Separation of major and minor deoxyribonucleoside monophosphates by reverse-phase high-performance liquid chromatography: a simple method applicable to quantitation of methylated nucleotides in DNA.
Separation of major and minor deoxyribonucleoside monophosphates by reverse-phase high-performance liquid chromatography: a simple method applicable to quantitation of methylated nucleotides in DNA.
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通过反相高效液相色谱分离主要和次要脱氧核糖核苷单磷酸：一种适用于 DNA 中甲基化核苷酸定量的简单方法。
DOI：
10.1016/0003-2697(82)90662-5
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发表时间：
1982
影响因子：
2.9
通讯作者：
Christman,JK
中科院分区：
文献类型：
--
作者：
Christman,JK
本文介绍了一种分离脱氧核苷单磷酸的简单反相色谱系统。在室温下使用等度洗脱，可以在不到2小时内实现原核或真核DNA中存在的七种脱氧核苷单磷酸的清晰分离。因此，该方法可以灵敏、快速地分析亚微克量的32P标记脱氧核苷一磷酸，这些脱氧核苷一磷酸来源于体内用32Pior标记的DNA，这些DNA来自体外酶标记的DNA的5′或3′末端。研究哺乳动物DNA甲基化的方法的适用性说明了其应用的例子（a）定量的主要和次要的核苷酸在新合成的DNA，（B）在体外甲基化的DNA的特异性的测定，和（c）在何种程度上，具体的限制性内切酶位点在体内甲基化的定量。
A simple reverse-phase chromatographic system for separating deoxyribonucleoside monophosphates is described. Using isocratic elution at room temperature, clear separation of seven of the deoxyribonucleoside monophosphates that occur in either procaryotic or eucaryotic DNAs can be achieved in less than 2 h. Thus, this method allows a sensitive and rapid analysis of submicrogram quantities of32P-labeled deoxyribonucleoside monophosphates derived from DNA labeled in vivo with32Pior from DNA labeled enzymatically in vitro at the 5′ or 3′ ends. The suitability of the method for studying methylation of mammalian DNAs is illustrated by presenting examples of its application to (a) quantitation of major and minor nucleotides in newly synthesized DNA, (b) determination of the specificity of in vitro methylation of DNA, and (c) quantitation of the extent to which specific restriction endonuclease sites are methylated in vivo.