Shaken, not stirred – breaking up with traditional DNA fragmentation

- - -

- - -

- - -

- - -

no conflict of interests

Next generation sequencing has become an integral part of modern molecular diagnostics. Regardless of the sequencing method employed fragmentation of DNA is an indispensable step within most library preparation protocols. To avoid the obstacles of enzymatic treatment, usually applied in high-throughput procedures, the objective was to establish a reliable, cost-efficient and safe method of mechanical fragmentation in a 96-well format.

High molecular weight DNA isolated from either peripheral blood or saliva were utilised as starting materials. The method is based on fragmentation by shear forces generated during the implosion of cavitation bubbles. A vibrating mill with corresponding glass beads was deployed to generate these bubbles. The fragmentation degree was monitored using agarose gel electrophoresis.

The results demonstrate that a high-throughput homogeneous fragmentation of human DNA across the entire 96-well plate is feasible using a vibrating mill. The digestion time, oscillation rate, material, geometry, size and quantity of the glass beads used as well as the plates employed can be optimised depending on the desired fragment size range. It is important to note that the sample itself also exert an influence on the fragmentation result, due to parameters such as volume, viscosity, pH value, presence of salts and temperature.

The use of a vibrating mill for mechanical DNA fragmentation represents an innovative approach that is in direct competition with established conventional methods. The major advantage of this method is that no enzymes are employed, obviating the necessity for the creation of a corresponding milieu. Furthermore, no subsequent inactivation and any requisite buffer exchange is required. Therefore, DNA fragmentation utilising a vibrating mill is a safe, time- and cost-efficient method.