Use of elemental analysis to determine comparative performance of established DNA quantification methods.
Quantification of genomic DNA is critical for many analyses in molecular biology. Current methods include optical density (OD) measurements or fluorescent enhancement but both approaches have limitations on achievable accuracy. In this study we performed an elemental analysis to quantify genomic DNA to provide an independent value for comparing the performance of four quantification methods. Specifically ICP-OES (inductively coupled plasma-optical emission spectroscopy) was used to assign a concentration value to a DNA stock solution, based on the stoichiometry of phosphorus within the molecule. Two absorbance- and two fluorescence-based methods were then used to quantify the same DNA solution using replicate analyses. The precision of each method was assessed by measurement of replicate spread (coefficient of variation) and trueness by t-test. Results showed that performance of the methods was variable, both in terms of concordance with the independent ICP-OES value and repeatability of data. While need for expensive equipment and technical expertise may preclude widespread replacement of more traditional methods for DNA quantification, use of primary methods such as ICP-OES analysis for production of accurate calibrants may increase quantitative accuracy and give greater appreciation of the true performance of current methods.
English
Claire A
CA
Bio-Molecular Innovation, LGC, Queens Road, Teddington, TW11 0LY, UK.
Merson
Sheila
S
Keer
Jacquie T
JT
D004247:DNA / Q000032:analysis*; D015203:Reproducibility of Results; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D004247:DNA