DNA methylation drift as a marker of lifespan in mammals [mouse]

We analyzed levels of 5-methyl cytosine at CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of mouse whole blood genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the mouse genome.

本研究通过SmaI与XmaI酶的顺序限制性酶切，向限制性片段连接Illumina接头，并借助双端Illumina高通量测序读取限制性片段末端的甲基化特异性特征序列，以此分析CCCGGG靶位点处的5-甲基胞嘧啶(5-methyl cytosine)水平。本研究采用数字化甲基化限制性酶分析（Digital restriction enzyme analysis of methylation，DREAM）技术，对小鼠全血基因组DNA的甲基化谱进行测定。将未甲基化、部分甲基化及完全甲基化的标准品掺入基因组DNA后，使用甲基化敏感酶SmaI（产生平末端）及其耐受甲基化的新同裂酶(neoschizomer)XmaI（产生5'CCGG突出末端）在CCCGGG位点进行顺序酶切，由此生成可反映CCCGGG位点甲基化状态的不同末端序列。随后通过Illumina高通量测序对这些末端序列进行分析。将测序读段比对至小鼠参考基因组后，通过统计读段起始处带有CCGG特征序列的甲基化读段与带有GGG特征序列的未甲基化读段数量，即可确定全基因组范围内各CCCGGG位点的甲基化状态。