O'Connor Gavin, Dawson Carol, Woolford Alison, Webb Kenneth S, Catterick Tim
LGC Limited, Queens Road, Teddington, Middlesex, UK.
Anal Chem. 2002 Aug 1;74(15):3670-6. doi: 10.1021/ac0255375.
The importance of DNA as a regulatory analyte is well-known. Recent years have seen an increased interest in the quantitation of this analyte. Accurate quantitative measurements have been hampered by the lack of well-characterized standards and pure materials for this large-molecular-weight analyte. Outlined here is an approach for the accurate and reproducible quantitation of an oligonucleotide that is solely reliant on the availability of pure, well-characterized deoxynucleotides and not a sequence-specific pure DNA standard. The proposed procedure is intended to provide an accurate and definitive method for the quantitation of DNA for reference measurements as an improved alternative to the more conventional UV absorbance-based methods. For proof of concept, a gravimetrically prepared oligonucleotide solution was enzymatically digested to its constituent monomer-deoxynucleotide monophosphates (dNMPs), of which there are four different types. Qualitative mass spectrometry was used to confirm the 100% successful completion of the enzymatic digestion step. The dNMPs were then separated by liquid chromatography (LC) before being detected by electrospray ionization (ESI) mass spectrometry (MS). The method of quantitation was based on isotope dilution mass spectrometry (IDMS) analysis of the four different monomer units. The concentrations of the four dNMP residues were then summed to obtain the original concentration of the oligonucleotide. The concentrations determined by liquid chromatography/mass spectrometry (LC/MS) and also by liquid chromatography-tandem mass spectrometry (LC/MS/MS) differed by <2.5 and 1%, respectively, from the gravimetrically assigned value. These differences were well within the uncertainty of the gravimetrically assigned value. This highly accurate method, suitable for the definitive quantitation of oligonucleotides, should be ideal for characterizing primary calibration standards and certified reference materials that can then be used to underpin the more conventional quantitative techniques of UV and fluorescence spectroscopy.
DNA作为一种调控分析物的重要性是众所周知的。近年来，人们对这种分析物的定量分析兴趣日益浓厚。由于缺乏针对这种大分子分析物的特征明确的标准品和纯物质，准确的定量测量受到了阻碍。本文概述了一种仅依赖于纯的、特征明确的脱氧核苷酸（而非序列特异性纯DNA标准品）来对寡核苷酸进行准确且可重复定量的方法。所提出的程序旨在提供一种准确且确定的DNA定量方法，用于参考测量，作为更传统的基于紫外吸光度方法的改进替代方法。为了验证概念，将通过重量法制备的寡核苷酸溶液酶解为其组成的单体脱氧核苷酸单磷酸（dNMP），共有四种不同类型。采用定性质谱法确认酶解步骤100%成功完成。然后通过液相色谱（LC）分离dNMP，再通过电喷雾电离（ESI）质谱（MS）进行检测。定量方法基于对四种不同单体单元的同位素稀释质谱（IDMS）分析。然后将四种dNMP残基的浓度相加，以获得寡核苷酸的原始浓度。通过液相色谱/质谱（LC/MS）以及液相色谱-串联质谱（LC/MS/MS）测定的浓度与重量法指定值的差异分别小于2.5%和1%。这些差异完全在重量法指定值的不确定度范围内。这种高度准确的方法适用于寡核苷酸的确定定量，对于表征一级校准标准品和有证参考物质应该是理想的，然后这些标准品可用于支持更传统的紫外和荧光光谱定量技术。
