Flow cytometric investigation on degradation of macro-DNA by common laboratory manipulations

Abstract

The degree and characteristics of physical degradation of macro-DNA molecules by common laboratory manipulations are reported. With linearized lambda-phage viral DNA as the model DNA, fragmentation of macro-DNA by various indispensable laboratory manipulations were investigated using a high sensitivity flow cytometric setup. Investigated manipulations included pipetting, vortexing, rocking, freezethawing, ultrasonication and ultrafiltration. "Exhaustive counting" of the intact lambda DNA molecules following such manipulations enabled a quantitative assessment of the resulting fragmentation, which also revealed the type of degradation reflected in the fragmentation patterns. The use of high sensitivity flow cytometry was especially suited to investigate the degradation of dilute DNA solutions that may not be suitable for analysis using traditional methods. Notable findings of this study included: the boarderline-size of DNA chains in terms of susceptibility to shear stresses by such manipulations; discernable instability of nicked DNAs; shattering-fragmentation of DNAs by freezethawing or ultrasonication; effectiveness of some protection media; marked "self-protection effect" of concentrated DNA solutions. These findings support and refine our traditional knowledge on how to maintain the physical integrity of macro-DNA molecules against inevitable laboratory manipulations.

A comprehensive model of DNA fragmentation for the preservation of High Molecular Weight DNA

“…Calculations by Vanapalli et al have proven that the pull of a DNA molecule stuck in two vortices at the same time is sufficient to break the strand in half and that this is the primary cause of fragmentation under most hydrodynamic conditions (Vanapalli et al, 2006). During sample processing fragmentation occurs over time until all DNA molecules have been degraded to a length where all fragments are short enough to remain within a single eddy, which is consistent with experimental results with commonly used laboratory equipment, such as vortex mixers and pipettes (Yoo et al, 2011), and results suggest that the breakage rate is a function of the shear rate rather than the shear stress (Bowman and Davidson, 1972;Meacle et al, 2007).…”

DNA stability: a central design consideration for DNA data storage systems

“…Several studies have repeatedly frozen and thawed DNA samples, generally observing exponential degradation. An approximately 10% degradation of lambda DNA in Tris-EDTA buffer was observed after 1 freeze-thaw, and 75% degradation was observed after 20 freeze thaws 39 . An exponential fit modeled this process relatively closely (% intact DNA = 0.9484*e −0.068*freeze-thaws ).…”

“…With a relatively rapid single pipetting action through a 1 mL tip, 70% of long lambda phage DNA was fragmented. Slower and gentler pipetting still led to more than 50% of lambda DNA being fragmented 39 . This is somewhat concerning given liquid handling often uses even smaller 200, 20, and 2 μL tips that would result in higher shear forces.…”

Serial profiling of cell-free DNA and nucleosome histone modifications in cell cultures

“…Therefore, the level of cfDNA association with other protein complexes should also be assessed; (4) Necrotic DNA may be digested into DNA fragments that resemble apoptotic DNA; (5) Extracellular DNA degradation and digestion activities may fluctuate over time (e.g., endonucleases can be released by dead or dying cells, and activity may increase in response to an increase in cfDNA levels 81 ); (6) cfDNA may be taken up by cells or adhere to cell surfaces, and there may differences in the behavior of apoptotic and necrotic DNA in this respect; (7) Various processes can inhibit the release of DNA, while other processes can speed up the degradation of DNA; (8) In addition to apoptosis and necrosis, various other forms of cellular demise can contribute to cfDNA release; (9) Different types of cell death result in the presence of differently sized cfDNA populations. As different preanalytical steps differently affect different cfDNA size populations (e.g., size bias of extraction kits 29,82 and mechanical vulnerability of long fragments during processing 83 ), this can potentially affect cfDNA measurements 29 ; (10) It is difficult to determine the amount of apoptotic and necrotic cells that have completely disintegrated and/or newly emerged between incubation periods; (11) Apoptosis and necrosis can occur in the same cell 76 .…”