Dept. Medical Biochemistry and Biophysics, Umeå University, SE-901 87 Umeå, Sweden.
Nucleic Acids Res. 2022 Feb 22;50(3):e18. doi: 10.1093/nar/gkab1117.
Information about the cellular concentrations of deoxyribonucleoside triphosphates (dNTPs) is instrumental for mechanistic studies of DNA replication and for understanding diseases caused by defects in dNTP metabolism. The dNTPs are measured by methods based on either HPLC or DNA polymerization. An advantage with the HPLC-based techniques is that the parallel analysis of ribonucleoside triphosphates (rNTPs) can serve as an internal quality control of nucleotide integrity and extraction efficiency. We have developed a Freon-free trichloroacetic acid-based method to extract cellular nucleotides and an isocratic reverse phase HPLC-based technique that is able to separate dNTPs, rNTPs and ADP in a single run. The ability to measure the ADP levels improves the control of nucleotide integrity, and the use of an isocratic elution overcomes the shifting baseline problems in previously developed gradient-based reversed phase protocols for simultaneously measuring dNTPs and rNTPs. An optional DNA-polymerase-dependent step is used for confirmation that the dNTP peaks do not overlap with other components of the extracts, further increasing the reliability of the analysis. The method is compatible with a wide range of biological samples and has a sensitivity better than other UV-based HPLC protocols, closely matching that of mass spectrometry-based detection.
有关脱氧核苷三磷酸 (dNTP) 的细胞浓度的信息对于 DNA 复制的机制研究以及对于理解由于 dNTP 代谢缺陷引起的疾病非常重要。dNTP 可通过基于 HPLC 或 DNA 聚合的方法进行测量。基于 HPLC 的技术的一个优点是，核糖核苷三磷酸 (rNTP) 的平行分析可以作为核苷酸完整性和提取效率的内部质量控制。我们开发了一种无氟三氯乙酸基方法来提取细胞核苷酸，并开发了一种等度反相 HPLC 技术，该技术能够在单次运行中分离 dNTP、rNTP 和 ADP。测量 ADP 水平的能力提高了核苷酸完整性的控制，并且使用等度洗脱克服了以前开发的用于同时测量 dNTP 和 rNTP 的基于梯度的反相协议中的基线漂移问题。可选的 DNA 聚合酶依赖性步骤用于确认 dNTP 峰不会与提取物的其他成分重叠，从而进一步提高分析的可靠性。该方法与广泛的生物样品兼容，并且灵敏度优于其他基于 UV 的 HPLC 方案，与基于质谱检测的灵敏度非常匹配。
