分析教案成果professor tus

Chapter5 Whatistheroleof binationingenomeevaluation?(Answerinshortanswerquestion17.1)binationallowsformajorchangesandextensiverestructuringofgenomes.Intheabsenceof genomeswouldundergolittlechangeandbefairlystaticstructures.Howcantheresolutionofahollidaystructureyieldtwodifferentresults?ResolutionofaHollidayjunctioncangenerateparentalor duplexes,dependingonwhichstrandsarenicked.BothtypesofproducthavearegionofheteroduplexDNA.Describehowthedouble-strandbreakmodelexinshowgeneconversionoccurs?(AnswerInthedouble-strandbreakmodel,homologous binationinitiateswithadouble-strandbreakinoneofthemolecules.Onestrandineachhalfofthebrokenchromosomeisshortened,leaving3'overhangs.OneoftheoverhangsinvadestheotherintactDNAmoleculetosetupaHollidayjunction.TheshortenedDNAstrandsareextendedbyDNApolymerase,withtheDNAsynthesisintheregionbeingconvertedusingtheDNAmoleculethatdidnotundergotheoriginaldouble-strandbreakasatemte.SomeE.colistrainsthatareusedforpropagating binantsmidscontainrecAdefectstobeusefulforresearchersworkingwith binantsmids?EstablishmentofheteroduplexduringHRbyformingaproteinRecA-coatedDNAfilamentthatishelixandformshydrogenboundwithbasepairsitencounters.WhatarethepropertiesoftheattPandattBsitesthatmediateintegrationofλDNAintotheE.coligenome?(Answer17.7)Theattsiteseachcontainanidentical15basepaircoresequence.Thecoresequencesareflankedbyvariablesequences:BandB'(eachjust4hpinlength)inthebacterialgenome,andPandP'inthephageDNA.PandP'arebothover100byinlength.Mutationsinthecoresequenceinactivatetheattsitesothatitcanalsonolongerparticipatein HowisthenewcopyofaretroelementinsertedintoaInsertionofnewcopyofretroelementintogenomeoccurspreferentiallyatcertainposition.Insertioninvolvesremovaloftwonucleotidesfrom3'endofdouble-strandedretroelementbyintegraseIntegrasealsomakesastaggeredcutingenomicDNAsothatbothretroelementandintegrationsitenowhavethe5'overhang.Theseoverhangmightnothavecomplementarysequencesbuttheystillappeartoinctinsomewaysothatretroelement esinsertedintogenomicDNA.Inctionresultsinlossofretroelementoverhangsandfillinginofgapsthatareleft,whichmeansthatintegrationsite esduplicatedintoapairofdirectrepeats,oneateitherendofinsertedretroelement.WhatistheroleoftRNAmoleculesinthereplicationofretroelements?(Answer17.9)ThefirststepinreplicationofaretroelementissynthesisofanRNAcopy.ThisRNAmoleculeisthenconvertedintodouble-strandedDNA.Thefirststageinthisconversionissynthesis,byreversetranscription,ofasingle-strandedDNAcopyoftheRNAmolecule.Thistoasitewithinthe5'longterminalrepeatoftheRNAcopyoftheGiveexamplesoftheharmfuleffectsthattransposonscanhaveonaTheobviousdisruptionofgeneactivitythatwilloccurifatransposableelementtakesupanewpositionthatlieswithinthecodingregionofagene.轉(zhuǎn)座因子在組的編碼區(qū)域就可能導致這種有害的活動)Someelements,notablyretrotransposonscontainpromoterandenhancersequencesthatcanmodifytheexpressionpatternsofadjacentgenes,transpositionofteninvolvescreationofdouble-strandbreakswhichcanhaveseriouslyharmfuleffectsontheintegrityofagenome.逆轉(zhuǎn)錄轉(zhuǎn)座子有啟動子和增強序列，能影響相鄰的表達，能破壞雙鏈破壞組的完整性)Interspersedrepeats,atleastsomeofwhichare ,makeupalmosthalfofeachm genomeandasubstantiallylargerfractionofsomentgenomes散布重復序列能移動組成哺乳動物和一些植物的組)OnewayinwhichtranspositionofbothDNAtransposonsandretroelementscouldbepreventedbymethylationoftheirDNAsequences.(DNA甲基化能轉(zhuǎn)座子和逆轉(zhuǎn)錄轉(zhuǎn)座子)Chapter6StudyingWhyaremapsrequiredforthesequencingofgenomes?Ifamapofagenomewasunavailable,whatwouldbethemajordifficultiesinobtainingagenomesequence?(Answerinshortanswerquestions3.1)Agenomemapprovidesaguideforthesequencingexperimentsbyshowingthepositionsofgenesandotherdistinctivefeatures.Ifamapisunavailablethenitislikelythaterrorswillbemadeinassemblingthegenomesequence,especiallyinregionsthatcontainrepetitiveDNA.HowhasPCRmadetheysisofRFLPsmuchfasterandeasier?WhatwasrequiredtomapRFLPspriortotheutilizationofPCR?(Answer3.3)TheprimersforthePCRaredesignedsothattheyannealeithersideofthepolymorphicsite,andtheRFLPistypedbytreatingtheamplifiedfragmentwiththerestrictionenzymeandthenrunningasampleinanagarosegel.BeforetheinventionofPCR,RFLPsweretypedbySouthernhybridization,whichistime-consuming.Howdoesthelinkagebetweengenesprovideacriticalcomponenttogeneticmap?Discusshowgeneticmarkerscanbelinkedtoprovidemapsofindividualchromosomes.(Answer3.5)Ifapairofgenesdisylinkagethentheymustbeonthesamechromosome.Ifcrossing-overisarandomeventthenthe binationfrequencybetweenapairoflinkedgenesisameasureoftheirdistanceapartonthechromosome.The binationfrequenciesfordifferentpairsofgenescanbeusedtoconstructamapoftheirrelativepositionsonthechromosome.Whyisadoublehomozygoteusedfortestcrossesinlinkageysisexperiments?Whyisitpreferablethatthehomozygoteallelesberecessiveforthetraitsbeingtested?(Answer3.7)Thedoublehomozygotewillproducegametesthatareallthesamegeneticallyandiftheyarerecessivethenthisparentwillnotcontributetothephenotypeoftheoffspring.Geneticmaptechniquesrequireatleasttwoallelesforagivenmarker,whilephysicalmaptechniquesdonotrelyonthepresenceofallelestomapgenomes.Discusshowthetechniqueoffluorescentinsituhybridizationcanbeusedtomapgenomelocationsevenifthereisnogeneticvariationpresentatagivenposition.FISHusesafluorescentlylabeledDNAfragmentasaprobetobindtoallintactchromosome.ThebindingpositioncanbedeterminedandthisinformationusedtocreateaphysicalmapoftheHowdoesascientistprepareaclonelibraryofDNAfromjustasingleIndividualchromosomescanbeseparatedbyflowcytometly.Dividingcellsarecarefullybrokenopensothatamixtureofintactchromosomeisobtained.Thechromosomesarethenstainedwithafluorescentdye.Theamountofdyethatachromosomebindsdependsonitssize,solargerchromosomesbindmoredyeandfluorescemorebrightlythansmallerones.Thechromosomepreparationisdilutedandpassedthroughafine,producingastreamofdroplets,eachonecontainingasinglechromosome.Thedropletspassthroughadetectorthatmeasurestheamountoffluorescence,andhenceidentifieswhichdropletscontaintheparticularchromosomebeingsought.Anelectricchargeisappliedtothesedrops,andnoothers,enablingthedropletscontainingthedesiredchromosometobedeflectedandseparatedfromtherest.WhataretheidealfeaturesofaDNAmarkerthatwillbeusedtoconstructageneticmap?TowhatextentcanRFLPs,SSLPs,orSNPsbeconsidered"ideal"DNAmarkers?Thetextindicatesthattheidealfeaturesincludehighfrequencyinthegenomebeingstudied,easeofty,andthepresenceofmultiplealleles.ThisimpliesthatSSLPsshouldbethe"ideal"markers,butinrealitySNPsaremorepopular.Adiscussionofthisapparentparadoxoftherelativeimportanceofeachofthethreecriteria,demandsconsiderationandinparticulararealizationthatthecriticalfeatureofan"ideal"markerishighWhichismoreuseful-ageneticoraphysicalmap?(AnswerThisisaveryopen-endedquestionthatisdesignedtopromptdiscussofanumberoftopicsthatarecoveredinlaterchapters.Thediscussionmightbeginbyaskingwhatpurposethemapisintendedtofulfill.Amapdesignedtoaidasequencingprojectmightnotbethesameasonedesignedtoenableindividualgenestobecloned.Ifitisconcludedthatforsequencingpurposesaphysicalmapismoreuseful,andinfactageneticmaphaslittleornodirectvalue(whichisareasonableinferencetomakefromareadingofChapter4),thenthediscussioncouldturntohoweasyorotherwiseitwouldbetolocatethegenesinagenomesequence,andtoassignfunctionstothosegeneswithoutanypriorknowledgeofwherethegenesare.TheseissuesarediscussedinChapter5.Howdoesadye-quenchingexperimentdetermineifanoligonucleotidehashybridizedtoaDNAmoleculecontainingasinglenucleotidepolymorphism?(AnswerinFiguretests3.1)Whentheoligonucleotideisnothybridizedtothetargetsequence,thefluorescentlabelandquenchingmoleculearenexttoeachotherandthefluorescenceisquenched.Whentheoligonucleotidebindstoatargetsequence,thefluorescentlabelislocatedawayfromthequenchingmolecule.Bycontrollingthehybridizationconditions,theoligonucleotidewillonlybindtothetargetsequenceifallthenucleotidesarecomplementary.Chapter7GenomeWhatisthefunctionofthedideoxynucleotidesinachainterminationsequencingreaction?(Answerinshortanswerquestions4.1)intotheDNA,strandsynthesisstops.WoulditbepossibletosequenceaPCRproductdirectly(withoutcloningthePCRproduct)?Howwouldthisbe plished?(Answer4.3)Yes,thisispossible.ThePCRproductispurifiedandthermalsequencingperformedwithoneofthePCRprimersusedastheprimersforthesequencingreactions.(Answer4.5)Automatedsequencerswithmultiplecapillariesworkinginparallelcanreadupto96differentsequencesina2-hourperiod,whichmeansthatwithanaverageof750byperindividualexperiment,864kbofinformationcanbegeneratedpermachineperday.Thisenablesdataneededtosequenceanentiregenometobegeneratedinaperiodofweeks.Whydoestheshotgunsequencingmethodrequirethatthenumberofnucleotidessequencedisseveraltimeslargerthanthesizeofthegenome?(Answer4.7)Theredundancyisrequiredastheclonesforthesequencingprojectarerandomlygeneratedandsequenced;thus,toensurecompletecoverageofthegenomeitisnecessarytosequencealargenumberofnucleotides.WhatmethodcanbeusedtoperformDNAfingerprintingwithclonesfromalargeDNAfragmentthatistobesequenced?(Answer4.9)Clonefingerprintingcanbebasedonrestrictionenzymepatterns,repetitiveDNAfingerprints,repetitiveDNAPCR,andSTScontentmap.(Answerinin-depthproblems4.3)Section4.2.2coverstheclonecontigapproach,butacriticalevaluationacomparisonbetweentheclonecontig（群）andwholegenomeshotgunapproaches,inparticularasthesehavebeenappliedtothehumangenome.Fromthiscomparisonit esobviousthatarigorousclonecontigprojectisrelativelytime-consuming,butthatitiscurrentlyonlywaytoensureanerrorrateoflessthanonein104nucleotides,thefiguresetastheacceptable umfora"finished"sequence.Howthefragmentlibrariesareobtained?WhyissocriticalforwholegenomeThefragmentlibrariesareobtainedbyannealingtform-specificlinkerstoblunt-endedfragmentsgenerateddirectlyfromagenomeorDNAsourceofinterest.BecausethepresenceofadaptersequencesmeansthatthemoleculesthencanbeselectivelyamplifiedbyPCR,nobacterialcloningstepisrequiredtoamplifythegenomicfragmentinabacterialintermediateasisdoneintraditionalsequencingapproaches.Chapter8FunctionalWhyisitrelativelyeasytoidentifyORFsinprokaryoticgenomesbycomputerysis?(Answerinshortanswerquestions5.1)ComputerscanreadilyscanallsixreadingframesofaDNAsequenceforORFs.Inaddition,asarandomDNAsequencewouldpossessastopcodonatleastevery100-200byandmostgenescontainmorethanthisnumberofcodons,itisfairlystraightforwardtoidentifycodingsequencesinbacterialgenomesthatlackintronsandothersignificantnoncodingsequences.WhatarethethreeprimarymodificationsthatcanbeusedtoimprovethelocationofORFsbycomputerysis?(Answer5.3)Computerprogramscanbemodifiedtoscreenforcodonbias,exon-intronboundaries,andupstreamregulatorysequencesofgenes.WhatarethetwomajorproblemsthatoccurwhenattemptingtousecomputerysistoidentifyORFsinthegenomesofhighereukaryotes?SimpleORFscanislesseffectivewithDNAofhighereukaryotes.Thisispartlybecausethereissubstantiallymorespacebetweentherealgenesinaeukaryoticgenome,increasingchancesoffindingspuriousORFs.Butmainproblemswithgenomesofhighereukaryotesingeneralisthattheirgenesareoftensplitbyintronsandsodonotappearascont