Chapter 10 - Recombinant DNA

Abstract

Recombinant DNA is the method of joining two or more DNA molecules to create a hybrid. The technology is made possible by two types of enzymes, restriction endonucleases and ligase. A restriction endonuclease recognizes a specific sequence of DNA and cuts within, or close to, that sequence. By chance, a restriction enzyme's recognition sequence will occur every (¼)n bases along a random DNA chain. DNA fragments generated by digestion with a restriction endonuclease can be joined together again by the enzyme ligase. The likelihood that two DNA molecules will ligate to each other is dependent on the concentration of their ends; the higher the concentration of compatible ends, the greater the likelihood that two termini will meet and be ligated. This parameter is designated by the term i and is defined as the total concentration of complementary ends in the ligation reaction. Recombinant clones can be identified by restriction digest that removes the insert (or a characterized piece of it). The generated fragments are sized by electrophoresis on a gel also carrying a size ladder. The ladder is used to generate a standard curve and regression line equation that can be used to determine the size of the fragments from the recombinant clones.