Genetically Modified Herbicide-Tolerant Crops and Sugar Beet—Environmental and Health Concerns - Sweeteners: Nutritional Aspects, Applications, and Production Technology - page 378

site-speciic natural isotope fractionation-nuclear magnetic resonance (SNIF-NMR) and more
updatedtechnologymicrosatellitemarkers,restrictionfragmentlengthpolymorphism(RFLP),and
single-strandconformationpolymorphism(Varzakasetal.2008).
Currently,thetwomostimportantapproachesareimmunologicalassaysusingantibodiesthat
bindtothenovelproteinsandPCR-basedmethodsusingprimersthatrecognizeDNAsequences
uniquetotheGMcrop.Thetwomostcommonimmunologicalassaysareenzyme-linkedimmu-
nosorbentassay(ELISA)andimmunochromatographicassay(lateral-lowstriptests).ELISAcan
producequalitative,semiquantitative,andquantitativeresultsin1-4hoflaboratorytime.
NestedPCRconirmsthePCRproduct,allowingdiscriminationbetweenspeciicandnonspe-
ciicampliicationsignals.Hence,thePCRproductisreampliiedusinganotherprimerpair,located
intheinnerregionoftheoriginaltargetsequence(Anklametal.2002).ItincreasesPCRsensitivity,
allowinglowlevelsofGMOtobedetected(Zimmermannetal.1998b).Inordertodetectthepres-
enceofRRsoybean,anestedPCRmethodwasappliedtocommerciallyavailablesoylour,infant
formulacontainingsoyproteinisolate,andsoymilkpowdersamples.Greineretal.(2005)analyzed
soylour,infantfoods,andsoyproteinisolates.
Electrochemiluminescence (ECL), where light-emitting species are produced by reactions
betweenelectrogeneratedintermediates,hasbecomeanimportantandpowerfulanalyticaltoolin
recent years. An ECL reaction using tripropylamine (TPA) and tris (2,2ʹ-bipyridyl)ruthenium(II)
(TBR)hasbeendemonstratedtobeahighlysensitivedetectionmethodforquantifyingampliied
DNA.TPAandTBRareoxidizedatapproximatelythesamevoltageontheanodesurface.After
deprotonation,TPAchemicallyreactswithTBRandresultsinanelectrontransfer.Theresulting
TBRmoleculerelaxestoitsgroundstatebyemittingaphoton.TPAdecomposestodipropylamine,
isthereforeconsumedinthisreactionand,ontheotherhand,isrecycled.Becausebothreactants
areproducedattheanode,luminescenceoccursthere.ECLhastheadvantagesthatnoradioisotopes
areused,detectionlimitsareextremelylow,theextensionofthedynamicrangeforquantiicationis
oversixordersofmagnitude,labelsareextremelystablecomparedtothoseofmostotherchemilu-
minescencesystems,andsimpleandrapidmeasurementrequiresonlyafewsecondscomparedwith
otherdetectiontechniques—theECLmethodisachemiluminescentreactionofspeciesgenerated
electrochemically on an electrode surface. It is a highly eficient and accurate detection method.
Liuetal.(2005)appliedECLPCRcombinedwithtwotypesofnucleicacidprobeshybridization
todetectGMOs.WhethertheorganismscontainGMcomponentswasdiscriminatedbydetecting
theCaMV35Spromoterandnopalinesynthaseterminator.Theexperimentalresultsshowedthat
thedetectionlimitis100fmolofPCRproducts.Thepromoterandtheterminatorcanbeclearly
detectedinGMOs.ThemethodmayprovideanewmeansforthedetectionofGMOsduetoitssim-
plicityandhigheficiency.Theinstrumentusedwascomposedofanelectrochemicalreactioncell,
apotentiostat,anultrahighsensitivitysinglephotoncountingmodule,amultifunctionacquisition
card,acomputer,andtheLabVIEWsoftware.Theelectrochemicalreactioncellcontainsaworking
electrode(platinum),acounterelectrode(platinum),andareferenceelectrode(Ag/AgCl 2 ).
Therearealsoin-processmethodsaimedatcomparingtheprimalandGMplantsdividedinto
targeted and nontargeted approaches. Targeted approaches monitor directly the consequence of
novelgeneproductpresenceontheGMplantphenotype.Moreover,changesinchemicalcomposi-
tion are being detected. Nontargeted approaches consist of three basic levels: functional genom-
ics,proteomics,andmetabolomics.FunctionalgenomicscontainmethodssuchasmessengerRNA
(mRNA)ingerprintingandDNAmicroarray.Consideringproteomics,theproteincompositionof
originalandGMplantsisbeingcomparedusingmethodssuchastwo-dimensionalelectrophoresis
(2-DELPO;Gorgetal.1999)anditsmodiicationor2-DELPOinconnectionwithmatrix-assisted
laserdesorption-ionizationtime-of-lightmassspectrometryanalysis(AndersenandMann2000).
Difference gel electrophoresis is being employed for the testing of two samples on the same gel
(Unlu 1999). The metabolomics level of analysis identiies and quantiies the maximum amount
of particular components. This involves separating methods like gas chromatography, liquid