The Integration of Focused Ultrasonication, ddPCR, and Flow Cytometry Effectively Estimates Genome Copies per Cell...

Microbiology researchers rely on nucleic acid measurement techniques, such as the quantitative polymerase chain reaction (qPCR) and DNA sequencing, to address diverse scientific and practical challenges. These applications range from detecting microbial contaminants in regenerative medicine and biotherapeutic products to advancing waste remediation, pathogen detection, biosurveillance, and microbiome studies. A critical step in these techniques is DNA extraction, which involves breaking cells to release their DNA as the required input for downstream analyses. The efficiency of this process, known as DNA extraction efficiency (DEE), directly impacts the accuracy of quantitative measurements and, therefore, the interpretation of results. Unfortunately, most DNA extraction methods suffer from suboptimal efficiency that varies across microbial strains, potentially leading to inaccurate results. In this paper, we present a highly efficient DNA extraction protocol leveraging adaptive focused acoustics (AFA) technology to achieve a balance between cell lysis and DNA integrity. Using Escherichia coli as the model organism, the protocol delivers nearly 100% DEE, setting a benchmark for performance. A key innovation in this protocol is the integration of focused ultrasonication, droplet digital polymerase chain reaction (ddPCR), and flow cytometry to estimate genome copies and the corrected DNA extraction efficiency (cDEE), which accounts for the number of genome copies. The proposed protocol addresses the need for an accurate assessment of DEE and DNA quantification, as demonstrated here with E. coli, for various DNA-based techniques, including metagenomic analysis of complex microbial communities and the development of new DNA extraction protocols. This novel protocol addresses a longstanding limitation in microbiological research and has the potential to significantly enhance accuracy and reproducibility across various applications. While there is significant potential for applying this approach, the authors acknowledge that further studies using microorganisms with thicker cell walls will enhance the utility of this framework. However, the knowledge generated in this study can be readily applied and tailored to the specific objectives of individual research groups.