Section 10.4   

Tools of Molecular Biology:

Restriction Digestion and Gel Electrophoresis of DNA

Restriction Digestion

Biochemists often take DNA from an organism's genome, such as a gene that codes for a protein, and paste this DNA of interest into a circular piece of DNA called a plasmid for use in the lab. This process, called sub-cloning, involves cutting double-stranded DNA with enzymes termed restriction endonucleases that recognize and cut the DNA only at very specific sequences. Both the gene that is being subcloned and the plasmid are digested with restriction enzymes, making it easy to ligate the two together and generate a recombinant plasmid containing the inserted gene.

Gel Electrophoresis

Restriction site map for the plasmid

Agarose or polyacrylamide gels are used to separate DNA fragments based on size. This process is called gel electrophoresis. The DNA is placed in a gel matrix and then subjected to an electric field. Because DNA in negatively charged at a neutral pH it migrates toward the positive electrode. The gel retards the movement of the DNA, as the molecules must navigate through its complex matrix. Because smaller molecules can maneuver more easily than large ones, smaller fragments of DNA migrate more quickly than larger fragments.

Interactive Excercise

In the following demonstration you can choose which restriction enzymes to use to cut your plasmid. The cut DNA will be run in lane 1 of the gel. A size standard consisting of fragments of size 3000 bp, 2000 bp, 1500 bp, 1000 bp, 500 bp, and 100 bp will be run in lane 2 so that you can determine the length of your digested fragments. Remember smaller DNA fragments will run more quickly than larger fragments. You can cut the plasmid with more than one enzyme at a time.

Select the enzymes and press the start button