A Simple Mixer-Mill Method for Fragmentation of Genomic DNA Templates for Droplet Digital PCR

Droplet digital PCR (ddPCR) has gained popularity for applications such as CNV assessment, rare target quantitation and gene expression analysis. In procedures involving genomic DNA, template viscosity can be challenging because it may interfere with droplet formation, potentially decreasing the accuracy of quantitative measurements. Fragmentation has been recommended as a means to reduce sample viscosity and may also enhance assay performance by improving template accessibility. Researchers have achieved fragmentation of ddPCR templates using a number of techniques including enzymatic digestion, hydrodynamic shearing, nebulization, sonication and passage through a biopolymer spin column (QiaShredder). Although effective, some of these approaches require specialized instrumentation and all will add considerable time and expense to analyses involving large numbers of samples.

We propose agitation together with a steel ball in a mixer-mill as a simple means to achieve template fragmentation and improve ddPCR performance. We extracted DNA from F₁ hybrid barley leaves and compared template performance in ddPCR (RainDrop, RainDance Technologies) of heterozygous targets following fragmentation using either a Hydroshear Plus (Digilab) or TissueLyser II (Qiagen) or with no treatment. Droplet generation failed with untreated template whereas templates sheared by either technique yielded allele ratio estimates consistently very close to expectations for heterozygous loci (50:50). Higher occupancy rates were attained using mixer-mill treated template, suggesting that there may be greater target loss with the Hydroshear method. The mixer-mill approach also offers high throughput; up to 192 samples may be processed at one time, with an instrument already commonly found in many molecular biology laboratories.