Quantification of phosphorus in DNA using capillary electrophoresis hyphenated with inductively coupled plasma mass spectrometry.
We have analyzed phosphorus in an enzymatically digested DNA molecule using capillary electrophoresis (CE) hyphenated with inductively coupled plasma mass spectrometry (ICP-MS). The DNA concentration was quantified by the phosphorus value obtained in the CE-ICP-MS analysis. The CE-ICP-MS measurement, for which the interface device AIF-01 equipped three layered nebulizer was adopted, was achieved with limited μL/min nebulizing without loss of sample in the vaporizing chamber. The samples of nucleotides and free phosphate were separated well in the CE-ICP-MS measurement, and the calibration curve (0.1-10μg/mL) of the phosphorus showed a linear (R(2)=0.999) increase in intensity. After digestion of the 100-bp double-strand DNA sample to deoxyribonucleotide-5'-monophosphates (dNMPs) by phosphodiesterase-I, phosphorus was detected by CE-ICP-MS without further purification steps. In this study, we applied two calculation schemes of DNA analysis using a dNMP concentration obtained from CE-ICP-MS. Comparative CE-ICP-MS analysis with DNA digested to dNMPs showed that the assay gave an equal value obtained from the total DNA quantification using fluorescence detection. The detection limits of the DNA sample obtained from these species and phosphorus in nucleotides using CE-ICP-MS were 3.1-26ng/mL. These LOD values were equal to the conventional fluorescence determination of DNA.
National Metrology Institute of Japan (NMIJ), National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki 305-8563, Japan. s-fujii@aist.go.jp
Shin-Ichiro
S
Fujii
Kazumi
K
Inagaki
Koichi
K
Chiba
Akiko
A
Takatsu
D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D019075:Electrophoresis, Capillary / Q000379:methods*; D016014:Linear Models; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D043264:Phosphodiesterase I / Q000378:metabolism; D010758:Phosphorus / Q000032:analysis*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
D010758:Phosphorus; D004247:DNA; D043264:Phosphodiesterase I