Chapter 10 - Characterization of Nucleic Acids and Proteins

Abstract

Once pure samples of nucleic acids have been prepared, manipulative enzymes can be used to process DNA or RNA for further diagnostic analysis. Commonly used nucleic acid manipulative enzymes can be grouped into four broad groups, depending on the type of reaction that they catalyze. (1) Nucleases are enzymes that cut, shorten, or degrade nucleic acid molecules. (2) Ligases join nucleic acid molecules together. (3) Polymerases make copies of molecules. (4) Modifying enzymes remove or add chemical groups. As such, nucleic acid manipulative enzymes play important role in nucleic acids analysis. Furthermore, the ability of one single-stranded nucleic acid to form a double helix with another single-stranded nucleic acid through complementary base sequence is known as hybridization. This is the backbone of the current diagnostic molecular techniques. On the other hand, identifying and measuring specific proteins in complex biological mixtures (such as blood) through antibody binding have also been important in diagnostic practice. To identify nucleic acids and proteins, blotting methods have been developed. These techniques are fairly simple and usually consist of four separate steps: separation by electrophoresis, transfer, hybridization, and visualization. Three main blotting techniques have been developed for DNA, RNA, and protein analysis, and are called Southern, Northern, and Western blotting, respectively. These blotting techniques (as well as array-based hybridization techniques) are commonly used for clinical analysis. Because combination of the above applications has spawned powerful diagnostic techniques, we discuss details of the aforementioned techniques in this chapter.