Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification.
Selective detection of N6-methyladenine in DNA via metal ion-mediated replication and rolling circle amplification.
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通过金属离子介导的复制和滚环扩增选择性检测 DNA 中的 N6-甲基腺嘌呤
DOI：
10.1039/c6sc02271e
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发表时间：
2017-01-01
期刊：
影响因子：
8.4
通讯作者：
Zhou X
中科院分区：
文献类型：
--
作者：
Hong T;Yuan Y;Wang T;Ma J;Yao Q;Hua X;Xia Y;Zhou X
由于A-Ag+-C与6 mA-Ag +-C在稳定性上的显著差异，6 mA在DNA中可与A区分开。N6-甲基腺嘌呤（6 mA）是真核生物基因组中一个潜在的表观遗传标记。然而，准确识别6 mA在DNA中的位置仍然是一项具有挑战性的任务。在这里，我们表明Ag+可以选择性地稳定A-C错配，并有效地促进引物延伸。相反，6 mA-Ag +-C的复合物是不稳定的，因此不能被DNA聚合酶识别，导致引物延伸终止。基于这一发现，我们成功地确定和定量6 mA在单碱基水平上通过分析的凝胶带的延伸引物和荧光测量结合滚环扩增。该方法的高选择性和高灵敏度为今后DNA中6 mA的高效分析提供了新的平台。
6mA can be discriminated from A in DNA due to the remarkable difference in stability between A–Ag+–C and 6mA–Ag+–C. N6-methyladenine (6mA) is reported as a potential epigenetic marker in eukaryotic genomes. However, accurate identification of the location of 6mA in DNA remains a challenging task. Here, we show that Ag+ can selectively stabilize the A–C mismatch and efficiently promote primer extension. In contrast, the complex of 6mA–Ag+–C is instable and therefore cannot be recognized by DNA polymerases, resulting in the termination of primer extension. Based on this finding, we successfully identified and quantified 6mA at the single-base level through the analysis of gel bands of extended primers and fluorescence measurements combined with rolling circle amplification. The high selectivity and sensitivity of this strategy may provide a new platform for the efficient analysis of 6mA in DNA in the future.