A Recombinant DNA Lab Exercise Based On Racing Endonuclease and Ligase Enzymes

Abstract

Recombinant DNA methods underlie much of modern biochemical and molecular biology research and industrial and pharmaceutical manufacturing. This laboratory exercise introduces these methods using a novel competition experiment in which restriction endonuclease and DNA ligase enzymes are “raced” against each other. The exercise is based on a common protein expression plasmid and the relatively inexpensive restriction enzymes EcoR V and Hind III. The digestions and ligations produce DNA fragments that are well‐resolved by agarose gel electrophoresis. In addition, ATP and DNA ligase concentrations were optimized to enable reproducible control of the number of different types of ligation products formed. This facilitates the identification of the composition of all products formed in the race experiment, including which DNA fragments have been covalently joined to form each ligation product. The distribution of products is further quantified by analysis of gel images using freely available image analysis software. Successful determination of the “winner” of the race experiment requires student conceptual mastery of both the DNA digestion and the ligation processes. The experiment is highly efficient in that the basic methods of DNA digestion and ligation can be covered within a single 3 hour laboratory period. In addition, the direct visualization of ligation products by gel electrophoresis and the quantification of product formation give the exercise a distinct biochemical flavor. This distinguishes it from many molecular biology exercises in which DNA ligation is detected indirectly by changes in the phenotype of transformed bacteria.