Use of Elemental Analysis To Determine Comparative Performance of Established DNA Quantification Methods

Abstract

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.

Impacts of Inter- and Intralaboratory Variations on the Reproducibility of Microbial Community Analyses

“…It is difficult to say which assays perform better. There are reports that above concentrations of 10 ng l Ϫ1 both assays perform similarly, while below this concentration PicoGreen performs better (1,15). The reproducibility is in the same range for both assays, as indicated by the coefficient of variation (see Fig.…”

Analysis of mono-phosphate nucleotides as a potential method for quantification of DNA using high performance liquid chromatography–inductively coupled plasma-mass spectrometry

“…Elemental analysis is preferred over other techniques as it offers lower detection limits, a larger dynamic range, a response that is to a first approximation independent of molecular form, and potentially more accurate quantification at low concentrations by isotope dilution analysis. Both ICP-MS and ICP-OES have been utilised for the quantification of nucleotides, DNA, oligonucleotides and DNA adducts [3][4][5][6][7][8][9][10][11][12][13][14][15]. The quantification of oligonucleotides has been achieved by two different methods by Yang et al [4] and Donald et al [7], with the latter group digesting the oligonucleotides to mono-nucleosides and quantifying by isotope dilution electrospray mass spectrometry (ES-ID-MS), and comparing that value to the total 31 P signal obtained from ICP-OES and the gravimetric value.…”

Development of Metrology for Modern Biology

“…This method has been used to determine the quantity of DNA materials for international comparison studies for DNA quantification. Several publications have been made in an attempt to establish the ICP-OES based phosphorous quantification as a primary analytical method for DNA quantification (English et al, 2006;Holden et al, 2007;Brennan et al, 2009).…”