21Techniques技术.docx

21Techniques技术.docx

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21Techniques技术

TechniquesofMolecularBiology

Chapter21

I.NucleicAcids

A.Gelelectrophoresis

B.Restrictionenzymes

C.DNAcloning

D.Hybridization

E.DNAlibrary

F.Expressionvectors

G.Polymerasechainreaction

H.Site-directedmutagenesis

I.Chemicallysynthesizedoligonucleotides

J.DNASequencing

II.Proteins

A.Polyacrylamidegelelectrophoresis

B.Two-dimensionalgelelectrophoresis

C.ColumnChromatography

D.Proteinsequencing

E.GreenFluorescentProtein

III.NucleicAcid-ProteinInteractions

A.Electrophoreticmobilityshiftassay

B.Nucleaseprotectionfootprinting

C.Chromatinimmunoprecipitation

I.NucleicAcids

WhattechniquecanbeusedtoseparateDNAorRNApiecesbysize?

A.gelelectrophoresis

DNAandRNAcanbeseparatedbysizeorlengthinanelectricfieldwiththetechniquecalledgelelectrophoresis.

WhichdirectionwillDNAmoveinanelectricfield?

Nucleicacidscanbeseparatedingelsmadeofagaroseoringelsmadeofpolyacrylamide.

AgarosegelscanseparatemuchlargerDNApieces,butthebandsarenotaswellresolved.

PolyacrylamidegelsaregoodforseparatingDNAfragmentsof100-700basepairs,butnotpiecesthatarelarger.Polyacrylamidegelshavegoodresolvingpowerandbandsthatdifferinsizebyonly1basepaircanberesolved.

SeparatingDNAorproteinthiswayiscalledpolyacrylamidegelelectrophoresisorPAGE.

LargerpiecesofDNAthatare30kbtoseveralmegabasescanbeseparatedbypulse-fieldgelelectrophoresis.Inthistechnique,theelectricfieldisappliedforashorttimeorpulseandthedirectionofthefieldchangeswitheachpulse.

Inpulse-fieldgelelectrophoresis,pulsesofelectricityfromdifferentdirectionsareusedtoseparateverylargepiecesofDNAinagarosegels.

VerylargepiecesofDNAcanbeseparatedbypulse-fieldgelelectrophoresisinagarosegels.

Beforeitisruninagel,DNAisusuallycutintosmallerpieces.

WhatisusedtocutDNA?

restrictionenzymesorrestrictionendonucleases

B.Restrictionenzymes

Restrictionenzymes（RE）:

endonucleasespurifiedfrombacteriathatcutdouble-strandedDNAatspecificnucleotidesequences.

Eachrestrictionenzymesisnamedbytheorganismfromwhichitcame.

Forexample,EcoRIcamefromEscherichiacoliRY13.

EachREcutsataspecificnucleotidesequence.Thesequenceisusually4-8bpslongandaninvertedrepeat.

EcoRIcutsatGAATTC

CTTAAG

ThisDNAiscut6timeswithEcoRItoproduce7DNAfragmentsofvaryingsizes.

ManyrestrictionenzymesmakeastaggeredcutinthetwostrandsofDNA,leavingsinglestrandedoverhangs.Theseoverhangsareoftencalledstickyends.

Thereare3kindsofendsproducedbyrestrictionenzymecutting.

1.5’overhangs

2.3’overhangs

3.bluntends

WhentherestrictionenzymedoesnotmakeastaggeredcutintheDNA,theendsarecalledbluntends.

Thereareseveralhundreddifferentrestrictionenzymesthatrecognizeavarietyofdifferentsequences.

Thesequencerecognizedbyarestrictionenzymeiscalledarestrictionsite.

ThepiecesofDNAthatareproducedbycuttingwithrestrictionenzymesarecalledrestrictionfragments.

Oftenthesequencethatisrecognizedbyarestrictionenzymeisaninvertedrepeatorpalindrome.

Apalindromeisaninvertedrepeatthatreadsthesameforwardasbackward.

Palindrome:

invertedrepeat

Examples:

“Racecar”readsthesameforwardasbackward

“Gohangasalami.I’malasagnahog.”

Canyouthinkofanyotherpalindromes?

“Level”,“Rotor”and“Radar”areallpalindromes.

Thefollowingaresomecommonrestrictionenzymes.

ThestickyendsoftheDNAcanbeusedtojointwopiecesofDNA.BluntendsofDNAcanalsobejoined,butitismoredifficult（theligationreactiontakeslonger）.

RecombinantDNA:

twopreviouslyseparatepiecesofDNAlinkedtogether

Whatisthenamefortheenzymethatformsacovalentbondbetween2piecesofDNA?

DNAligase:

anenzymethatformscovalentbondsbetweentheendsofDNAstrands

C.DNAcloning

DNAcloningisisolatingtheDNAweareinterestedinandinsertingitintoavectorthatwillreplicateandmakemanycopiesoftheDNA.

1.Vector:

apieceofDNA（plasmidorphage）thatservesasacarrieringenecloningexperiments.

Acloningvectorhas3components:

1.anoriginofreplication

2.adominantselectablemarker

3.auniquerestrictionendonucleasesequence

1.Vectorsmusthaveanoriginofreplication.

Whatisanoriginofreplication?

Originofreplication:

asiteinDNAwherereplicationbegins

2.dominantselectablemarker

2.Avectorcontainsadominantselectablemarker.

Thisisusuallyresistancetoanantibiotic.Afterthevectoristransferredintobacteria,thepresenceofthevectorcanbeselectedforbygrowingthebacteriainthepresenceofanantibiotic.

Aplasmidvectorwastransferredintothesebacteriaandthentheyweregrownonaplatecontainingtheantibiotictetracycline.Onlybacteriathatcontainthevectorcangrowinthepresenceoftetracycline.

3.auniquerestrictionendonucleasesequence

ThecloningvectormusthaveaDNAsequencethatisrecognizedbyarestrictionendonuclease,butitmusthaveonly1site.Ifithas2ormoresites,thecloningvectorwillbecutintopieces.

DifferentkindsofvectorsareusedbasedonthesizeofDNAthatwillbeinsertedintothevector.

Therearedifferentkindsofvectors.Wewilllookat:

1.plasmids

2.bacteriophage

3.bacterialartificialchromosomes（BACs）

Whatisaplasmid?

1.plasmid

1.plasmid:

anextrachromosomalpieceofDNAthatreplicatesindependentlyofthebacterialchromosome

Thereare3methodsthatcanbeusedtotransferDNAintobacteria:

transformation

transduction

conjugation

transformation

Oftenplasmidsaretransferredintobacteriabytransformation.

TomakethebacteriacompetenttoreceivetheDNA,thebacteriaaretreatedwithCa++ionsorasolutionofCaCl2.

TransformationofbacteriaisveryinefficientandonlyasmallpercentageofthebacteriawilltakeuptheDNA.

Alternatively,highvoltagecanbeusedtomakesholesinthemembranessothattheDNAcanenter.Thisiscalledelectroporation.

2.Bacteriophage

bacteriophage:

bacterialvirusesareusefulforcloning

BacteriophagecanbedesignedtocontainpiecesofDNAthatyouareinterestedin.Whenthebacteriophageinfectsbacteria,theDNAwillbetransferredtothebacteriabytransduction.BecausethephageinjectstheDNAinthebacteria,thetransferofDNAismuchmoreefficientthattransformation.

3.Bacterialartificialchromosomes

Bacterialartificialchromosomes:

avectorbasedontheE.coliFplasmid.BACsarecapableofholdinginsertsofupto300kb（averagesizeof150kb）

2.Selection

WhenwetransferDNAintobacterianotallofthebacteriawilltakeuptheDNA.

WeneedawaytoseparatethebacteriathathavetheDNAfromthosethatdonot.

selection:

addingageneforantibioticresistancetoaplasmidsothatbacteriathatcontaintheplasmidcangrowonmediacontainingtheantibiotic,butbacteriathatdonothavetheplasmiddie.

NotalltheplasmidswillcontaintheDNAthatwewanttoinsert.SomeoftheplasmidswillreligatetothemselveswithouttheinsertDNA.

ToscreenforclonesthatcontaintheinsertedDNA,wecantestthebacteriafortheirabilitytogrowonampicillin.

Thevectorhasagenethatmakesbacteriaresistanttoampicillin.

ThecutsiteforPstIisinsidetheampicillinresistancegene.Clonesthatcontaininsertwillnotgrowonampicillin.

InsertingDNAintotheampicillingeneinactivatesit.ThebacteriathathaveinsertedDNAwillnolongerberesistanttoampicillin.

Velvetisatypeofsoftcloththathasfibersthatstickout.

4.Multiplecloningsite

Mostvectorsusedtodayhavemanychoicesfortherestrictionenzymesyoucanusetocutthembecausetheyhavemultiplecloningsites.

Multiplecloningsite:

aregioninmostcloningvectorsthatcontainsseveralrestrictionsitesthatcanbeusedforinsertingforeignDNA.

5.DirectionalCloning

Multiplecloningsitesallowforthevectortobecutwith2differentrestrictionenzymes.

ThisiscalleddirectionalcloningbecausetheinsertedDNAcanbeinthevectoronlyinonedirection.

Directionalcloning:

DNAinsertandvectormoleculesaredigestedwithtwodifferentrestrictionenzymestocreatenoncomplementarystickyendsateitherendofeachrestrictionfragment.Thisallowstheinserttobeligatedtothevectorinaspecificdirectionandpreventsthevectorfromreligatingtoitself.

D.Hybridization

WhathappenswhenDNAisheated?

HeatingDNAcancausethetwostrandstoseparate.TheDNAissaidtodenature.

WhathappenstoDNAthatiscooled?

SlowlycoolingdenaturedDNAallowsittorenatureorhybridize.

TwopiecesofDNAthatarecomplementarywillbasepairwitheachotherundertheproperconditions.ThisallowsustofindaparticularsequenceofDNAbymakingaprobe—apieceofDNAthatiscomplementarytotheDNAwearetryingtoisolate.

AprobehasaknownDNAsequence.Thiscanbeapurifiedfragmentoritcanbechemicallysynthesized.

TheprobeisusedtosearchamixtureofDNAmoleculesforthosethatcontaincomplementarysequences.

Theprobeislabeledsoitcanbelocated.

Theprobecanbelabeledwithradioactivityorafluorescentmolecule.

Thereare2waystolabelaprobe.

TheendoftheDNAprobecanbelabeled

LabeledbasepairscanbeincorporatedintotheDNAstrandoftheprobe.

Wewilllookat4techniquesthatusehybridization:

1.Southernblotanalysis

2.northernblotanalysis

3.insituhybridization

4.DNAmicroarrays

1.Southernblotanalysis

InSouthernblotanalysisaradioactiveprobeishybridizedtoDNA.