分子生物学实验室技术ppt课件.ppt

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1、Chapter3TechniquesofMolecularBiologyMethodsfortheanalysisofDNAandRNA;Thelarge-scaleanalysisofgenomicDNA;Analysisofproteins;Large-scaleanalysisofproteins;Theanalysisofnucleicacid–proteininteractions.1Thelivingcellisanextraordinarilycomplicatedentity,p

2、roducingthousandsofdifferentmacromoleculesandharboringagenomethatrangesinsizefromamilliontobillionsofbasepairs.Understandinghowthegeneticprocessesofthecellworkrequiresavarietyofchallengingexperimentalapproaches.Theseincludemethodsforseparatingindivid

3、ualmacromoleculesfromthemyriadmixturesfoundinthecellandfordissectingthegenomeintomanageablysizedsegmentsformanipulationandanalysisofspecificDNAsequences,aswellastheuseofsuitablemodelorganismsinwhichthetoolsofgeneticanalysisareavailable.Thesuccessfuld

4、evelopmentofsuchmethodshasbeenoneofthemajordrivingforcesinthefieldofmolecularbiologyduringthelastseveraldecades.2Figure7-1NUCLEICACIDS:BASICMETHODSGelElectrophoresisSeparatesDNAandRNAMoleculesaccordingtoSize3Figure4-28Figure4-274Figure7-2Pulsed-field

5、gelelectrophoresis5Figure7-3RestrictionEndonucleasesCleaveDNAMoleculesatParticularSitesRestrictionenzymesusedinmolecularbiologytypicallyrecognizeshort(4–8bp)targetsequences,usuallypalindromic,andcutatadefinedpositionwithinthosesequences.EcoRI,sonamed

6、becauseitwasfoundincertainstrainsofEscherichiacoliandwasthefirst(I)suchenzymefoundinthatspecies.Thisenzymerecognizesandcleavesthesequence5′-GAATTC-3′.(BecausethetwostrandsofDNAarecomplementary,weneedspecifyonlyonestrandanditspolaritytodescribearecogn

7、itionsequenceunambiguously.)6Figure7-37Figure7-4Recognitionsequencesandcutsitesofvariousendonucleases8Figure7-6DNAHybridizationCanBeUsedtoIdentifySpecificDNAMolecules9Figure7-710IsolationofSpecificSegmentsofDNADNACloningTheabilitytoconstructrecombina

8、ntDNAmoleculesandmaintainthemincellsiscalledDNAcloning.ThisprocesstypicallyinvolvesavectorthatprovidestheinformationnecessarytopropagatetheclonedDNAinthereplicatinghostcell.KeytocreatingrecombinantDNAmoleculesaretherestrictionenzymesthatcutDNAatspeci