Guerrero-Bosagna Carlos, Jensen Per
Avian Behavioral Genomics and Physiology Group, IFM Biology, Linköping University, Linköping 58 183, Sweden.
MethodsX. 2015 Oct 19;2:432-9. doi: 10.1016/j.mex.2015.10.006. eCollection 2015.
Methylated DNA immunoprecipitation (MeDIP) is one of the most widely used methods to evaluate DNA methylation on a whole genome scale, and involves the capture of the methylated fraction of the DNA by an antibody specific to methyl-cytosine. MeDIP was initially coupled with microarray hybridization to detect local DNA methylation enrichments along the genome. More recently, MeDIP has been coupled with next generation sequencing, which highlights its current and future applicability. In previous studies in which MeDIP was applied, the protocol took around 3 days to be performed. Given the importance of MeDIP for studies involving DNA methylation, it was important to optimize the method in order to deliver faster turnouts. The present article describes optimization steps of the MeDIP method. The length of the procedure was reduced in half without compromising the quality of the results. This was achieved by:•Reduction of the number of washes in different stages of the protocol, after a careful evaluation of the number of indispensable washes.•Reduction of reaction times for detaching methylated DNA fragments from the complex agarose beads:antibody.•Modification of the methods to purify methylated DNA, which incorporates new devices and procedures, and eliminates a lengthy phenol and chloroform:isoamyl alcohol extraction.
甲基化DNA免疫沉淀法（MeDIP）是在全基因组范围内评估DNA甲基化最广泛使用的方法之一，它涉及用一种针对甲基胞嘧啶的抗体捕获DNA的甲基化部分。MeDIP最初与微阵列杂交相结合，以检测沿基因组的局部DNA甲基化富集情况。最近，MeDIP已与下一代测序相结合，这突出了其当前和未来的适用性。在之前应用MeDIP的研究中，该方案执行大约需要3天时间。鉴于MeDIP对涉及DNA甲基化研究的重要性，优化该方法以更快得出结果很重要。本文描述了MeDIP方法的优化步骤。在不影响结果质量的情况下，该程序的时长缩短了一半。这是通过以下方式实现的：
在仔细评估必需洗涤次数后，减少方案不同阶段的洗涤次数。
减少从复合琼脂糖珠：抗体上分离甲基化DNA片段的反应时间。
改进纯化甲基化DNA的方法，该方法采用了新设备和程序，并省去了冗长的酚和氯仿：异戊醇提取步骤。
