2.3 基因型表现型分析

RandomlyAmplifiedPolymorphicDNA(RAPD)

• RandomlyAmplifiedPolymorphicDNA(RAPD)by(DNAKAFFE)RAPDanalysishasbeensuccessfullyusedinmappingandfingerprintingapplications.RAPDcanefficientlyidentifydifferencesthatconstituteasignificantfractionofagenome.Thisisaveryniceprotocoldescribingingreatdetailthebackgroundknowledge,methodandtroubleshooting.
• RAPDReactionandCyclingConditions(KimMarshall)
• ARapidCTABDNAIsolationTechniqueUsefulforRAPDFingerprintingandOtherPCRApplications(KimMarshall)
• RandomAmplifiedPolymorphicDNA(RAPD)Protocol(GrahamCasey)

AmplifyedFragmentLengthPolymophism(AFLP)

• AmplifyedFragmentLengthPolymophism(AFLP)by(DNAKAFFE)AFLPoriginallyknownasselectiverestrictionfragmentamplification(SRFA)produceshighlycomplexDNAprofilesbyarbitraryamplificationofrestrictionfragmentsligatedtodouble-strandedadaptorswithhemi-specificprimersharboringadaptor-complementary5"termini.TheAFLPprotocolcanbedevidedinthefollowingsteps:(1)DNAdigestionwithtwodifferentrestrictionenzymes,(2)ligationofdouble-strandedadapterstotheendsoftherestrictionfragments,(3)optionalDNApre-amplificationofligatedproduct,and(4)DNAamplificationofsubsetsofrestrictionfragmentsusingselectiveAFLPprimersandlabellingofamplifiedproducts.
• AmplifiedFragmentLengthPolymorphisms(AFLPs)(OregonStateU)
• AmplifiedFragmentLengthPolymorphism(AFLP)protocolforWheat(GrahamCasey)

Single-StrandConformationPolymorphism(SSCP)

AmethodfordistinguishingbetweensimilarsizedDNAfragmentsaccordingtothemobilityofthesingle-strandedDNAunderpolyacrylamidegelelectrophoresis.(BirgidSchlindwein"sHypermediaGlossaryOfGeneticTerms)

• SSCP(FMC)providesgeneralguideforPCRandgelresolution.
• SSCP(PEBiosystems)ThisprotocolprovidesdetailedinstructionsforperformingSSCPmutationscreening.

HeteroduplexAnalysis

ThestudyofthemobilitiesofheteroduplexDNAunderpolyacrilamidegelelectrophoresis.ThereducedmobilityofheteroduplexDNAcomparedwithhomoduplexDNAisproportionaltothedegreeofdivergenceofthesequences.(BirgidSchlindwein"sHypermediaGlossaryOfGeneticTerms)

• HeteroduplexAnalysis(FMC)GuideforpreparingPCRforheteroduplexanalysis

MicrosatelliteAnalysis

• MicrosatelliteAnalysis(PEBiosystems)MicrosatellitelociarePCRamplifiedandthePCRproductsarethenanalyzedbyelectrophoresistoseparatetheallelesaccordingtosize.PCR-amplifiedmicrosatelliteallelescanbedetectedusingusr/localiousmethods,suchasfluorescentdyelabeling,silverstaining,orfluorescentdyestaining.

Others

• DNASeparationby(DNAKAFFE)ThescanningofnucleicacidsbyamplificationproducesanarrayofDNAproductsthatcanberesolvedusingausr/localietyofmethods.Asoriginallydescribed,DAFandAP-PCRusePAGE,whileRAPDanalysisseparatesamplificationproductsbyelectrophoresisinagarosegels.Thesetechniquesdifferalsointhewayhownucleicacidfragmentsaredetected;DAFusessilverstaining,AP-PCRusesautorADIography,andRAPDusesstainingwithethidiumbromide.ThisProtocolfocusesonseveralpolyacrylamide-basedseparationtechniquesthatcanbeusedtoseparateDAFordifferentiallydisplayedRNAprofiles.ItalsodescribesthestainingofnucleicacidfragmentswithsilverandrecoveryofDNA.
• DenaturingGradientGelElectrophoresis(DGGE)(Donis-Kellerlab)Denaturinggradientgelsareusedtodetectnon-RFLPpolymorphisms.Thesmall(200-700bp)genomicrestrictionfragmentsarerunonalowtohighdenaturantgradientacrylamidegel;initiallythefragmentsmoveaccordingtomolecularweight,butastheyprogressintohigherdenaturingconditions,each(dependingonitssequencecomposition)reachesapointwheretheDNAbeginstomelt.Thepartialmeltingseverelyretardstheprogressofthemoleculeinthegel,andamobilityshiftisobserved.Itisthemobilityshiftwhichcandifferforslightlydifferentsequences(dependingonthesequence,aslittleasasinglebpchangecancauseamobilityshift).Allelesaredetectedbydifferencesinmobility.
• 3%TrevigelProtocolfor3bpresolutionofSSRPCRproducts(KimMarshall)
• SSRGelandSilverStainingProtocol(KimMarshall)