Comparison of Methods for Accurate Quantification of DNA Mass Concentration with Traceability to the International System of Units

摘要

Accurate estimation of total DNA concentration (massnconcentration, e.g., ng/μL) that is traceable to the InternationalnSystem of Units (SI) is a crucial starting pointnfor improving reproducible measurements in many applicationsninvolving nucleic acid testing and requires anDNA reference material which has been certified for itsntotal DNA concentration. In this study, the concentrationsnof six different lambda DNA preparations were determinednusing different measurement platforms: UV Absorbancenat 260 nm (A260) with and without priornsodium hydroxide (NaOH) treatment of the DNA,nPicoGreen assay, and digital polymerase chain reactionn(dPCR). DNA concentration estimates by A260 with andnwithout prior NaOH treatment were significantly differentnfor five of the six samples tested. There were nonsignificant differences in concentration estimates basednon A260 with prior NaOH treatment, PicoGreen analysis,nand dPCR for two of the three samples tested usingndPCR. Since the measurand in dPCR is amount (copynnumber) concentration (copies/μL), the results suggestnthat accurate estimation of DNA mass concentrationnbased on copy number concentration is achievablenprovided the DNA is fully characterized and in thendouble-stranded form or amplification is designed tonbe initiated from only one of the two complementarynstrands.