离子交换色谱法检测人血白细胞基因组DNA整体甲基化水平  

Determination of global DNA methylation in human leukocyte by isocratic cation exchange high-porformance liquid chromatography

作　　者：李峥[1] 李瑛[1] 杨永佳[1] 刘伏友[1] 李伍秋[2] 

机构地区：[1]中南大学湘雅二医院肾脏病研究所,长沙410011 [2]中南大学湘雅二医院药剂科,长沙410011

出　　处：《中国医师杂志》2010年第1期55-58,共4页Journal of Chinese Physician

摘　　要：目的改进现有方法，能快速、稳定、简便地测定人基因组DNA整体甲基化水平。方法采用美国HPAgilent1100色谱工作站，利用离子交换液相色谱法对健康人外周血白细胞基因组DNA整体甲基化水平进行检测。色谱柱采用德国MN公司的强阳离子交换色谱柱（250mm×4．6mm5μm）；流动相为60mM醋酸＋15％乙腈，用氢氧化钠调节pH为4．6；流速为1．0ml／min；检测器波长为276nm；柱温为28℃；进样量为50μl。基因组DNA整体甲基化水平用5甲基脱氧胞嘧啶核苷占DNA样品中总脱氧胞嘧啶核苷的百分数来表示。结果在上述色谱条件下，约10min可以将DNA中的脱氧胞嘧啶核苷和5甲基脱氧胞嘧啶核苷完全分离。并测得健康成人血白细胞基因组DNA整体甲基化水平为（4．389±0．0159）％。结论本方法能快速、有效地对基因组DNA整体甲基化水平进行检测，可供广大实验室采用。Objective Improving the existed HPLC methods in order to detect the levels of global DNA methylation rapidly, stably and conveniently. Methods The HP1100 high performance liquid chrom- atographic （HPLC）system was used in this study. The analytic column was Macherey-Nagel （MN） EC 250/4.6 Nucleosil 100- 5SA （250mm×4.6mm5μm） cation exchange chromatographic column. We used 60raM acetic acid ＋ 15% acetonitrile as mobile phase （adjust to pH = 4.6 by NaOH）. The flow rate was 1.0 ml/min, detective UV wavelength was set to 276 nm and column temperature was set to 28℃. The injection volume was 50μl. The global DNA methylation was expressed as 5mdC/（dC ＋ 5mdC） × 100%. Results Under these conditions, we can isolate dC and 5mdC completely in ten minutes. The level of leukocyte global DNA methylation in healthy people is （4. 389 ± 0. 0159）%. Conclusions This method can determine the levels of global DNA methylation rapidly, and it can be widely applied in some laboratories.

关 键 词：色谱法 离子交换 基因组 DNA甲基化 

分 类 号：R440[医药卫生—诊断学]