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对高效液相色谱法测定DNA总甲基化水平的关键因素进行研究,即基因组DNA的提取及纯化和高效液相色谱条件的选择。结果表明:CTAB法Ⅰ提取和纯化效果优于CTAB法Ⅱ;较优高效液相色谱条件为:采用Diamonsil C18(2)(250 mm×4.6 mm,5μm)的色谱柱,以甲醇-10 mmol/L磷酸二氢钾(10-90,v/v)为流动相构成,流动相pH为4.7,流速为0.5 mL/min,柱温为30℃,紫外检测器波长为285 nm时,是分离胞嘧啶和5-甲基胞嘧啶的较优条件。以试验优化的DNA提取方法和HPLC色谱条件,基因组DNA水解液的胞嘧啶(C)和5-甲基胞嘧啶(5 mC)可得到较好的分离效果。
The key factors that determine the total DNA methylation level of high performance liquid chromatography (HPLC) were studied, ie the extraction and purification of genomic DNA and the selection of high performance liquid chromatography (HPLC) conditions. The results showed that the extraction and purification of CTAB Ⅰ was superior to that of CTAB Ⅱ. The optimal HPLC conditions were as follows: Diamonsil C18 (2) (250 mm × 4.6 mm, 5 μm) L potassium dihydrogen phosphate (10-90, v / v) as mobile phase, mobile phase pH 4.7, flow rate 0.5 mL / min, column temperature 30 ℃, UV detector wavelength 285 nm, Pyrimidine and 5-methylcytosine. Optimized DNA extraction methods and HPLC chromatographic conditions, genomic DNA hydrolyzate of cytosine (C) and 5 - methylcytosine (5 mC) can get better separation results.