Development of gene-in-plasmid DNA reference materials certified by single-molecule counting

Abstract

The mole, the SI unit for measuring the amount of a substance, was redefined as a fixed number of entities. This definition enables straightforward quantification of substances by counting individual entities. Counting proves particularly effective for quantifying large and discrete biological entities such as DNA, proteins, viruses, and cells, which are challenging to quantify via traditional physical or chemical methods. In this study, we detail our approach to develop gene reference materials certified through single-molecule counting, which enables mole-traceable measurements. We quantified three plasmid DNA constructs, each carrying a specific gene of interest, via single-molecule counting. The resulting values were cross-validated via digital PCR and LC‒MS. Sequence impurities in the certified reference materials were quantified via single-molecule real-time sequencing, whereas fragment impurities were quantified via two-color digital PCR analysis. We precisely accounted for various sources of uncertainty, including measurement precision, weighing, homogeneity, and impurities, when estimating the total uncertainty of the reference materials. In conclusion, a practical format for gene-based DNA reference materials, a measurement method to achieve metrological traceability, and methods for quantifying fragments and sequence impurities were developed and implemented in this study. We anticipate that our gene-based DNA reference materials will serve as valuable higher-order standards for the calibration of other methods or reference materials for DNA quantification in a variety of bioanalytical applications.