Fraga Mario F, Uriol Esther, Borja Diego L, Berdasco María, Esteller Manel, Cañal María Jesús, Rodríguez Roberto
Laboratorio de Fisiología Vegetal, Dpt. B.O.S., Facultad de Biología, Universidad de Oviedo, Oviedo, Spain.
Electrophoresis. 2002 Jun;23(11):1677-81. doi: 10.1002/1522-2683(200206)23:11<1677::AID-ELPS1677>3.0.CO;2-Z.
A new approach to the evaluation of the relative degree of genomic DNA methylation through the quantification of 2'-deoxynucleosides is proposed. Detection and quantification of 5-methyl 2'-deoxycytidine in genomic DNA has been performed using micellar high-performance capillary electrophoresis (HPCE) with UV-Vis detection. This approach has been demonstrated to be more sensitive and specific than other HPCE methods for the quantification of DNA methylation degree and also to be faster than other HPLC-based methods. The detection and quantification of nucleosides through enzymatic hydrolyses notably increases the specificity of the technique and allows its exploitation in the analysis of poorly purified and/or concentrated DNA samples such as those obtained from meristematic plant regions and paraffin-embedded tissues.
提出了一种通过定量2'-脱氧核苷来评估基因组DNA甲基化相对程度的新方法。使用胶束高效毛细管电泳(HPCE)结合紫外-可见检测对基因组DNA中的5-甲基2'-脱氧胞苷进行了检测和定量。已证明该方法在DNA甲基化程度定量方面比其他HPCE方法更灵敏、更特异,并且比其他基于高效液相色谱(HPLC)的方法更快。通过酶促水解对核苷进行检测和定量显著提高了该技术的特异性,并使其能够用于分析纯化程度低和/或浓度低的DNA样品,例如从植物分生组织区域和石蜡包埋组织中获得的样品。