反相高效液相色谱法定量测定DNA中的主要和修饰脱氧核糖核苷。
Quantitative reversed-phase high performance liquid chromatographic determination of major and modified deoxyribonucleosides in DNA.
作者信息
Kuo K C, McCune R A, Gehrke C W, Midgett R, Ehrlich M
出版信息
Nucleic Acids Res. 1980 Oct 24;8(20):4763-76. doi: 10.1093/nar/8.20.4763.
Abstract
We have developed a method to accurately determine (< 3% RSD) the complete major and modified base composition of a few micrograms of unlabeled DNA. The DNA samples were quantitatively hydrolyzed with DNase 1, Nuclease P1, and bacterial alkaline phosphatase. The resulting deoxyribonucleosides were directly separated in 70 min by reversed-phase high performance liquid chromatography with detection by ultraviolet absorption at 254 nm and 280 nm (RP-HPLC). The highly sensitive and selective dual wavelength quantitation greatly enhances the precision and accuracy of the chromatographic analysis. Contamination of DNA preparations with RNA does not interfere with the DNA analysis due to the high resolution of the chromatography. We have used this method for the quantitation of m5dCyd in 5 microgram of calf thymus and salmon sperm DNA in which the m5dCyd comprises only 1 to 2% of the total bases. This method should be a useful research tool in studies on various DNAs and DNA subfractions and should help to elucidate the functions of methylation of DNA.
摘要
我们开发了一种方法，可准确测定（相对标准偏差<3%）几微克未标记DNA的完整主要碱基和修饰碱基组成。DNA样品用DNase 1、核酸酶P1和细菌碱性磷酸酶进行定量水解。所得脱氧核糖核苷通过反相高效液相色谱在70分钟内直接分离，采用254 nm和280 nm紫外吸收检测（RP-HPLC）。高灵敏度和选择性的双波长定量大大提高了色谱分析的精密度和准确性。由于色谱分辨率高，DNA制剂中RNA的污染不会干扰DNA分析。我们已使用该方法对5微克小牛胸腺DNA和鲑鱼精子DNA中的5-甲基脱氧胞苷进行定量，其中5-甲基脱氧胞苷仅占总碱基的1%至2%。该方法应是研究各种DNA和DNA亚组分的有用研究工具，并应有助于阐明DNA甲基化的功能。
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