLoSONE3(10):e3584.doi:10.1371/journal.pone.0003584 Editor:RobertDeSalle,AmericanMuseumofNaturalHistory,UnitedStatesofAmerica ReceivedJuly31,2008;AcceptedOctober7,2008;PublishedOctober31,2008 Copyright: ? 2008 Telle, Thines. This is an open-