DNA Recovery from Droplet Digital™ PCR Emulsions Using Liquid Nitrogen

Abstract

Droplet microfluidics is a technology that enables the production and manipulation of small volumes. In biosciences, the most popular application of this technology is Droplet Digital™ PCR (ddPCR™), where parallel nanoliter-scale PCR assays are used to provide a high sensitivity and specificity for DNA detection. However, the recovery of PCR products for downstream applications such as sequencing can be challenging due to the droplets' stability. Here we compared five methods for disrupting the droplets to recover DNA. We found that rapid freezing in liquid nitrogen results in a clear phase separation and recovery of up to 70% of the DNA content. Liquid nitrogen freezing can thus offer a simple and environmentally friendly protocol for recovering DNA from ddPCR.

Single-cell resolution genetic association analysis of heterogeneous bacterial communities by utilizing droplet digital PCR

“…The taxonomic marker is the sole product in droplets containing non-carrier bacterial cells. C) The produced amplicons are recovered from droplets by breaking the emulsion using liquid nitrogen method (Dutra et al . 2020).…”

“…The amplified bla_16S concatemers were recovered from droplets and enriched before preparing the sequencing libraries. The recovery was done by freeze-thawing the ddPCR emulsion using liquid nitrogen (LN2), a method we described recently (Dutra et al . 2020).…”

“…The taxonomic marker is the sole product in droplets containing non-carrier bacterial cells. C) The produced amplicons are recovered from droplets by breaking the emulsion using liquid nitrogen method (Dutra et al 2020). D) The recovered amplicons are used to construct sequencing libraries and E) sequenced by massive parallel sequencing.…”