Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry
Introduction
A demand for the quantification of nucleic acids, such as DNA and RNA, has been increased in many areas, such as clinical diagnosis, food analysis, microbial analysis, and molecular biology. These nucleic acids have conventionally been determined with a photometric method such as UV adsorption (OD 260 nm) or fluorescence detection. The analytical results obtained from these photometric analyses were different depending on these analytical and sample conditions such as salt levels and pH. Though these methods essentially require a well-characterized or quantified reference material of nucleic acid, a well-established calibrant such as certified reference materials for quantification of nucleic acids does not exist. Although an accurate analysis of nucleic acid determined by phosphorus content in acid-digested nucleotides has developed, accurate quantification of each molecule of nucleic acids and intact DNA sequence has not been achieved yet [1]. DNA is composed of deoxynucleotides (dNMPs), which consist of deoxyribose, a base, and phosphate. Therefore, the determination of phosphorus is one of the most effective methods to quantify nucleotides. The determination of phosphorus can be used for the quantification of macromolecules, such as DNA, due to the fixed stoichiometry of these elements in that molecule. The quantification of a DNA sample using inductively coupled plasma optical emission spectrometry (ICP-OES) on the basis of the precise measurement of the phosphorus content of DNA has been reported [2], [3], [4]. Although the ICP-OES method is effective for the analysis of total phosphorus in a nucleic acid sample, care is required to avoid contamination of the materials that include phosphorus. Furthermore, this method requires a large volume of sample material. On the other hand, capillary electrophoresis (CE) has the advantages of achieving good separation and requiring small volume of samples; numerous studies have used CE methods for the analysis of dNMPs, which are easily separated due to their negative charge in a widely pH range [5]. In the case of the quantification of DNA, which was digested to dNMPs, the quantification value is obtained from each dNMP. Therefore, the base composition, which was not obtained in photometric analysis such as UV or fluorescence determination, is even obtained from sequence unknown sample of DNA. The composition of bases is available for the monomeric analysis such as guanine and cytosine (GC) content [6], [7], [8], [9] and modification of bases [10], [11]. And then, the phosphorus measurement provides a highly accurate quantification of mass for both dNMPs and DNA using higher order reference material of phosphate (e.g. distributed from the Japan Calibration Service System (JCSS) and NIST). Moreover, the contaminants including phosphorus are also separated using CE, and inductively coupled plasma mass spectrometry (ICP-MS) represents a highly sensitive technique for the determination of an element. Then, we have developed an interface device for CE hyphenated with ICP-MS [12]. There are several reports on the interface device for CE hyphenated with ICP-MS [13], [14], [15], [16]. These reports demonstrate the advantages regarding the high efficiency of the sample injection and ease of handling for the maintenance of the interface device. Although a few studies on dNMP analysis using CE-ICP-MS have been reported [12], [16], quantitative DNA analysis was not realized in these reports. In the present article, to improve the accuracy of phosphorus quantification for DNA analysis, we applied the quantification of phosphorus in nucleotides obtained from an enzymatically digested DNA molecule using CE-ICP-MS equipped with our interface device. The content of nucleotides content in DNA sample was separated by CE, and the concentration of DNA was then obtained from the measured value of phosphorus in each nucleotide without intact DNA reference materials. Moreover, the composition of each nucleotide and impurity in DNA sample can be obtained in this method.
Section snippets
Chemical reagents
The free acid forms of deoxyadenosine-5′-monophosphate (dAMP), deoxyguanosine-5′-monophosphate (dGMP), deoxycytidine-5′-monophosphate (dCMP), and thymidine-5′-monophosphate (dTMP) were purchased from Sigma–Aldrich Japan (Tokyo, Japan) at >98% purity. The nitric acid solution was adjusted to 0.1% by dissolving an appropriate amount in ultra-pure water. Potassium hydroxide (1.25%) was prepared by dissolving potassium hydroxide in methanol. A phosphate ion standard solution (Kanto, Tokyo, Japan),
Phosphorus analysis
Phosphorus determination using CE-ICP-MS was performed to investigate the ability to quantify the phosphorus in nucleotides. The interface achieved nebulizing at 4.5 μL/min and effective introduction of the sample to ICP-MS. Using ICP-MS, the following elements were measured in this experiment: 31P, 82Se, and 133Cs. The element of 133Cs was used for the monitoring of the flowing of the sheath liquid. The electropherograms of the phosphorus analysis monitoring 31P and 82Se as used for the
Conclusions
CE hyphenated with ICP-MS using the developed interface has proved to be a suitable technique for the separation and element-specific determination of phosphorus in dNMPs. Phosphorus was determined with good repeatability and linearity in intensity with CE-ICP-MS equipped with the interface device. And four kinds of dNMPs, which were enzymatically digested from DNA, were separated at the baseline with CE and detected as phosphorus in ICP-MS. In this study, we applied two calculation schemes of
Acknowledgments
We would like to thank Dr. N. Noda in Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST) and Dr. H. Tani in Radioisotope Center; The University of Tokyo for the instruction on PCR preparation of DNA sample. A part of this study was sponsored by Grants-in-Aid for Scientific Research (21550094) from Japan Society for the Promotion of Science (JSPS).
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