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

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...

Full description

Saved in:
Bibliographic Details
Published in:Analytical chemistry (Washington) 2019-03, Vol.91 (6), p.3936-3943
Main Authors: Kwon, Ha-Jeong, Jeong, Ji-Seon, Bae, Young-Kyung, Choi, Kihwan, Yang, Inchul
Format: Article
Language:English
Subjects:
Analytical chemistry
Calibration
Chemistry
Chromatography
Deoxyribonucleic acid
Dilution
DNA
E coli
International System of Units
Ions
Liquid chromatography
Mass spectrometry
Mass spectroscopy
Monomers
Purification
Scientific imaging
Spectroscopy
Stable isotopes
Citations: Items that this one cites
Items that cite this one
Online Access:Get full text
Tags: Add Tag
No Tags, Be the first to tag this record!
Description
Summary: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.
ISSN:0003-2700
1520-6882
DOI:10.1021/acs.analchem.8b04940