Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi'an 710062, China.
Biosens Bioelectron. 2017 May 15;91:328-333. doi: 10.1016/j.bios.2016.12.039. Epub 2016 Dec 16.
5-hydroxymethylcytosine (5hmC) is the sixth base of DNA. It is involved in active DNA demethylation and can be a marker of diseases such as cancer. In this study, we developed a simple and sensitive 2-(4-boronophenyl)quinoline-4-carboxylic acid modified poly (glycidyl methacrylate (PBAQA-PGMA) fluorescent probe to detect the 5hmC content of genomic DNA based on T4 β-glucosyltransferase-catalyzed glucosylation of 5hmC. The fluorescence-enhanced intensity recorded from the DNA sample was proportional to its 5-hydroxymethylcytosine content and could be quantified by fluorescence spectrophotometry. The developed probe showed good detection sensitivity and selectivity and a good linear relationship between the fluorescence intensity and the concentration of 5 hmC within a 0-100nM range. Compared with other fluorescence detection methods, this method not only could determine trace amounts of 5 hmC from genomic DNA but also could eliminate the interference of fluorescent dyes and the need for purification. It also could avoid multiple labeling. Because the PBAQA-PGMA probe could enrich the content of glycosyl-5-hydroxymethyl-2-deoxycytidine from a complex ground substance, it will broaden the linear detection range and improve sensitivity. The limit of detection was calculated to be 0.167nM after enrichment. Furthermore, the method was successfully used to detect 5-hydroxymethylcytosine from mouse tissues.
5-羟甲基胞嘧啶（5hmC）是 DNA 的第六种碱基。它参与了活性 DNA 去甲基化过程，并且可以作为癌症等疾病的标志物。在本研究中，我们开发了一种简单灵敏的 2-(4-硼苯基)喹啉-4-羧酸修饰的聚（甲基丙烯酸缩水甘油酯）（PBAQA-PGMA）荧光探针，基于 T4 β-葡萄糖基转移酶催化的 5hmC 的葡萄糖基化作用，用于检测基因组 DNA 中的 5hmC 含量。从 DNA 样品中记录到的荧光增强强度与其 5-羟甲基胞嘧啶含量成正比，可以通过荧光分光光度法进行定量。所开发的探针具有良好的检测灵敏度和选择性，并且荧光强度与 5 hmC 浓度在 0-100nM 范围内呈良好的线性关系。与其他荧光检测方法相比，该方法不仅可以从基因组 DNA 中测定痕量的 5 hmC，还可以消除荧光染料的干扰和纯化的需要。它还可以避免多重标记。由于 PBAQA-PGMA 探针可以从复杂的基质中富集糖苷-5-羟甲基-2-脱氧胞苷的含量，因此会拓宽线性检测范围并提高灵敏度。富集后的检测限计算为 0.167nM。此外，该方法成功用于检测小鼠组织中的 5-羟甲基胞嘧啶。
