Traceable phosphorus measurements by ICP-OES and HPLC for the quantitation of DNA.
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.
Holden
Marcia J
MJ
Chemical Science and Technology Laboratory, National Institute of Standards and Technology, 100 Bureau Drive, Gaithersburg, Maryland 20899-8311, USA. marcia.holden@ nist.gov
Rabb
Savelas A
SA
Tewari
Yadu B
YB
Winchester
Michael R
MR
D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004247:DNA / Q000032:analysis* / Q000737:chemistry*; D009711:Nucleotides / Q000737:chemistry; D010758:Phosphorus / Q000032:analysis*; D012680:Sensitivity and Specificity; D013054:Spectrophotometry, Atomic / Q000379:methods*
D009711:Nucleotides; D010758:Phosphorus; D004247:DNA