17、分子生物學(xué)課件4個(gè)mb14

Student’spresentation(nextweek)Paper6:

LiXY,QianQ,FuZM,WangYH,XiongGS,ZengDL,WangXQ,LiuXF,TengS,HiroshiF,YangM,,LuokD,HanB,LiJY(2004)Controloftilleringinrice.Nature,422:618-621

Group6andGroup12

3.Mapbasedcloning2.Mutationongenomics1.GenomicssequenceprojectGenomics

Finished：S.cerevisiae

(Yeast),C.Elegans,Dmelanogaster(Fruitfly),Athaliana,H.Sapiens(Human,2001）,Danio

rerio(Zebrafish),O.Sativa(rice,2002),M.musculus(mouse,2002),S.scrofa(Pig,2005),R.norvegicus(Rat)Aredoing：chimp,baboon,chicken,cattle,wheat,maize,barley,soybean,cotton,parasite,pathogenicmicroorganism

GenomicssequenceprojectWilldoinfutureDomesticanimals、Tree,Halobios,allrepresentativespeciesinexperimentalmodelorganism世界大型基因組研究中心USA：NationalHumanGenomeResearchInstitutioninNIHGenomeCenteratWhiteHead/MITWashingtonUniversityGenomeCenterJointGenomeInstitutionatDOEGenomeCenteratBaylorMedicalCollageUK：SangerCenterJapan：RIKENChina：華大基因研究中心（北京、杭州） 國(guó)家人類(lèi)基因組中心（北京、上海）InstitutionsworkingonGenomesequenceprojectTechniquesupportforgenomesequenceprojectSangerchain-terminatormethod

PCRAutomatedDNAsequencingmachineBioinformaticsoftwareandfacilitydideoxynucleotidedeoxynucleotideSangerchain-terminatormethod

Sangerchain-terminatormethod

ClonebyClone（逐步克隆法）WholeGenomeShot-gun(全基因組鳥(niǎo)槍?zhuān)睆棧┓?Twostrategiesingenomesequenceproject………ATGCCGTAGGCCTAGCTAGGCCTAGCTCGGA……………ATGCCGTAGGCCTAGCTCGGA……BAClibraryAssembleBACcontigsaccordingtophysicalmapABACcloneusedinshot-gunsequenceSub-cloneinshot-gunClonebyCloneWholeGenomeShot-gunGenomicDNAshot-guncloningSequenceandassemblyWholegenomesequenceWholegenomesequenceGenomicDNASequenceandassembly

Item

strategyWholegenomeshot-gunClonebycloneGeneticbackgroundNorequirementRequirephysicalmapSpeedquickslowExpenselowhighThecapacityofcomputerRequirehighcapacitylowcapacityApplytoWithouthugerepeatedsequenceWithhugerepeatedsequenceRepresentativespeciesDrosophila,Arabidopsis,riceHuman,C.elegansComparingtwostrategiesingenomesequenceprojectBACbyBACWholeGenomeShotgun“WorkingDraft”(90%;4X)FinishedGenome(99.99%;8X)Gap1Gap2Chromosome“WorkingDraft”and“FinishedGenome”BACbyBACAnexampleofhumangenomicsequenceprojectThesequenceofthehumangenomeC.Venteretal.Science16Feb.291:1304–1351,2001tBeijingGenomicsInstituteClonebyCloneConstructphysicalmapConstruct

BAClibraryAssembleBACcontigsaccordingtoSTSmarkerAssemblingshotgunsequenceAssemblingwholegenomesequenceandfinishingPhysicalmap:

AgeneticmapbasedonphysicalcharacteristicsoftheDNA,suchasrestrictionsitesratherthanonlocationsofgenes.Precondition:aPhysicalmapSTS（SequenceTaggedSite):AshortstretchofDNAthatcanbeidentifiedbyamplifyingitusingPCRwithdefinedprimers.EachSTSmarkerhasitsspecificlocationonchromosome.Physicalmap=STSmap1.ConstructphysicalmapIntroductionoftwovectorsforlarge-scalegenomeprojectsYAC(YeastArtificialChromosomes)：biggestcapacity,butnotstableBAC(BacterialArtificialChromosomes)2.Construct

BAClibraryNotI、SacIInsertintovectorpulsed-fieldgelelectrophoresis,

toget200KbDNAElectroporationintoE.coliScreenonantibioticmediumEachcolonycarryoneinsertion2.Construct

BAClibrary3.HowtoassembleclonecontigFillinterclonegapsbyBACendsequencingwalkingorFingerprintingwalkingScreenBACclonesby“STS-PCRpooling”,assembleclonecontigScreenBACclonesScreenclonesby“STS-PCRpooling”STSmarkersBACclonesareassembledascontigaccordingtotheSTSinformationonphysicalmapContigEachclonecarryoneinsertion“STS-PCRPooling”superpools

Columnpools

Rowpools

123456789101112EighthplateSecondplatePlatepoolsFirstplate

platepools，rowpools，columnpoolsAssembleBAConchromosomeaccordingtoSTSThedensityofSTSmarkersonchromosomeisnotenough,manyregionswouldnotbecoveredbyclones,WalkingbyEndSequenceorWalkingbyFingerprintingcanhelptoselectnewclonescovertheemptyregions.WalkingbyEndSequence（末端序列步行法）

Sequencetheclonelocatesontheendofcontig,thespecificnewsequencecanberegardedasanewSTSmarkertoscreennewclonescovertheemptyregionHowtwoFill“interclonegaps”Filling“interclonegaps”>20kb~300bpMolecularweightmarkerevery5laneBACclonesareculturedin96plate

HindIIIdigest1%agarosegelelectrophoresisWalkingbyFingerprinting（指紋圖譜法）

Fingerprint:ThespecificelectrophoresispatternofDNAfragmentsformedwhenaDNAiscutintopieceswitharestrictionendonucleaseHindIII完全酶切HindIII完全酶切ComparewithFingerprintingdatabaseCloneACloneBCloneCCABMK2MK1MK2MK115clonesdigestedbyHindIIIselectminima