Background

Many biotechnological applications, such as recombinant protein purification, require lysing the cells releasing host oligonucleotides in the process. However, intact oligonucleotides in crude cellular lysates rapidly increase the viscosity of biological samples, which can complicate sample handling and downstream processes (Prazeres et al., 1999), or may completely impede routine analytical methods, such as SDS-PAGE (Kurien and Scofield, 2012). While some cell lysis methods such as sonication lead to some DNA shearing and decreased viscosity, the addition of purified nucleases that hydrolyze intact oligonucleotides in lysates is a more commonplace solution in industrial settings (Boynton et al., 1999). Alternatively, we recently developed a method using an E. coli autolysis/autohydrolysis strain where, after cell growth and autoinducible protein expression, cell autolysis and DNA/RNA autohydrolysis can easily be triggered upon addition of a 0.1% detergent and a freeze-thaw cycle. This method leverages a chromosomally integrated genetic circuit that encodes the co-expression of a lysozyme and a nuclease (Menacho-Melgar et al., 2020).

In this context, a method to quantify oligonucleotide hydrolysis may be desirable. DNA quantification methods, such as fluorescence-based and qPCR, may be used for this purpose but require expensive equipment and reagents and do not differentiate based on DNA fragment size (Nakayama et al., 2016). A simpler alternative is to directly measure the viscosity of the lysates. While the viscosity of lysates is dependent on various factors, intact oligonucleotides are the major contributor. Still, viscosity can only be used to measure oligonucleotide hydrolysis within equivalent samples and not to make comparisons across different strains or cell cultures of different cell densities. Importantly, this method will not work in lysates obtained using sonication, as this shears the DNA. Here, we provide a detailed protocol for measuring DNA hydrolysis using viscosity measurements, only requiring the use of common laboratory supplies and a cell phone camera.