Department of Chemistry and Institute for Biophysical Dynamics, University of Chicago, 929 E 57th Street, Chicago, Illinois 60637, USA.
Nat Commun. 2013;4:1517. doi: 10.1038/ncomms2527.
5-methylcytosine is an epigenetic mark that affects a broad range of biological functions in mammals. The chemically inert methyl group prevents direct labelling for subsequent affinity purification and detection. Therefore, most current approaches for the analysis of 5-methylcytosine still have limitations of being either density-biased, lacking in robustness and consistency, or incapable of analysing 5-methylcytosine specifically. Here we present an approach, TAmC-Seq, which selectively tags 5-methylcytosine with an azide functionality that can be further labelled with a biotin for affinity purification, detection and genome-wide mapping. Using this covalent labelling approach, we demonstrate high sensitivity and specificity for known methylated loci, as well as increased CpG dinucleotide coverage at lower sequencing depth as compared with antibody-based enrichment, providing an improved efficiency in the 5-methylcytosine enrichment and genome-wide profiling.
5- 甲基胞嘧啶是一种表观遗传标记，影响哺乳动物的广泛的生物学功能。化学惰性的甲基基团阻止了直接标记，从而无法进行后续的亲和纯化和检测。因此，目前大多数分析 5- 甲基胞嘧啶的方法要么存在密度偏差，要么缺乏稳健性和一致性，要么无法特异性地分析 5- 甲基胞嘧啶。在这里，我们提出了一种方法，TAmC-Seq，它可以选择性地用叠氮化物功能基团标记 5- 甲基胞嘧啶，该基团可以进一步用生物素标记进行亲和纯化、检测和全基因组作图。使用这种共价标记方法，我们在已知的甲基化基因座上表现出高灵敏度和特异性，并且与基于抗体的富集相比，在较低的测序深度下增加了 CpG 二核苷酸的覆盖度，从而提高了 5- 甲基胞嘧啶的富集和全基因组分析的效率。
