Chapter 7 - Quantification and analysis of nucleic acids

Abstract

Nucleic acid-based molecular diagnosis is a promising laboratory technique because of its ability to identify disease and/or pathogens accurately. Although nucleic acid extraction and purification are the critical steps, the accurate quantification of nucleic acid is extremely important for the downstream modern molecular diagnosis application. Downstream applications often have a specific target or window of nucleic acid concentration that is required in order to perform optimally. While most automated extraction systems ensure that DNA purification is optimized for most protocols, there is variability in systems, specimen types, and microbes. Sometimes, it is essential to know the exact concentration of a nucleic acid sample. Knowing the accurate concentration can help to prevent unnecessary consumption, enhance reproducibility, enhance amplification of difficult targets, and standardize downstream protocols, such as sequencing. Inaccurate quantification can increase variability in downstream assays, which leads to reduced confidence in results. Gel electrophoresis has been commonly used in the separation and analysis of nucleic acids. Therefore, molecular separation can help to visualize the quality and quantity of nucleic acids. Furthermore, the molecular separation can also be used to detect diseases caused by bacteria, viruses, or fungi. Here, we discuss commonly used techniques to quantify and analyze nucleic acid molecules. The principle of gel electrophoresis as well as the applications of different electrophoresis techniques are also discussed in this chapter.