Selective Derivatization of Cytosine and Methylcytosine Moieties with 2-Bromoacetophenone for Submicrogram DNA Methylation Analysis by Reversed Phase HPLC with Spectrofluorimetric Detection
Selective Derivatization of Cytosine and Methylcytosine Moieties with 2-Bromoacetophenone for Submicrogram DNA Methylation Analysis by Reversed Phase HPLC with Spectrofluorimetric Detection
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DOI：
10.1021/ac2020799
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发表时间：
2011-10-15
影响因子：
7.4
通讯作者：
Wrobel, Kazimierz
中科院分区：
文献类型：
--
作者：
Lopez Torres, Adolfo;Yanez Barrientos, Eunice;Wrobel, Kazimierz
在真核生物中，实际的DNA甲基化模式提供了生物学上重要的信息，因此，胞嘧啶残基的全基因组和基因座特异性甲基化已经被广泛研究。这项工作的原始贡献依赖于胞嘧啶部分与2-溴苯乙酮的选择性衍生化，通过反相高效液相色谱荧光检测测定全球DNA甲基化。拟议程序的重要特点如下：（1）无需消除RNA，（2）胞苷、2 '-脱氧胞苷、5-甲基胞苷和5-甲基-2'-脱氧胞苷的检测限在14.4-22.7 fmol范围内，（3）检测的可行性少量DNA（80 ng）中0.06%的甲基化，（4）用于评估RNA甲基化的潜在可行性，和（5）在分析仪器和人员培训方面相对简单。鲑鱼睾丸DNA和核酸的分析中获得的结果从植物，人的血液，蚯蚓）证明了生物学研究中的实用程序，特别是环境因素对实际的DNA甲基化在不同类型的生物体的潜在影响的评估。
In eukaryotes, actual DNA methylation patterns provide biologically important information, for which both, genome-wide and locus-specific methylation at cytosine residues have been extensively studied. The original contribution of this work relies on the selective derivatization of cytosine moieties with 2-bromoacetophenone for the determination of global DNA methylation by reversed phase high performance liquid chromatography with spectrofluorimetric detection. The important features of the proposed procedure are as follows: (1) no need for the elimination of RNA, (2) detection limits for cytidine, 2'-deoxycytidine, 5-methylcyticline, and 5-methyl-2'-deoxycytidine in the range of 14.4-22.7 fmol, (3) feasibility for the detection of 0.06% of methylation in a low amount of DNA (80 ng), (4) potential viability for the evaluation of RNA methylation, and (5) relative simplicity in terms of analytical instrumentation and personnel training. The results obtained in the analysis of salmon testes DNA and nucleic acids from plant, human blood, and earthworms)demonstrate the utility of the proposed procedure in biological studies and, in particular, for evaluation of the potential effect of environmental factors on actual DNA methylation in different types of living organisms.