Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units Somanath Bhat,* Natalie Curach, Thomas Mostyn, Gursharan Singh Bains, Kate R. Griffiths, and Kerry R. Emslie National Measurement Institute, Lindfield, New South Wales, Australia Accurate estimation of total DNA concentration (mass concentration, e.g., ng/μL) that is traceable to the Inter- national System of Units (SI) is a crucial starting point for improving reproducible measurements in many ap- plications involving nucleic acid testing and requires a DNA reference material which has been certified for its total DNA concentration. In this study, the concentrations of six different lambda DNA preparations were deter- mined using different measurement platforms: UV Ab- sorbance at 260 nm (A 260 ) with and without prior sodium hydroxide (NaOH) treatment of the DNA, PicoGreen assay, and digital polymerase chain reaction (dPCR). DNA concentration estimates by A 260 with and without prior NaOH treatment were significantly dif- ferent for five of the six samples tested. There were no significant differences in concentration estimates based on A 260 with prior NaOH treatment, PicoGreen analy- sis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/μL), the results suggest that accurate estimation of DNA mass concentration based on copy number concentration is achievable provided the DNA is fully characterized and in the double-stranded form or amplification is designed to be initiated from only one of the two complementary strands. Accurate estimation of total DNA concentration (mass con- centration) is a critical component for many analytical processes involving nucleic acids, including various DNA manipulations (e.g., digestion, ligation, cloning, sequencing, gene expression, etc.) and molecular analyses (e.g., polymerase chain reaction (PCR), real- time PCR). However, several studies 1-3 have highlighted the lack of standards which leads to difficulties in comparing results from different laboratories or between different methods. DNA refer- ence materials certified for their DNA mass concentration, with an estimated measurement uncertainty and traceable to the International system of units (SI), are currently unavailable, yet are necessary for effective comparison of quantitative measure- ments, method validation, and quality control in routine analysis. Measuring absorbance of light at a wavelength of 260 nm (A 260 ) remains the simplest and most familiar way of estimating DNA mass concentration, 4 based on the Beer-Lambert’s law (A 260 ) εcl), where ε is the molar absorption coefficient, c is the concentration, and l is the path length of the cuvette. 5 The presence of nucleotides, RNA, single stranded DNA (ssDNA), and impurities such as proteins and phenols may significantly affect the final A 260 reading 5,6 and could lead to a bias in DNA concentration estimates. When the molar absorption coefficient for double-stranded DNA (dsDNA) is used, the presence of significant amounts of ssDNA could lead to an overestimation of the DNA concentration. 5,7 For this reason, ISO21571 “Foodstuffs- methods of analysis for the detection of genetically modified organisms and derived products-nucleic acid extraction” 8 recom- mends denaturing dsDNA to its single stranded form using NaOH prior to measuring the A 260 and using the molar absorption coefficient for ssDNA to estimate the DNA concentration. The A 260 method is applicable to DNA concentrations in the range of 2-50 μg/mL. 1,8–10 More recently, fluorescent dyes have been used for quantifying total dsDNA. One such dye is PicoGreen which is more sensitive than the A 260 assay, so suitable for quantifying lower concentra- tions of dsDNA. The dye fluoresces upon intercalating into dsDNA, with limited binding to RNA and ssDNA. 11 However, the PicoGreen assay relies on an external standard (e.g., lambda DNA supplied by manufacturers) which has been quantified using A 260 and is not currently traceable to the SI. 2,4,6 * Corresponding author. E-mail: Somanath.Bhat@measurement.gov.au. Tel: +61 2 9449 0111. Fax: +61 2 9449 1653. (1) Nielsen, K.; Mogensen, H. S.; Hedman, J.; Niedersta¨tter, H.; Parson, W.; Morling, N. Forensic Sci. Int.: Genet. 2008, 2, 226–230. (2) Holden, M. J.; Rabb, S. A.; Tewari, Y. B.; Winchester, M. R. Anal. Chem. 2007, 79, 1536–1541. (3) Stevenson, J.; Hymas, W.; Hillyard, D. J. Clin. Microbiol. 2005, 43, 2391– 2398. (4) Corbisier, P.; Broothaerts, W.; Gioria, S.; Schimmel, H.; Burns, M.; Baoutina, A.; Emslie, K. R.; Furui, S.; Kurosawa, Y.; Holden, M. J.; Kim, H. H.; Lee, Y. M.; Kawaharasaki, M.; Sin, D.; Wang, J. J. Agric. Food Chem. 2007, 55, 3249–3257. (5) Cavaluzzi, M. J.; Borer, P. N. 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