分子期末復(fù)習(xí)-基因序列工程

GenomicssequenceTechniquesupportforgenomesequenceSangerchain-terminatormethodAutomatedDNAsequencingmachineBioinformaticsoftwareandfacilityTwostrategiesingenomesequenceCloneby WholeGenomeShot- Wholegenomeshot-ClonebyNoRequirephysicalThecapacityRequirehighlowApplyWithouthugeWithhugeHuman,C.ClonebyConstructphysical→ConstructBAC→AssembleBACcontigsaccordingtoSTS→Assemblingshotgun→AssemblingwholegenomesequenceandPhysicalmap:AgeneticmapbasedonphysicalcharacteristicsoftheDNA,suchasrestrictionsitesratherthanonlocationsofgenes.STS(SequenceTagged ashortstretchofDNAthatcanbeidentifiedbyamplifyingitPCRwithdefinedprimers.EachSTSmarkerhasitsspecificlocationonchromosome.Physicalmap=STSConstructBACNotISacIareenzymesusedtocutgenomicgenesinto200kbDNA.Andtheywillbeinsertedintovectors.AftergrowinginE.coil,thegenescanbedetectedbyscreeningonantibioticmedium.Eachcolonyhasoneinsertion.AssemblecloneScreentheBACcolonsby“STS-PCRpooling”assembleclonecontigs,andfillinterclonegapsbyBACendsequencingwalkingorfingerprintingwalking.AssembleBAConchromosomeaccordingtoThedensityofSTSmarkersonchromosomeisnotenough,manyregionswouldnotbecoveredbyclones,WalkingbyEndSequenceorWalkingbyFingerprintingcanhelptoselectnewclonescovertheemptyregions.WalkingbyEndSequence（Sequencetheclonelocatesontheendofcontig,thespecificnewsequencecanberegardedasanewSTSmarkertoscreennewclonescovertheemptyregionHowtwoFill“interclonegaps”Filling“interclonegaps”Molecularweightmarkerevery5laneBACclonesareculturedin96plateHindIIIdigest1%agarosegelWalkingbyFingerprinting（Fingerprint:ThespecificelectrophoresispatternofDNAfragmentsformedwhenaDNAiscutintopieceswitharestrictionendonucleaseHindIIIHindIIIComparewithFingerprintingdatabaseCloneACloneBCloneC15clonesdigestedbyselectminimaltilingpathforshotgunsequencing.WalkingbyFingerprintingAssemblingshotgunsequenceandFinishingShotgunSequencingI:RandomphaseBacClone:100-200ShearedDNA:1.0-2.0kbShotgunSequencingII:AssemblyShotgunSequencingIII:FinishingHighAccuracySequence:<1error/10,000basesStudent’spresentationPaper5:MarkCigan,A.,Unger-Wallace,E.&Haug-Collet,K.(2005).Transcriptionalgenesilencingasatoolforuncoveringgenefunctioninmaize.PlantJ.43:947-951MutationonTheeffectofmutationonReplicationslippage(滑移):InDNAreplicationwhenparentalstrandhasshortrepeatsequence,theslippagewilloccasionallyoccurbetweenparentalstrandanditscomplementarydaughterstrand,thatcausetherepeatunitsequencereplicateseveraltimesorloseseveraltimes.EffectofmutationongeneLoss-of–function:themutationcausesthereducedproteinactivityorlossproteinactivity.Inmostcasesitisrecessivebecauseinheterozygous(雜合體)anothernormalhaploidchromosomeencodecorrectproteins.Butsometimesitcanalsobedominant(dominantnegative).Gain-of–function:Itgainsabnormalproteinactivity.Sometimesthatisbecauseofectopicexpression(異位表達(dá)),andinspecifictissuetheexpressionwillbeAndmutationoccurringindestructive cancauseproteinabnormallyHowtoProducemutation?Spontaneous,E.coli1/1010 Deaminatingagents:IntercalatingUVradiation(TT),ionizingTwostrategiesinfunctional

Phenotypedrivenapproach:forwardgeneticstrategyInvestigatethefunctionofaspecialgeneGenedrivenapproach:ReversegeneticstrategyTILLING(TargetingInducedLocalLesionsinGenomes,定向誘導(dǎo)組局部突變PCRtoamplifytargetDNAfrombothmutantandwild-typereferencehybridizationtoformheteroduplexesbetweenmutantandwild-typereferencetreatmentofannealedDNAwithsurveyornucleasetocleaveysisofdigestedDNACommonmutagenesismethodsinT-DNAinsertion T-DNAcarriedonvectorcanbetaggedintoplantgenomeandexpressedTransposableelementsinsertion TEmovesfromonelocationinthegenometoanotherAutonomouselementscanencodetransposaseenzyme,andnon-autonomouselementscanbemobilisedbytheautonomouselementofthesametransposonsystem.MapbasedMapbasedcloninginvolvesisolatingamarkerpieceofDNAwhoselocationisknownandwhichistightlylinkedtothegeneyouaretryingtoclonelinkagemapgeneticmap遺傳圖譜RFLP,RestrictionfragmentlengthPolymorphism:ageneticlocushasdifferentforms,oralleles.DigestingDNAfromtwoindividualswithrestrictionendonucleasesmayyieldfragmentsofdifferent4PCRbasedmarkers:SSLP,STS,CAPS,SSLP:SimpleSequenceLengthPolymorphism(=SSR,Simplesequencerepeat) SequenceTaggedSiteCAPS:CleavedAmplifiedPolymorphicProcedureofmapbasedF2populationconstruction.CrossthericemutantwithanotherGenotypericetogetF1,selfpollinateofF1togetF2progenyforma.Initialmap(初步定位).Screen30mutantphenotypeplantsinF2progeny.ExtractDNAthese30mutants.Detectthepolymorphicbandsamongthissmallpopulationusingmarkersondifferentchromosome.ThecompleylinkagemarkerarefindFinemap(精細(xì)定位).Screen1000-2000mutantsinamplifiedF2population.Extractofthese1000-2000mutantsbypooling.Detectthepolymorphicbandsamongthisbigpopulationusingmarkerswhicharoundthelinkagedmarkerwhichfindininitialma