JournalofVisualizedExperimentswww.jove.comCopyright©2010JournalofVisualizedExperimentsandAgilentTechnologies,Inc.April2010|38|e1871|Page1of7VideoArticleDNA-basedFishSpeciesIdentificationProtocolRachelFormosa1,HariniRavi1,ScottHappe1,DanielleHuffman1,NataliaNovoradovskaya1,RobertKincaid1,SteveGarrett11AgilentTechnologiesCorrespondenceto:RachelFormosaatrachel.formosa@agilent.comURL:http://www.jove.com/video/1871DOI:doi:10.3791/1871Keywords:CellularBiology,Issue38,seafood,fish,mislabeling,authenticity,PCR,Bioanalyzer,food,RFLP,identityDatePublished:4/28/2010Citation:Formosa,R.,Ravi,H.,Happe,S.,Huffman,D.,Novoradovskaya,N.,Kincaid,R.,Garrett,S.DNA-basedFishSpeciesIdentificationProtocol.J.Vis.Exp.(38),e1871,doi:10.3791/1871(2010).AbstractWehavedevelopedafast,simple,andaccurateDNA-basedscreeningmethodtoidentifythefishspeciespresentinfreshandprocessedseafoodsamples.ThisversatilemethodemploysPCRamplificationofgenomicDNAextractedfromfishsamples,followedbyrestrictionfragmentlengthpolymorphism(RFLP)analysistogeneratefragmentpatternsthatcanberesolvedontheAgilent2100BioanalyzerandmatchedtothecorrectspeciesusingRFLPpatternmatchingsoftware.Thefishidentificationmethodusesasimple,reliable,spincolumn-basedprotocoltoisolateDNAfromfishsamples.ThesamplesaretreatedwithproteinaseKtoreleasethenucleicacidsintosolution.DNAisthenisolatedbysuspendingthesampleinbindingbufferandloadingontoamicro-spincupcontainingasilica-basedfibermatrix.Thenucleicacidsinthesamplebindtothefibermatrix.Theimmobilizednucleicacidsarewashedtoremovecontaminants,andtotalDNAisrecoveredinafinalvolumeof100μl.TheisolatedDNAisreadyforPCRamplificationwiththeprovidedprimersthatbindtosequencesfoundinallfishgenomes.ThePCRproductsarethendigestedwiththreedifferentrestrictionenzymesandresolvedontheAgilent2100Bioanalyzer.ThefragmentlengthsproducedinthedigestionreactionscanbeusedtodeterminethespeciesoffishfromwhichtheDNAsamplewasprepared,usingtheRFLPpatternmatchingsoftwarecontainingadatabaseofexperimentally-derivedRFLPpatternsfr...
