Chapter 6 - Extraction and purification of nucleic acids

Abstract

Genes are the principal functional units of heredity and the specific sequences of nucleic acid that they contain encode most of the proteins required for organism function. Different diseases and cancers may result from genetic disorder within cells, in that the relevant genes contained in such cells lose their ability to carry out normal physiological metabolic processes. Molecular diagnostics is a powerful technique for detecting genetic abnormality and it can also detect the presence of pathogens with high sensitivity. As such, nucleic acid (DNA and RNA)-based molecular diagnosis is a promising laboratory technique. However, one of its disadvantages is the inevitable purification and detection of nucleic acids from other contaminated entities. To avoid the downstream error, the extraction and purification of nucleic acids are the most critical steps in the process. This is because they are the key components for current high-throughput nucleic acid analysis. Nucleic acids can be isolated from any biological material such as living or conserved tissues, cells, virus particles, or other samples for analytical or preparative purposes. In general, successful nucleic acid purification requires four important steps: effective disruption of cells or tissue, denaturation of nucleoprotein complexes, inactivation of nucleases, and removal of contamination. In the end, the target nucleic acid should be free of contaminants including protein, carbohydrate, lipids, or other nucleic acids. The quality and integrity of the isolated nucleic acid will directly affect the results of all succeeding downstream diagnoses. Based on cost-effectiveness, time-efficiency, and technical instruments, the best-suited methods should be chosen based on their purpose. Here, we discuss different strategies to prepare contaminant-free nucleic acids from different sources.