13 an introduction to genetic analysis.docx
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13 an introduction to genetic analysis.docx
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13anintroductiontogeneticanalysis
Chapter13
ApplicationsofRecombinantDNATechnology
KeyConcepts
Invitromutagenesisallowshighlyspecificchangestobemadeatspecificpositionswithinagene.
Inthechromosomesofanindividualorganism,specificrestrictionsitescanbeeitherpresentorabsent,resultinginrestrictionfragmentlengthpolymorphisms(RFLPs).
RFLPscanbeusedaslociforgenomemapping,aswellasinthediagnosisoflinkeddiseasegenes.
Reversegeneticsworksinadirectionoppositethatoftraditionalgenetics;itenablesthefunctionofanunknownDNAorproteinsequencetobeidentifiedatthephenotypiclevel.
Agenewithaneasilydetectableproductcanbesplicedtotheregulatoryregionsofanothergeneandtherebyactasareporterforthatgene'sfunction.
RecombinantDNAtechnologyisusedtomakemicrobes,plants,andanimalsthatcarrygenesfromotherspecies.
RecombinantDNAtechnologycanbeusedintheprenataldiagnosisofhumangeneticdisease.
Introduction
Intheprecedingchapter,weexaminedthebasictechniquesforusingrecombinantDNAtechnologytoisolateandcharacterizegenes.Inthischapter,weexpandthisdiscussiontoseehowhavinganisolatedgeneinhandasaDNAcloneopensupdiverseexperimentalpossibilities.Forexample,aclonedgenecanbemutatedinahighlyspecificwayinthetesttubeandthenreintroducedintoitsoriginalhosttostudythegene'snormalfunctionalproperties.Thisproceduremirrorsthestandardgeneticprocedureofgeneticdissection.However,inspontaneousandinducedmutations,thenatureoftheDNAchangeislargelyunpredictable,andthesitesofthemutationscanbeinanynumberoflocationswithinthegene.Incontrast,mutagenesisinvitrocanbedirectedtowardaveryspecificlocationandtypeofchange.Henceinvitromutagenesisisapowerfulapproachtostudyinggenefunction.
ThetechniquesofDNAcloningandrestriction-enzymeanalysishavebeencombinedtocreateapowerfulnewmethodusedingeneticmapping.Inthismethod,thevariationofrestriction-enzymetargetsiteswithinaspeciesprovideslargenumbersofmolecular“alleles,”calledmolecularmarkers,forchromosomemapping.SuchmarkersaredetectedbyprobingwithclonedDNAfragments.Theabilitytodetectsuchmolecularmarkersinlargenumbershasrevolutionizedmappinginmostorganisms,includinghumans.
Clonedgenes(wildtypeormutated)canbemovedintoanewhostorganismtocreatetransgenicmicrobes,plants,andanimalsthatcouldneverhavebeenmadebyusingstandardbreedingprocedures.Thistechniquenotonlyfindsextensiveuseinbasicresearch,butalsoopensupaplethoraofapplicationsinplantandanimalbreeding,industrialmicrobiology,andmedicine.
Theavailabilityofclonedgenestobeusedasprobesalsoopensupnewfrontiersinthediagnosisofhumangeneticdiseasesinprospectiveparentsandinfetusesinutero.
First,weexaminesomewaysinwhichspecificmutationscanbemadeinclonedgenesinthetesttube(invitro);inotherwords,howtofabricate“designergenes.”
Invitromutagenesis
Oneofthemostestablishedtechniquesissite-directedmutagenesis.Byusingthismethod,wecancreatemutationsatanyspecificsiteinagenewhosewild-typesequenceisalreadyknown.ThisknownsequenceisusedtochemicallysynthesizeshortDNAsegmentscalledoligonucleotides.Throughsingle-strandhybridization,theseoligonucleotidescanbedirectedtoanychosensiteinthegene.Inoneapproach,thegeneofinterestisinsertedintoasingle-strandedphagevector,suchasthephageM13.Asyntheticoligonucleotidecontainingthedesiredmutationisdesigned.Thisoligonucleotideisallowedtohybridizetothemutantsitebycomplementarybasepairing.TheoligonucleotideservesasaprimerfortheinvitrosynthesisofthecomplementarystrandoftheM13vector(Figure13-1a).Anydesiredspecificbasechangecanbeprogrammedintothesequenceofthesyntheticprimer.AlthoughtherewillbeamispairedbasewhenthesyntheticoligonucleotidehybridizeswiththecomplementarysequenceontheM13vector,afewmismatchedbasescanbetoleratedwhenhybridizationtakesplaceatalowtemperatureandahighsaltconcentration.AfterDNAsynthesishasbeenmediatedbyDNApolymeraseinvitro,theM13DNAisallowedtoreplicateinE.coli,inwhichcasemanyoftheresultingphageswillbethedesiredmutant.Thesyntheticoligonucleotidecanbeusedasalabeledprobetodistinguishwild-typefrommutantphages.Although,atlowtemperature,themismatchedbasewillnotpreventtheprimerfromhybridizingwithbothtypesofphage,athightemperature,theprimerwillhybridizeonlywiththemutantphage.Oligonucleotideswithdeletionsorinsertionswilldirectcomparablemutationsintheresidentgene.Thesite-directedmethodcanalsobeusedongenesclonedindouble-strandedvect
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