Measurement of Mitochondrial dNTP Pools
Measurement of Mitochondrial dNTP Pools
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DOI：
10.1007/978-1-61779-504-6_9
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发表时间：
2012-01-01
期刊：
影响因子：
--
通讯作者：
Hirano, Michio
中科院分区：
文献类型：
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作者：
Marti, Ramon;Dorado, Beatriz;Hirano, Michio
由于脱氧核苷三磷酸（dNTP）是DNA复制和修复的关键底物，因此已在多种基本生化过程的背景下研究了INTP池。在过去的12年中，对dNTP，特别是线粒体dNTP库的兴趣已经扩展到生物医学科学，因为已经发现几种线粒体疾病是由参与dNTP催化或还原的几种酶的功能障碍引起的。可靠地测量线粒体dNTPs的技术应该是敏感和特异的，以避免由丰富的核糖核苷酸引起的干扰。在这里，我们描述了详细的协议来测量线粒体dNTPs从两个特定的样本，培养的皮肤成纤维细胞和小鼠肝脏。该方法可以很容易地适用于其他类型的样品。该方案遵循基于聚合酶的方法，这是测量dNTP池最广泛使用的方法。我们的描述是基于该技术的最新更新，该技术最大限度地减少了核糖核苷酸的潜在干扰。
Because deoxyribonucleoside triphosphates (dNTPs) arc the critical substrates for DNA replication and repair, (INTP pools have been studied in context of multiple basic biochemical processes. Over the last 12 years, interest in dNTPs, and specifically the mitochondrial dNTP pools, has expanded to biomedical science because several mitochondrial diseases have been found to be caused by dysfunctions of several enzymes involved in dNTP catabolism or anabolism. Techniques to reliably measure mitochondrial dNTPs should be sensitive and specific to avoid interference caused by the abundant ribonucleotides. Here, we describe detailed protocols to measure mitochondrial dNTPs from two specific samples, cultured skin fibroblasts and mouse liver. The methods can be easily adapted to other types of samples. The protocol follows a polymerase-based method, which is the most widely used approach to measure dNTP pools. Our description is based on the latest update of the technique, which minimizes the potential interference from ribonucleotides.