作者：高运华¹, 陈鸿飞², 盛灵慧¹, 武利庆¹, 王晶¹

作者单位：1. 中国计量科学研究院, 北京 100029;
2. 南京市计量监督检测院, 江苏 南京 210037

关键词：DNA甲基化;同位素稀释质谱;Sanger测序;国际比对

摘要：

建立基因组DNA基质中CDKN2A基因甲基化含量的液相色谱同位素稀释质谱法和Sanger序列测定法两种定量方法, 应用于国际物质量咨询委员会(consultative committee for amount of substance, CCQM)开展的DNA甲基化定量测量国际比对(CCQM P94.2)研究。考察DNA甲基化效率、酶解条件、色谱分离条件等对测定结果的影响, 评定测量结果的不确定度。液相色谱同位素稀释质谱定量两个比对未知样的结果分别为未知样1:64.5%6.1%;未知样2:46.7%8.0%。Sanger序列测定法测定两个比对未知样的结果分别为未知样1:48.9%1.0%;未知样2:48.9%1.1%。与主导实验室标准值未知样1:51%5.9%, 未知样2:46.9%4.9%十分符合, 为我国DNA甲基化定量测量标准物质的研制奠定坚实的基础。

International comparison CCQM P94.2:quantitative analysis of DNA methylation

GAO Yunhua¹, CHEN Hongfei², SHENG Linghui¹, WU Liqing¹, WANG Jing¹

1. National Institute of Metrology, Beijing 100029, China;
2. Nanjing Institute of Measurement and Technology, Nanjing 210037, China

Abstract: Two quantitative methods namely liquid chromatography-isotope dilution mass spectrometry (HPLC-IDMS) and Sanger sequencing method(SS) have been established to analyze and determine the CDKN2A gene methylation in the matrix of genomic DNA. The two methods, applied to the international comparison (CCQM P94.2) on quantitative measurement of DNA methylation carried out by the Consultative Committee for the Amount of Substance (CCQM), has investigated the influence of the DNA methylation efficiency, enzymolysis conditions and chromatographic separation conditions upon the measurement results and evaluated the uncertainty in measurement results. The DNA methylation of two unknown comparison samples measured were respectively U1:64.5%±6.1% and U2:46.7%±8.0% by HPLC-IDMS and U1:respectively 48.9%±1.0% and U2:48.9%±1.1% by the SS method. These figures are very close to the reference values from the leading laboratory, i.e. U1:51%±5.9% and U2:46.9%±4.9%. The established HPLC-IDMS and SS method have laid a solid foundation for China to quantitatively measure the DNA methylation in reference materials.

Keywords: DNA methylation;isotope dilution mass spectrometry;Sanger sequencing;international comparison

2015, 41(6): 47-51  收稿日期: 2014-11-3;收到修改稿日期: 2015-1-17

基金项目: 国家质检行业公益专项项目资助(201310008)中央级公益性科研院所基本科研业务费项目资助(AKY1311)

作者简介: 高运华(1972-),男,山东定陶县人,副研究员,主要从事核酸计量及相关的法制计量工作。

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