Stable Isotope Labeled DNA: A New Strategy for the Quantification of Total DNA Using Liquid Chromatography-Mass...

Conventional DNA quantification methods require a DNA purification step that limits their reliability in estimating the original DNA amount, especially in complex matrix. To overcome this limitation, we developed a method to calibrate the variable DNA extraction efficiencies during the purification process, allowing for the accurate quantification of DNA in complex matrix. This method is based on isotope dilution-liquid chromatography-mass spectrometry using stable isotope labeled DNA (SILD) as an internal standard. Steps include spiking prepared SILD into samples, purification, enzymatic hydrolysis, and detection of DNA monomers via mass spectrometry, where the spiked SILD is expected to behave the same as the target DNA throughout the entire procedure. We show that the mean recoveries of four different DNA purification kits were dramatically improved by using the SILD internal standard, both for Escherichia coli and human genomic DNA. As standards for calibration, deoxyribonucleoside monophosphates and purified genomic DNA were tested, with genomic DNA from corresponding species found to calibrate the variable extraction efficiencies more effectively. With this successful calibration, our newly developed procedure enables International System of Units-traceable quantification of total DNA in complex matrix.