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ID:38345414
大小:140.82 KB
页数:5页
时间:2019-06-10
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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
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