Stevens Aaron J, Kennedy Martin A
Department of Pathology, School of Medicine, University of Otago, Christchurch, New Zealand.
PLoS One. 2017 Jan 4;12(1):e0169433. doi: 10.1371/journal.pone.0169433. eCollection 2017.
The promoter region of the imprinted gene MEST contains several motifs capable of forming G-quadruplex (G4) structures, which appear to contribute to consistent allelic dropout during polymerase chain reaction (PCR) analysis of this region. Here, we extend our previous analysis of MEST G4 structures by applying fluorescent footprinting techniques to assess non B-DNA structure and topology in dsDNA from the full MEST promoter region, under conditions that mimic PCR. We demonstrate that the buffer used for PCR provides an extremely favourable milieu for G4 formation, and that cytosine methylation helps maintain G4 structures during PCR. Additionally, we demonstrate G4 formation at motifs not previously identified through bioinformatic analysis of the MEST promoter, and provide nucleotide level resolution for topological reconstruction of these structures. These observations increase our understanding of the mechanisms through which methylation and G4 contribute towards allelic drop-out during PCR.
印记基因MEST的启动子区域包含几个能够形成G-四链体（G4）结构的基序，这些基序似乎在该区域的聚合酶链反应（PCR）分析过程中导致一致的等位基因缺失。在此，我们通过应用荧光足迹技术扩展了之前对MEST G4结构的分析，以评估模拟PCR条件下来自完整MEST启动子区域的双链DNA中的非B-DNA结构和拓扑结构。我们证明用于PCR的缓冲液为G4形成提供了极其有利的环境，并且胞嘧啶甲基化有助于在PCR过程中维持G4结构。此外，我们证明在MEST启动子的生物信息学分析中先前未鉴定的基序处形成G4，并为这些结构的拓扑重建提供核苷酸水平的分辨率。这些观察结果增加了我们对甲基化和G4在PCR过程中导致等位基因缺失的机制的理解。
