Restriction enzymes have become an indispensable tool of molecular researchers over the past
fifty years. This unique group of enzymes function as molecular scissors when applied to nucleic acids, in this case DNA. Forms of this special class of protein have been isolated from several species of bacteria and are employed to make predictable, precise cuts in experimental DNA samples. Currently, over two hundred different restriction enzymes are available to researchers, each keying on a unique nucleotide recognition sequence. The mode of action of a restriction enzyme is to attach and scan a strand of DNA looking for the presence of a specific nucleotide sequence. When found, the DNA is cleaved at two opposite positions of the recognition site on the sugar-phosphate backbone.
During this lab activity, prepared samples of bacteriophage lambda DNA (λ DNA) are used to perform agarose gel electrophoresis. The samples result from λ DNA being digested with different restriction enzymes. The individual digests of this bacteriophage, a 48,502 base pair linear DNA segment, use two common restriction enzymes; EcoRI and HindIII. One of samples is digested by EcoRI while another is cut by HindIII. There is a sample that is formed from a dual digest that has both enzymes acting simultaneously on λ DNA. As a control, an uncut form of λ DNA is also used.
The technique of agarose gel electrophoresis relies on an electric field being applied to a charged gel matrix containing polar molecules. The response of these molecules to the electric field induces them to migrate through the gel to the pole with an opposite charge. The rate of molecular movement in a gel is determined by the charge, shape, structure and weight of the molecule being studied. Negatively charged phosphate groups are present in DNA nucleotides causing the molecule to migrate toward the positive end of the gel chamber. DNA fragments maintain the same charge, shape, and structure, so base pair number differentiates the molecules migration through the gel.
During this exercise, gel electrophoresis will be performed using the E-Gel Pre-Cast Agarose Electrophoresis System with SYBR Safe stain. The Blue Digital Bioimaging System and Logger Pro software will also be used to capture and analyze a digital photograph of your electrophoresis results.
Recommended for Grades 9-16.
Requires: White Digital Bioimaging System, Blue Digital Bioimaging System