DNA quantification approach by GE-ICP-SFMS and complementary total phosphorus determination by ICP-SFMS

Abstract

Quantification of DNA is still a great challenge for scientists in many fields. Here, we present the on-line coupling of gel electrophoresis (GE) and inductively coupled plasma-sector field mass spectrometry (ICP-SFMS) for quantitative purposes. GE conditions are chosen for optimised separations depending on the target analyte composition in terms of DNA chain length. In particular, agarose concentrations are varied in the range 0.6–2.2%, which corresponds to a separation range of DNA from 100 base pairs (bp) to genomic DNA (approximately 3 Mbp). Separated DNA compounds are directly transported at a flow rate of 100 μL min⁻¹ to a Micromist nebuliser which is followed by ICP-SFMS with ³¹P detection at a mass resolution of 4000. For their quantification different approaches are tested using external calibration by PO₄³⁻ standards as well as by a commercially available QuantLadder standard. Precisions lower than 3% can be achieved in any analysis: however, the indicative values for DNA concentrations given by the manufacturers cannot be found in all the samples. As to our best knowledge no certified quantitative DNA reference material exists, unfortunately we could not prove the accuracy of the developed method. Despite this, we then applied the GE-ICP-SFMS coupling to the analysis of genomic DNA in order to confirm its practicability for the separation and detection of these large biomolecules.