1、 HYPERLINK mailto: Chapter21TechniquesofMolecular HYPERLINK mailto: Chapter21TechniquesofMolecularI.Gel GelSeparate DNA and RNA molecules according to size, shape and topological Stainingthegelwithfluorescent dyes, suchas Polyacrylamide and agarose Pulsed-fieldgelelectrophoresisMigrationspeed:superc

2、oiled linear ElectrophoresisisalsousedtoseparateRNAcan be treated with reagents, such as glyoxal ,that react with the RNAin such a way as to prevent the formation of base pairs.II.Restriction endonucleasesDef. Enzymes typically recognize short target sequences, usually palindromic, and cut at a defi

3、ned position within those sequences.Eg.EcoRI Targetsite Nature of DNA ends Cleavage activity Methylationsensitivity.DNAUsedtoidentifyspecificDNAProbe: a piece of defined sequence,either a purified fragment or a chemically synthesized DNA molecule, used to search mixtures of nucleic acids for molecul

4、es containing a complementary sequence.( defined, labeled)Twomethodsforlabelingaddingalabel tothe endofanintact DNAmolecule.Eg. Labeling by incorporation (using PCR), synthesizing new DNAin the presence of a labeled precursor.NicktranslationlabelingofRandomlabelingof DNAthroughDNaseelectrophoretical

5、lyseparatedProcess: electrophoresis - blotting - membrane - hybridization - visualization Northern blot hybridization-used to identify a particular mRNA in a population of RNAs.Detection: HYPERLINK mailto: .DNA DNA cloning: the ability HYPERLINK mailto: .DNA DNA cloning: the ability to construct rec

6、ombinant DNA molecules and maintain theme in cells.Vectors and insert DNA Keys:therestrictionenzymesCharacteristicsofvectorOrigin of replication Selectable marker MultiplecloningsitesEasytobeisolatedfrom hostExpressionCloningin aplasmid Fig.21-VectorDNAcanbeintroducedintohostorganismsbyDNA library:

7、a population of identical vectors that each contains a different DNA Fig.21-8 constructionofaDNAGenomic libraries: a population of identical vectors contains total genomic DNA cleaved with a restriction enzyme.cDNA library: a collection of vectors contains cDNAs converted from mRNA Fig.21-9 construc

8、tionofacDNAHybridizationcan beused toidentifya specific cloneinaDNAColony hybridization: the process by which a labeled DNAprobe is used to screen a library .AmplifiesDNAsbyrepeatedroundsofDNAreplicationinvitro. Material: DNA polymerases, ssDNA template, dNTP, primer Process: denaturation, primer an

9、nealing, polymerization. Nucleotide sequencing Chain-terminationmethodddNTPS as substrates, prevent the addition of further nucleotides causing elongation to terminate.Mostextractpreparation andproteinpurification isperformedatSeparatingproteinsusingcolumnColumn chromatograph: protein fractions are

10、passed through glass columns filled with appropriately modified small acrylamide or agarose beads.ThreebasicFig.21-1.Ion-exchangechromatography HYPERLINK mailto: Gel-filtrationchromatography(sizeand HYPERLINK mailto: Gel-filtrationchromatography(sizeandAffinity chromatography Immunoaffinitychromatog

11、raphyEpitopes: a sequence of 7-10 amino acids recognized by an antibody. Immunoprecipitation(IP) is used to rapidly purify proteins or protein complexes from crude extracts.SeparationofproteinsonpolyacrylamideSodiumdodecylsulfate(SDS)gives theproteinanegativeCoomassiebrilliantAntibodies are used to

12、visualize electrophoretically separated proteins. Immunoblotting: by which and individual protein is visualized amid thousands of other proteinsProteinmoleculescanbedirectly1. Edman degradation: a chemical reaction in which the amino acids residues are sequentially released from the amino terminus o

13、f a polypeptide chain.Process: Modify N-terminal with PITC,cleave off identify the released amino acid by HPLC.N-terminal by acid 2. Tandem mass spectrometry: a method in which the mass of very small samples of a material can be determined with great accuracy.Process: digest target protein into shor

14、t peptides, subject mixture to MS, capture and fragment individual peptides into all components peptides, determine mass, collect and deconvolution.NUCLEICACID-PROTEINTheelectrophoreticmobilityofDNAisalteredbyproteinbinding. Electrophoretic mobility-shift assy(EMSA)Usedtodetect bindingtossDNAortoRNAandtomonitortheassociation ofmultiple proteins with the same DNA.DNA-boundproteinprotectstheDNAfromnucleasesandchemicalNuclease protection footprintingrevealthebindingposition Chemical protection footprintingC