Are Enzymes Required for the Extraction and Isolation of DNA in Conjunction with Mechanical Homogenization?

Abstract

Enzymes are biological catalysts that play a role in the isolation of nucleic acids from tissue and microbial sample types. In 1973, Maria Gross‐Bellard was one of the first researchers to use Proteinase K in the isolation of DNA from mammalian cells1. Today, enzymes are commonly provided in DNA isolation kits to enable cellular and organelle disruption and for the removal of contaminating proteins. DNA kit enzymes vary based on the target sample. While Proteinase K is commonly used in the isolation of DNA from mammalian cells and tissues, lyticase and lysozyme are enzymes used to degrade the cell walls of yeast and bacteria and are frequently included in microbial DNA isolation kits. While effective, enzymes represent a significant percentage of DNA kit cost and require special storage and handling conditions. While standard lysis buffers coupled with enzymes can be used to disrupt soft tissues and cells, mechanical homogenizers are routinely employed for disruption of tougher tissues and cells with robust cell walls. In these cases, are enzymes necessary to obtain high concentrations of DNA?In this study, DNA extractions were performed using commercially available DNA extraction kits from three sample types: bacteria, yeast and tissues. DNA was extracted per the kit manufacturer's instructions in conjunction with mechanical homogenization via bead milling. The analysis was then repeated without the use of enzymes. DNA concentration and quality was evaluated by spectrophotometry and electrophoresis respectively for each sample type and processing method. High quality DNA was recovered with and without the use of enzymes. Although samples with enzyme digestion brought higher yields of DNA, the integrity of DNA was equivalent in samples without the enzyme digestion. Enzyme requirement in DNA isolation is based on down stream analysis.