The accurate mass determination of DNA and other nucleic acids is of critical importance in many clinical diagnoses and in forensic and molecular biological studies. In addition, these measurements could aid in the determination of transgenic material in agricultural and food products. NIST is currently developing inductively coupled plasma optical emission spectrometry (ICP-OES) methodology that allows for the mass of DNA to be traceable to the SI. This method, if successful, will aide in the development of Standard Reference Materials (SRMs) with certified nucleic acid content.
The biotechnology industry relies primarily on absorbance and fluorescence methodologies to assess DNA mass. However, these measurements can be seriously compromised by impurities in the DNA preparations or state of the DNA itself. These techniques are also not traceable to the SI because the calibrations cannot currently be performed using calibration standards whose values are traceable to the SI. Such techniques cannot achieve the high accuracy and precision required to produce SRMs for nucleic acid materials.
At NIST in collaboration with The George Washington University (GWU), a methodology that can be used to provide accurate measurements of DNA and nucleic acid mass that are traceable to the SI is being developed with the long-term goal of providing an SRM for DNA mass. Currently, the ICP-OES approach developed at NIST, referred to as high-performance ICP-OES (HP-ICP-OES) incorporating a ratio-based technique with drift correction, has been utilized for the measurement of phosphorus content of acid-digested nucleotides and DNA. The HP-ICP-OES measurement of phosphorus provides a highly accurate quantification of mass for both nucleotide monophosphate and DNA with relative uncertainties less than 0.1 % (95 % confidence level). However, the current approach requires a significant sample size (10 mL of solution containing a DNA concentration of 50 μg/mL), restricting its usefulness for the quantification of DNA. A demountable, direct-injection high-efficiency nebulizer (d-DIHEN), developed by GWU, is incorporated to limit sample consumption via the introduction of samples at low sample uptake rates.
An automated sample analysis system utilizing a d-DIHEN for ICP-OES was successfully incorporated for the accurate measurement of the phosphorus content of acid-digested nucleotides and DNA. For the first time, an automated procedure was developed for use with the d-DIHEN, without plasma shutdowns. The solution uptake rate was reduced from 170 µL/min to 30 µL/min and the required sample size was reduced from 10 mL to 2.4 mL, while providing relative expanded uncertainties (% U) in the range of 0.1 to 0.4 (95% confidence interval) for most analyses. The use of direct injection improves P (I) 213.617 nm sensitivity by a factor of 4 on average compared to a glass concentric nebulizer with cyclonic spray chamber arrangement. This successful method will aid in the development of SRMs with certified nucleic acid content, particularly for these samples that are typically limited in volume.
Start Date:September 1, 2005
Lead Organizational Unit:mml
Ryan G. Brennan
Marcia J. Holden
Michael R. Winchester
Related Programs and Projects:
Fundamental Chemical Metrology
Optimization of the Standard Additions Method Using Monte Carlo Simulation
Mercury Vapor Pressure Correlation
High-Precision Isotopic Measurements and Standards
Studies of Micro-Scale Heterogeneity of Solid Reference Materials
Improving High-Performance ICP-OES Determinations Using Exact Matching
Evaluation of Independent High-Precision Assay Procedures for a High-Purity Primary Standard Reagent
High-Resolution Separations Technologies
M. J. Holden, S. A. Rabb, Y. B. Tewari, and M. R. Winchester, Traceable Phosphorus Measurements by ICP-OES and HPLC for the Quantitation of
R. G. Brennan, S. A. Rabb, M. J. Holden, M. R. Winchester, and G. C. Turk, Potential Primary Measurement Tool for Quantification of DNA, Anal. Chem., 2009, 81(9), 3414-3420.