Harpers

MOLECULAR GENETICS, RECOMBINANT DNA, & GENOMIC TECHNOLOGY / 403Ampicillinresistance geneTetracyclineresistance geneEcoRIEcoRIHindIIIBamHITetracyclineresistance genePstIHindIIIBamHIPstISalICut open withPstIPstISalIAmp rTet rThen insertPstI-cut DNAAmp sTet rHost pBR322Chimeric pBR322Figure 40–4. A method of screening recombinants for inserted DNA fragments. Using the plasmid pBR322, apiece of DNA is inserted into the unique PstI site. This insertion disrupts the gene coding for a protein that providesampicillin resistance to the host bacterium. Hence, the chimeric plasmid will no longer survive when platedon a substrate medium that contains this antibiotic. The differential sensitivity to tetracycline and ampicillin cantherefore be used to distinguish clones of plasmid that contain an insert. A similar scheme relying upon productionof an in-frame fusion of a newly inserted DNA producing a peptide fragment capable of complementing aninactive, deleted form of the enzyme β-galactosidase allows for blue-white colony formation on agar plates containinga dye hydrolyzable by β-galactoside. β-Galactosidase-positive colonies are blue.Blotting & Hybridization Techniques AllowVisualization of Specific FragmentsVisualization of a specific DNA or RNA fragmentamong the many thousands of “contaminating” moleculesrequires the convergence of a number of techniques,collectively termed blot transfer. Figure 40–5illustrates the Southern (DNA), Northern (RNA), andWestern (protein) blot transfer procedures. (The first isnamed for the person who devised the technique, andthe other names began as laboratory jargon but are nowaccepted terms.) These procedures are useful in determininghow many copies of a gene are in a given tissueor whether there are any gross alterations in a gene(deletions, insertions, or rearrangements). Occasionally,if a specific base is changed and a restriction site is altered,these procedures can detect a point mutation.The Northern and Western blot transfer techniques areused to size and quantitate specific RNA and proteinmolecules, respectively. A fourth hybridization technique,the Southwestern blot, examines protein•DNAinteractions. Proteins are separated by electrophoresis,renatured, and analyzed for an interaction by hybridizationwith a specific labeled DNA probe.Colony or plaque hybridization is the method bywhich specific clones are identified and purified. Bacteriaare grown on colonies on an agar plate and overlaidwith nitrocellulose filter paper. Cells from each colonystick to the filter and are permanently fixed thereto byheat, which with NaOH treatment also lyses the cellsand denatures the DNA so that it will hybridize withthe probe. A radioactive probe is added to the filter,and (after washing) the hybrid complex is localized byexposing the filter to x-ray film. By matching the spoton the autoradiograph to a colony, the latter can bepicked from the plate. A similar strategy is used to identifyfragments in phage libraries. Successive rounds ofthis procedure result in a clonal isolate (bacterialcolony) or individual phage plaque.All of the hybridization procedures discussed in thissection depend on the specific base-pairing propertiesof complementary nucleic acid strands described above.Perfect matches hybridize readily and withstand hightemperatures in the hybridization and washing reac-