Comparison of methods for accurate quantification of DNA mass concentration with traceability to the international system of units.
Accurate estimation of total DNA concentration (mass concentration, e.g., ng/muL) that is traceable to the International System of Units (SI) is a crucial starting point for improving reproducible measurements in many applications 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 determined using different measurement platforms: UV Absorbance 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 different for five of the six samples tested. There were no significant differences in concentration estimates based on A(260) with prior NaOH treatment, PicoGreen analysis, and dPCR for two of the three samples tested using dPCR. Since the measurand in dPCR is amount (copy number) concentration (copies/muL), 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.
Bhat
Somanath
S
National Measurement Institute, Lindfield, New South Wales, Australia. Somanath.Bhat@measurement.gov.au
Curach
Natalie
N
Mostyn
Thomas
T
Bains
Gursharan Singh
GS
Griffiths
Kate R
KR
Emslie
Kerry R
KR
D004247:DNA / Q000032:analysis*; D005456:Fluorescent Dyes / Q000737:chemistry; D005470:Fluorometry / Q000379:methods*; D009930:Organic Chemicals / Q000737:chemistry; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D013056:Spectrophotometry, Ultraviolet / Q000379:methods*
D005456:Fluorescent Dyes; D009930:Organic Chemicals; C099476:PicoGreen; D004247:DNA