Chapter 11 - Characterization of nucleic acids and proteins

Abstract

Manipulative enzymes can be used to process DNA or RNA for 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. Nucleic acid manipulative enzymes play important roles in downstream molecular diagnosis. The ability of one single-stranded nucleic acid to form a double helix with another single-stranded nucleic acid through a complementary base sequence is known as hybridization. This is the backbone of the current diagnostic molecular techniques, and many important nucleic acid hybridization-based diagnostic techniques have been developed. For example, blotting techniques have been developed for DNA and RNA analysis including Southern blot, Northern blot, dot blot, and slot blot. These blotting techniques (as well as array-based hybridization techniques) are commonly used for clinical analysis. 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 specific proteins, immunodetection methods have been developed. Here, we also discuss several important techniques for current diagnostic applications, including Western blot, ELISA, and protein microarrays.