Southern、Northern印记

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1、SouthernandNorthernBlottingDNAandRNAblotting,nucleicacidblotting.Figure1Thesouthernandnorthernblottingtechniques.EnzymaticallydigestedDNAordenaturedRNAisfractionatedbygelelectrophoresis.AftertransferandfixationoftheDNAfragmentsorRNAtoamembranesupport,themembraneisincubatedwithalabeledDNAo

2、rRNAprobethatisspecificforthetargetofinterest.Becausetheprobehybridizestoonlythetargetfragment/molecule,aspecificbandisvisualizedfollowingdetection.Figure2CapillarytransferofDNAorRNAfromagarosegels.Bufferisdrawnfromareservoirviaapaperwickandpassesthroughthegelintoablottingmaterial(e.g.pap

3、ertowels,blottingpaper).TheDNAorRNAiselutedfromthegelbytheflowofbufferandisdepositedontoamembrane.Aweightappliedtothetopoftheblottingapparatusensuresatightjunctionbetweenthelayersofmaterialusedinthetransfersystem.ShortDescriptionNucleicaciddetectioncanbeperformedusingatechniqueknownasblot

4、ting,whichisthetransferofDNA(deoxyribonucleicacid)orRNA(ribonucleicacid)ontoamembranesupport.WhenthisprocedureisusedtoexamineDNAorRNAitisreferredtoasSouthernornorthernblotting,respectively.CharacteristicsSouthernblottingisthetransferofDNAfromanagarosegeltoamembranesupport(1).Sinceitsincep

5、tion,Southernblottinghasbeenusedtodeterminegenecopynumber,detectrestrictionfragmentlengthpolymorphisms,determinemodificationstoDNA(e.g.methylation),anddetectthepresenceofgeneticallyinsertedtransgenes.ItalsoisusedtostudyRNAsplicingandantibodyandTcellreceptorformation,andhasaidedintheidenti

6、ficationofgenerearrangementsassociatedwithavarietyofhumangeneticdisordersandcancers.NorthernblottingisthetransferofRNAfromanagarosegeltoamembranesimilartothatusedinSouthernblotting(2).Northernblottingisusedtoevaluategeneexpressioninamannerthatisbothqualitative(inwhichtissuesorcells,orunde

7、rwhichphysiologicalconditionsageneisexpressed)andquantitative(levelofgeneexpression).ThismethodalsoisusedtodetecttheexpressionofforeigngenesintransgenicorganismsandisausefuladjuncttocomplementaryDNA(cDNA)cloningbecausemRNAmolecularweightscanbecomparedwiththoseofclon