Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of DNA

Abstract

Measurement of the phosphorus content of nucleotides and deoxyribonucleic acid (DNA) offers an approach to the quantitation of nucleic acids that is traceable to the SI. Such measurements can be an alternative to the commonly used spectroscopic tools that are not traceable. Phosphorus measurements of thymidine 5'-monophosphate (TMP) and acid-digested plasmid and genomic DNA preparations were made using high-performance inductively coupled plasma optical emission spectroscopy (HP-ICP-OES) and high-performance liquid chromatography (HPLC) and compared for bias and uncertainty. A prerequisite for quality measurement is the purity of the materials. Quantitation with the two platforms was comparable for the TMP. However, the HPLC values had larger uncertainties and were all statistically different from the gravimetric values at the 95% confidence level. When using ICP-OES, the digestion of the nucleotide monophosphate can be eliminated, thus simplifying the procedure. The differences between the results obtained by using the two platforms, when measuring genomic or plasmid DNA, were dependent on the mass fraction of the digest. ICP-OES measurement of phosphorus provides a highly accurate quantitation for both nucleotide monophosphates and DNA with expanded uncertainties of less than 0.1%. Currently, ICP-OES requires a significant sample size restricting its usefulness for the quantitation of DNA but represents a valuable tool for certification of reference materials. HPLC requires smaller amounts of material to perform the analysis but is less useful for certification of reference materials because of lower accuracy and 10-fold higher expanded uncertainties.

Comparison of fluorometric and spectrophotometric DNA quantification for real-time quantitative PCR of degraded DNA

“…Absorbance at 260 nm has been the most commonly used method to calculate nucleic acid concentrations (Holden, Rabb, Tewari, & Winchester, 2007). More recently, however, methods that employ fluorescent intercalating DNA dyes are becoming more popular for quantification of genomic DNA for applications in qPCR.…”

International Standards and Reference Materials for Quantitative Molecular Infectious Disease Testing

“…Pure DNA can be accurately and precisely quantified based on phosphorus measurements using high-performance inductively coupled plasma-optical emission spectroscopy. 39 Once the HCMV standard reference material has been certified, interlaboratory studies will be conducted.…”

Fragmentation of DNA affects the accuracy of the DNA quantitation by the commonly used methods

“…The accuracy of DNA quantification is crucial for the success of following downstream applications such as (q)PCR, sequencing and cloning. Commonly used methods of DNA concentration measurements are the evaluation of the intensity of a band on an agarose gel, fluorescence measurements using various DNA-binding dyes and measurements of UV absorbance at 260 nm [5], with the latter being the most commonly used [6,7]. The disadvantages of the latter method are that the absorbance measurement at 260 nm includes signals of a double-stranded and single-stranded DNA oligonucleotides and free nucleotides, the fact that it does not distinguish between DNA and RNA, and that it has a low sensitivity, reaching 1 ng/μl [6,8,9].…”