基因工程课后题英文版.docx

基因工程课后题英文版.docx

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基因工程课后题英文版

第一章为什么基因克隆和DNA分析很重要

geneticengineering:

isthedirectmanipulationofanorganism’sgenomeusingmodernDNA

technology.ItinvolvestheintroductionofforeignDNAorsyntheticdenesintotheorganismofinterest.

1.HowtoCreateaGeneticallyModifiedPlant?

Ans:

①Createrecombinantbacteriawithdesiredgene.

②Allowthebacteriato“infect”theplantcells.

③Desiredgeneisinterestedintoplantchromosomes.

2.WhygenecloningandPCRaresoimpartant?

Ans:

becausebothtechniquescanprovideapuresampleofanindividualgene,separatedfromalltheothergenesinthecell.

第二章基因克隆的载体：

质粒和噬菌体

1.Pleaselistessentialpartsofacloningvetor.

（1）Self-replication:

orisite.

（2）Plasmidsize:

assmallaspossible.

（3）Insertsite:

MCS（Multi-cloningsite）.

（4）Selectablemarker.

（5）Notaffecthostcell.

2.Classificationofplasmid.

1）Basedonthemaincharacteristiccodedbytheplasmidgene.

①FertilityorFplasmids

②ResistanceorRplasmids

③Colplasmid

④Degradativeplasmids

⑤virulenceplasmids

2）dependonwhethertheycarrythetragenes:

①conjugativeplasmids

②non-conjugativeplasmids.

3）basedonthecopiestheycarrying:

relaxedplasmids；stringentplasmids

Words：

1.Plasmid：

AusuallycircularpieceofDNA,primarilyindependentofthehostchromosome,oftenfoundinbacteriaandsomeothertypesofcells.

2.cccDNA：

（covalentlyclosed-circularDNA）:

Acompletelydouble-standedcircularDNAmolecule,withnonicksordisconuities,usuallywithasupercoiledconformation.

3.ori（originofreplication:

ThespecificpositiononaDNAmoleculewhereDNAreplicationbegins.

4.episome:

Aplasmidcapableofintegrationintothehostcell'schromosome.

5.copynumber:

Thenumberofmoleculesofaplasmidcontainedinasinglecell.

6.bacteriophage:

Aviruswhosehostisabacterium。

BacteriophageDNAmoleculesareoftenusedascloningvectors。

7.2μmplasmid:

AplasmidfoundintheyeastSaccharomycesCerevisiaeandusedasthebasisforaseriesofcloningvectors.

8.cossite:

thebacteriophagecohesiveendsarecalledcossite。

9.Pilus：

oneofthestructurespresentonthesurfaceofabacteriumcontainingaconjugativeplasmid,throughwhichDNAisassumedtopassduringconjugation.

第3章从活细胞中纯化DNA

1.Whichstepsisincludedintheplasmidextractionfrombacterial?

Pleaselistthemainchemical

componentsanddescribetheirfunctionsinplasmidextractions.

①Acultureofbacteriaisgrownandthenharvested.

②Thecellsarebrokenopentoreleasetheircontents.

③ThiscellsextractistreatedtoremoveallcomponentsexcepttheDNA.

④TheresultingDNAsolutionisconcentrated.

Lysozyme：

digestthepolymericincellwall.。

EDTA:

removemagnesiumionsandinhibitenzymesthatdegradeDNA.

SDS:

removelipidmoleculesinthecellmembranes.

Removeproteinfromcellextractphenol（苯酚）or1:

1mistureofphenoland

chloroform（氯仿）;

Whenproteincontentisgreatincellextract:

treatwithaprotease

RNAintheextractwasmovedbyribonuclease

CollectingDNAbyethanolprecipitation

2.Techniquesforbreakingbacterialcells.

Physicalmethods：

ultrasonic

Chemicalmethods：

lysozyme/EDTA/SDS

3.whatisthemostfrequentlyusedmethodofconcentration?

P32Ethanolprecipitation

5.Pleasedescribethegenomeextractionprocedurefromyeast.①Acultureofbacteriaisgrownandthenharvested.

②Thecellsarebrokenopentoreleasetheircontents.

③ThiscellextractistreatedtoremoveallcomponentsexcepttheDNA.

④TheresultingDNAsolutionisconcentrated。

1.Brothculture:

Theculturemediummustprovideabalancedmixtureoftheessentialnutrientsatconcentrationsthatwillallowthebacteriatogrowanddivideefficiently.

2.alkalinedenaturation:

thebasisofthistechniqueisthatthereisanarrowPh.rangeatwhichnonsupercoiledDNAisdenatured,whereassupercoiledplasmidarenot.

3.densitygradientcentrifugation:

Separationofmoleculesandparticlesonthebasisofbuoyantdensity,bycentrifugationinaconcentratedsucroseorcesiumchloridesolution.

4.plasmidamplification:

Amethodinvolvingincubationwithaninhibitorofproteinsynthesisaimedatincreasingthecopynumberofcertaintypesofplasmidinabacterialculture.

第4章DNA纯化后的利用

1.whenpuresamplesofDNAareprepared,howdowedotheminthenextstep?

ANS:

constructionoftherecombinantDNAmolecule.

2.PleaselistclassificationofDNAmanipulativeenzymes.

①nucleases②ligases③polymerases

④DNAmodifyingenzymes⑤topoisomerases

3.PleaselistfourtypesofDNApolymerase,anddescribetheirfunction.

①DNApolymeraseI:

polymerizationanddegradationDNA.

②Klenowfragment：

polymerizationDNAbutnotdegradation.

③TaqDNApolymerase：

resistanttodenaturationbyheattreatment.

④Reversetranscriptase：

SynthesisofcomplementaryDNAstrandsbyasingleRNAtemplate.

4.whatisthecentralfeatureoftypeIIrestrictionendonucleases.

ANS:

eachenzymehasaspecificrecognitionsequenceatwhichitcutsaDNAmolecule.

5.puttingstickyendsontoablunt-endedmolecule.

①Linkers②Adaptors③Homopolymertailing

6.howtoincreasetheefficiencyofligation？

①Theligationofcomplementarystickyendsincreasestheefficiencyofligation.

②Increasetheperiodoftimethattheendsareincontactwithoneanother.

③BluntendligationshoudbeperformedathightDNAconcentrations.

1.nucleases:

nucleasesareenzymesthatcut,shorten,ordegradednucleicacidmolecules.

2.ligases：

joinnucleicacidmoleculestogether.

3.DNApolymerases:

areenzymethatsynthesizesanewstandofDNAcomplementarytoanexistingDNAorRNAtemplate.

4.Exonucleases：

removenucleotidesoneatatimefromtheendofaDNAmolecule.

5.Endonucleases：

areabletobreakinternalphosphodiesterbondswithinaDNA

molecule.

6.Alkalinephosphatase（碱性磷酸酶）whichremovesthephosphategrouppresentatthephosphategrouppresentat5’terminusofaDNAmolecule.

7.Polynucleotidekinase（多核苷酸激酶）addingphosphategroupsontofree5'termini.

8.Terminaldeoxyribonucleotidestransferase（末端脱氧核苷酸转移酶）whichaddsoneormoredeoxyribonucleotidesontothe3'terminusofaDNAmolecule.

9.Doubledigestions:

inwhichtheDNAiscutbytworestrictionendonucleasesatonce.

10.partialdigestion:

carriedoutunderconditionsthatresultinthecleavageofonlyalimitednumber

oftherestrictionsitesonanyDNAmolecule.

11.Linkers:

areshortpiecesofdouble-strandedDNAwithbluntends,ofknownnucleotidesequence

whichcontainingasiteofarestrictionendonuclease,thataresynthesizedinthetesttube.

12.Adaptors:

areshortsyntheticoligonucleotideswithonestickyendofarestrictionendonuclease.

第5章将DNA引入活细胞

1.pleaseexplainthemainpurposesofcloning?

①Amplification，itallowsalargenumberofrecombinantDNAmoleculestobeproducedfromalimitedamountofstartingmaterial.

②Purification.。

2.Whataretheligationproduction？

1）ThedesiredrecombinantDNAmolecule.

2）Unligatedvectormolecules.

3）UnligatedDNAfragments.

4）Self-ligatedvector.

5）WrongrecombinantDNAmolecules.

3.Pleaselistthemethodsofidentifyingrecombinants.

①Insertionalinactivation.pBR322

②Lacselection.pUC8

4.Pleaselistthemethodsoftransformingnon-bacterialcells.

1）MethodsforintroducingnewDNAintoanimalsandplantcells.

①PrecipitationofDNAontoanimalscells.

②IntroductionofDNAintoanimalcellsbyliposomefusion.

③Transformationofplantprotoplasts.

2）TwophysicalmethodsforintroducingDNAintocells.

①Microinjection.

②Bombardingthecellswithhigh-velocitymicroprojectiles.

Words：

1.transformation:

TheintroductionofanyDNAmoleculeintoanylivingcell.

2.competentcell:

AcultureofbacteriathathasbeentreatedtoenhancetheirabilitytotakeupDNAmolecules.

3.insertionalinactivation:

AcloningstrategywherebyinsertionofanewpieceofDNAintoavectorinactivatesagenecarriedbythevector.

4.lacselection:

AmeansofidentifyingrecombinantbacteriacontainingvectorsthatcarrythelacZ'gene.Thebacteriaareplatedonamediumthatcontainsananalogueoflactosethatgivesabluecolourinthepresenceofβ-galactosidaseactivity.

5.electroporation:

AmethodforincreasingDNAuptakebyprotoplaststhroughpriorexposuretoahighvoltage,whichresultsinthetemporaryformationofsmallporesinthecellmembrane.

6.microinjection:

AmethodofintroducingnewDNAintoacellbyinjectingitdirectlyintothemolecule.

7.protoplast:

Acellfromwhichthecellwallhasbeencompletelyremoved.

第6章大肠杆菌的克隆载体

1.therequirementsforaDNAmoleculeasacloningvehicles?

①Self-replication.

②Lowmolecularweight.

③selectablemarker.

④Multiplecloningsites.

⑤Noharmfultothehostcells.

2.Propertiesofanidealcloningvehicle?

1）Easyofpurification.

2）Hightransformationefficiency.

3）Convenientselectablemarkersfortransformantsandrecombinants.

4）TheabilitytoclonereasonablylargepiecesofDNA.

3.WhyhaspBR322beensuchapopularcloningvector?

①Sizeis4363bp.

②Twosetsofantibioticresistancegenes.

③Highcopynumber.

4.pleaselistTHREEtypicalE.coliplasmidcloningvectors.

①pBR322②pBR327③pUC8orpGEM3Z

1.Cosmid:

Ancloningvectorconsistingoftheλcossiteinsertedintoaplasmid,usedtocloneDNAfragmentsupto40kbinsize.

2.Genomiclibrary:

Acollectionofclonessufficientinnumbertoincludeallthegenesofaparticularorganism.

3.Bacterialartificialchromosome（BAC）：

AncloningvectorbasedontheFplasmid,usedforcloningrelativelylargefragmentsofDNAinE.coli.

4.P1-derivedartifcialchromosome（PAC）：

Acloningvect