遗传学新版课件 9

CHAPTER9TheDirectDetectionOfGenotypePolymorphism--aDNAlocusthathastwoormoresequencevariations,oralleles,eachpresentatafrequencyof1%ormoreinapopulation.MicrosatelliteDNA---DNAelementcomposedof50-500tandemrepeatsof2-6basesequences.STR(shorttandemrepeat)---inthechromosomesofseveralorganismsareareascontainingshortrepeatedsequenceofDNA.ThereareascanbeeasilyamplifiedbyPCRduetotheirpolymorphism.MinisatelliteDNA---DNAelementcomposedoflonger(15-50bp)tandemrepeatingunitsofidenticalsequencelineduplikeboxcarsinafreighttrain.VNTR(variablenumbertandemrepeat)---AshortDNAsequencethatispresentastandemrepeatsandinhighlyvariablecopynumber.

ConceptsConcepts(Continued)SNP(singlenucleotidepolymorphism)---Thegeneticpolymorphismduetobasesubstitutionsiscalledsinglenucleotidepolymorphism.SSCP---single-strandconformationpolymorphismAFLP(amplifiedfragmentlengthpolymorphism)---genomicanalysisbyPCRofrestrictiondigestsofgenomicDNA.RFLP(restrictionfragmentlengthpolymorphism)---individualDNAnucleotidesequencedifferencesthatleadtovariationinlengthsoffragmentswhenDNAisdigestedwithspecificrestrictionendonucleases.RAPD(randomamplifiedpolymorphicDNA)---basedmethodofDNAprofilingthatinvolvedamplificationofsequencesusingrandomprimers.Generatesatypeofgeneticfingerprintthatcanbeusedtoidentifyindividuals.

1.FiveclassesofDNApolymorphism

SNP

Thegeneticpolymorphismduetobasesubstitutionsiscalledsinglenucleotidepolymorphism(SNP).A:ATTCGCB:ATTAGC

a.SNP

SNPClassificationEcoRIrestrictionfragmentdetectedbyprobe

Restrictionfragmentlengthpolymorphism

IndividualDNAnucleotidesequencedifferencesthatleadtovariationinlengthsoffragmentswhenDNAisdigestedwithspecificrestrictionendonucleases.

RFLPUsingRFLPsasmarkerstofollowthe

inheritanceoflinkedallelesatdiseaseloci

UsinggelelectrophoresisofPCR

productstodetectmutationsinrestrictionsites

Shorthybridizationprobescandistinguishsingle-basemismatches,longerprobescannot

UsingPCRwithASOtodeterminegenotypeattheβ-globinlocus

Allele-specificoligonucleotideprobesSingle-strandconformationpolymorphisms(SSCP)enabledetectionofSNPalleles

SSCPTherandomamplificationofpolymorphicDNA(RAPD)enabledetectionofSNPalleles

RAPDUsingmassspectrometrytoanalysisofSNPalleles

MS

b.Microsatellites,orshorttandemrepeats,STRs

SatelliteDNAusedinforensicgeneticsDNAelementcomposedof50-500tandemrepeatsof2-6basesequences.

ThegenerationofSTRsFormicrosatellites:PCR-basedprotocolsdetectpolymorphisms

PCRprotocolsforSTRanalysisCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemSexTypingSexTypingSexTypingSexTypingMaleorFemale？

STRloci9CODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCarboxy-X-rhodamine(CXR)---Blue---Green---Yellow---RedCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystem--------------------------------------------------------------------------------------------------CODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemAnormalSTRmapCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemCODIS---CombinedDNAIndexSystemc.Minisatellites,orVNTR

DNAelementcomposedoflonger(15-50bp)tandemrepeatingunitsofidenticalsequencelineduplike

boxcarsinafreighttrain.

Minisatelliterepeatanalysis

Minisatelliterepeatanalysis

Minisatelliterepeatanalysis

(A)(B)d.DNADeletions,DuplicationsandInsertions

ThePCRprotocolcanidentify

largeinsertionsanddeletions

Deletionsandinsertions

canbedetectedasRFLPs

PCRandsequencingFormationofhaplotype

Haplotypeisasetofsinglenucleotidepolymorphisms(SNPs)onasinglechromosomeofachromosomepairthatareassociatedstatistically.

e.Haplotype2.ApplicationsofDNAPolymorphisms

CysticFibrosis(CF)

Cysticfibrosis(CF)affecting1/2000newbornsinnorthernEuropeanheritageCFisinheritedasanautosomalrecessivemutationThefrequencyofheterozygotesistobeabout1/25CaucasianpopulationsInUSA,over30000peoplesufferfromthedevastatingdisease

CysticFibrosis(Continued)1989,FrancisCollinsandLap-CheeTsuiidentifiedtheCFgeneTheCFgenewasfirstmappedtothelongarmofchromosome7ThreekindsofinformationwereusedtonarrowthesearchfortheCFgene:CpGislands,zooblots,colonyhybridizationThegeneishuge,spanning250kbandcontaining24exons.TheCFmRNAisabout6.5kbinlengthandencodesaproteinof1480aminoacids.Over170differentCFmutationshavebeenidentified,70%ofthemutantallelescontainthesamethreebasedeletion,ΔF508

CFGeneCloning

Chromosomewalksandjumps1989,FrancisCollinsandLap-CheeTsui

CFTRCFTR(cysticfibrosistransmembraneconductanceregulator)proteinandthedistributionandclassificationofthemutationsthatcausecysticfibrosis

Detectionofthecysticfibrosismutant

genebyASOProbeHybridizations

Detectingthecysticfibrosisgenotype

ofeukaryoticcells

Directdetectionofthemost

commoncysticfibrosismutation

(2)Huntington’sDisease(HD)Huntington’sDisease(HD),1983byJamesGusellaandNancy

Wexler

UseOfRecombinantDNATechnology

ToIdentifyHuntington’sDiseaseGenes1872,GeorgeHuntington-Huntington’sdisease(HD)

HDinanautosomaldominantmutation,whichoccursinabout1/10000individualsofEuropeandescentHDbeginsatageof30-50yrsandterminatingindeath10-15yrslaterTodate,HDisuntreatable1983,JamesGusellaandNancyWexlermappedthegeneonchromosome41993,HDproteinisolatedanddetermined

HuntingtonDiseasegeneandhuntingtinGuselladetectedexpandedCAGrepeatin72familieswithHD,identifiedthecorrectgeneGusellaandWexleridentifiedagene(firstcalledIT15andsubsequentlynamedhuntingtin),thatspansabout210kbneartheshortarmofchromosome4Thehuntingtingeneproducingalarge10-11kbmRNA,coding3144aminoacidsThefunctionofthehuntingtinproteinisunknown

ExonamplificationGusellaandWexler:IT15---huntingtin210kb,(CAG)nrepeatNormal:11-34Mutant:42-100

ExonamplificationTestingfortheexpandedtrinucleotiderepeat

regionsinthehuntingtingenethatareresponsible

forHuntington’sdiseasebyPCRCAGrepeat:11-34,normal42-100,mutant

(3)MolecularDiagnosisofHuman

Sickle-cellMutationMutation:ChangesintheGeneticMaterialoverTime---sickle-cellhemoglobinmutation

Sickle-cell

genotypesicklenormal

Diseasediagnosis

Sickle-cell

genotypesicklenormal

Diseasediagnosis

Detectionofthesickle-cellhemoglobinmutation

MutationMstIIsiteabsent

Normal

MstIIsitepresent(4)Diagnosisofhemophilia

DiseaseBclIsiteabsent

NormalBclIsitepresent

(5)Calculatingtheprobabilitiesofrelatednessbetweentwoindividuals

(6)DNAfingerprintingdistinguishindividuals

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