Multiplex amplification of ancient DNA

SAMPLINGANDANALYSISOFCOMMERCIALFATSANDOILS AOCSOfficialMethodCe1f-96Reapproved1997•Revised2001 Determinationofcis-andtrans-FattyAcidsinHydrogenatedandRefinedOilsandFatsbyCapillaryGLCDEFINITIONThismethodconsistsofthegas–liquidchromatography(GLC)conditionsoptimizedtoidentifyandquantifythetransfattyacidisomersinvegetableoilsandfats(References,1).Thefattyacidmethylesters(FAME)ofthesampleareseparatedonacapillarygaschromatographycolumnhavingahigh-lypolarstationaryphase,accordingtotheirchainlength(CL),degreeof(un)saturation,andgeome-tryandpositionofthedoublebonds[DB(s)].SCOPEThismethodisspeciallydesignedtoevaluate,byasinglecapillaryGLCprocedure,theleveloftransisomersasformedduring(high-temperature)refiningorduringhydrogenationofvegetableoilsorfats(seeNotes,1and2).Themethodmayalsobeusedtoreportallotherfattyacids,forexampletoobtainsaturatedfattyacid(SAFA),monounsaturatedfattyacid(MUFA),andpolyunsaturatedfattyacid(PUFA)levelsfromthesamesampleandsameanalysis.APPARATUS1.Gaschromatograph—equippedwithacapillaryinjectionsystem(preferablysplitmode,operatedatasplitratioofapproximately1:100)andflameionizationdetector(FID),capableofmeetingthefollowingrequirements:injectionporttemperature,250°C;detectortemperature,250°C;oventemperatureconditionsasgiveninTable1.Typicalresultswiththesedescribedconditionsareshowninexamplechromatograms(Figures1–5).2.Column—highlypolarstationaryphase,suchasoneofthefollowing:(a)CP™-Sil88,100or50m×0.25mmi.d.,0.20µmfilm(Chrompack,Middelburg,TheNetherlands).(b)SP-2650,100m×.025mmi.d.,0.20µmfilm(SupelcoInc.,Bellefonte,PA,USA).(c)SP-2340,60m×0.25mmi.d.,0.2µmfilm(SupelcoInc.).(d)BPX-70,120mor50m×0.22mmi.d.,0.25µmfilm(SGEInc.,Austin,TX,USA).3.Recordinginstrument.4.Electronicintegratororchromatographysoftware.REAGENTSUnlessotherwisestated,useonlyreagentsasspecifiedinISO6353(parts2and3)(References,2)iflistedthere;ifnot,thenusereagentsofrecognizedanalyticalgradeandwaterofatleastgrade3asdefinedinISO3696(References,3).1.Carriergas—helium,nitrogen,orhydrogen,GCquali-ty,dried,andoxygenremovedbysuitablefilters.2.Internalstandard(forcalculatingfattyaciddataasmgpergoil)—tridecanoin,5.0mg/mLinchloroform.Thissolutionisstableupto1weekifstoredinrefrigeratorinwellsealedamberbottle.(SeeNotes,3).PROCEDURE1.Samplepreparation—(a)Preparethemethylestersfromthetriglyceridesfromtheoilsorfatstobeanalyzed,usingtheborontrifluoridemethodasdescribed,forexam-ple,inAOCSOfficialMethodCe2-66orIUPAC2.301(References,5and6).(b)Beforetestportionsaretakenfromsamples,thesamplesshouldbemixedthoroughly.Solidsamplesshouldbemeltedtoensurepropermixing.2.Chromatography—(a)SetupthegaschromatographwiththetemperatureandcolumnasdescribedinTable1.MeasuretheaveragecarriergaslinearvelocityasindicatedinTable1,withasplitratioofapproximately1:100.(b)Inject0.5to1µLofthemethylesters(concentra-tionapproximately7mg/mL)fromthetestsampleintothegaschromatograph.Comparethe resultwiththeexamplechromatograms(Figures1–5).IftheseparationobtainedisTable1ProposedoptimalGLCconditionsforidentificationandquantificationoftransisomersinrefinedandhydrogenatedveg-etableoilsamples(seeReferences,3).StationaryphaseSP-2340SP-2560CP™-Sil88BPX-70TemperatureconditionsIsotherm192°CIsotherm170°CIsotherm175°CIsotherm198°CColumnheadpressure(kPa)125125130155Linearvelocityofcarriergas(He)15cm/sec16cm/sec19cm/sec17cm/sec