The Action of Restriction Endonucleases on Plasmid or Viral DNA

Restriction endonucleases catalyze the hydrolysis of specific phosphodi-ester bonds in double-stranded DNA. These enzymes are often used to linearize a circular plasmid for hybrid DNA construction. They have also found use in the analysis of DNA and the construction of restriction maps. In this experiment, students will incubate various restriction enzymes with plasmid or viral DNA and analyze the product DNA fragments by agarose gel electrophoresis. 

Experiment 14 introduced students to some principles and techniques involved in recombinant DNA research. Specifically, the experiment outlined the replication, isolation, and analysis of bacterial plasmid vehicles for molecular cloning experiments. This experiment describes another tool that is essential in hybrid plasmid construction and analysis-restriction enzyme action. The procedures introduced here have also found widespread use in the analysis and characterization of all DNA molecules. 

Bacterial cells produce many enzymes that act to degrade various forms of DNA. Of special interest are the restriction endonucleases, enzymes that recognize specific base sequences in double-stranded DNA and catalyze 

The site of action of EcoRI is a specific hexanucleotide sequence. Two phosphodiester bonds are hydrolyzed (see arrows), resulting in fragmentation of both strands. Note the twofold rotational symmetry feature at the recognition site and the formation of cohesive ends. The weak base pairing between the cohesive ends is not sufficient to hold the two fragments together. 

Several hundred restriction enzymes have been isolated and characterized. Nomenclature for the enzymes consists of a three-letter abbreviation representing the source (Eco — E. colt), a letter representing the strain (R), and a roman numeral designating the order of discovery. fcoRI is the first to be isolated from E. coli (strain R) and characterized. Table E15.1 lists several other restriction enzymes, their recognition sequence for cleavage, and optimum reaction conditions. 

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EXPERIMENT 15 The Action of Restriction Endonucleases on Plasmid or Viral DNA 431... 

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