real-time pcr copy number .pdf

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1、PlantCellRep(2004)23:263–271DOI10.1007/s00299-004-0859-yREVIEWBenBubner·IanT.BaldwinUseofreal-timePCRfordeterminingcopynumberandzygosityintransgenicplantsReceived:16March2004/Revised:25July2004/Accepted:26July2004/Publishedonline:11September2004Springer-Verlag2004AbstractThisreviewex

2、amineshowreal-timePCRcanplantscontainthetransgeneandinhowmanycopies.Forbeusedtodeterminecopynumberandzygosityintrans-thepurposeofthisreviewthetermcopynumberreferstogenicplants.Distinguishingbetweenplantsthatharborthenumberofinsertsperhaploidgenome,ahemizygousoneandtwocopiesofatransgen

3、eorarehemizygousandtwo-copyplantcontainstwogiventransgenicsequences,homozygousrequirestheabilitytoroutinelydistinguishahomozygoustwo-copyplantfour.Whilemultiplecopiestwofolddifferences,adetectiondifferencewhichap-ofthetransgeneareusefulforover-expressionexperi-proachestheresolutionofP

4、CR-basedquantificationments,single-copytransformationeventsarepreferredformethods.Afterexplainingthebasicprinciples,especiallymostapplicationsbecausetheyarestableoverseveralthethresholdcycle(Ctvalue)asthebasicmeasuringunitgenerationsofsubsequentbreeding(Meyer1998).Com-inreal-timePCR,w

5、eintroducethreequantitationmeth-plicatedinsertionstructures,suchasinvertedrepeats,odscurrentlyinuse.Whiletheabsoluteandrelativeconcatamers,andtruncatedexpressioncassettes,maystandardcurveapproachesarequalitativemethodsthatoccurinalltransformationprocedures(Smithetal.2001),distinguishh

6、igh-copyfromlow-copytransformants,theandadeterminationofcopynumbercandistinguishcomparative(2
DDCt)methodwithdouble-dyeoligonu-simpletransformationeventsfromcomplexones.Thecleotides(TaqManprobes)isabletodetecttwofolddif-mostcommonlyusedmethodofdeterminingcopynum-ferences.Inordertoobta

7、inreliableresults,CtvaluesforbersisSouthernanalysis,inwhichablotofdigestedanampliconshouldbebelow25andthestandarddevi-genomicplantDNAishybridizedwitharadioactiveDNAationbelow0.3.Althoughreal-timePCRcandeliverprobecorrespondingtothetransgenetoproduceanin-exactcopynumberdeterminations,t

8、hepro