Deep eutectic solvents for direct DNA extraction and their seamless integration into the quantitative polymerase...

Advancements in nucleic acid (NA) analysis rely on the development of efficient extraction and amplification techniques that deliver high sensitivity, reliability, and streamlined design. Traditional organic solvents used in NA extraction often present challenges related to their toxicity, volatility, and limited biocompatibility. Deep eutectic solvents (DESs) have emerged as a promising class of solvents due to their customizable properties and potential for biomolecular applications. This study addresses a fundamental challenge in molecular diagnostics by evaluating DESs as both extraction media for NAs and direct additives within quantitative polymerase chain reaction (qPCR) assays, thereby eliminating the need for intermediate purification.

In this study, fifty-three DESs from seven distinct classes were evaluated for their compatibility with qPCR and utility as extraction solvents for NA analysis. Incorporating DESs directly into qPCR revealed key benefits like enhanced DNA amplification efficiency and the ability to tailor conditions for diverse extraction requirements. Two DNA sequences, a 98 base pair (bp) BRAF and 121 bp uidA fragment, were analyzed to assess their amplification in the presence of DESs with varied chemical composition. A subset of thirty thermally stable DESs proved promising under simulated PCR conditions. However, when the DESs were added directly to qPCR, six permitted successful amplification, while the others interfered with amplicon generation. Three of these DESs yielded qPCR efficiencies of 97.22 ± 5.41 % for the 121 bp uidA fragment. Findings revealed that the nature of hydrogen bond acceptor and donor play a crucial role in maintaining qPCR enzymatic activity and preserving NA integrity.

This work is the first to systematically evaluate a broad range of DESs for integrated NA extraction and direct amplification with qPCR. The findings offer valuable insight into the DES structure-function relationships that govern assay compatibility. By eliminating traditional purification steps, DESs can simplify workflows and increase throughput for molecular diagnostics. This platform opens new opportunities for the development of automated, field-deployable NA testing systems using customizable solvent systems with enhanced analytical performance.