小鼠外周组织时钟基因启动子区甲基化分析

 摘要：目的 建立一种简单快速经济的检测八个主要时钟基因BMAL1、BMAL2、CLOCK、NPAS2、PER1、PER2、CRY1和CRY2启动子区甲基化状态的方法。同时检测小鼠外周组织肝、心、肾、胸腺、睾丸时钟基因启动子区甲基化状态。方法 用偏重亚硫酸氢钠和对苯二酚对基因组DNA进行脱氨基修饰。修饰后的DNA为模板，两套不同的引物对：甲基化特异性引物对和非甲基化特异性引物扩增小鼠肝、心、肾、胸腺、睾丸组织时钟基因启动子区。PCR产物进行电泳和测序。结果 扩增产物与预期片段大小相符合。PCR产物经过直接测序得到进一步证实。结论 成功建立了检测小鼠时钟基因启动子区甲基化的方法，为检测小鼠周围组织时钟基因启动子区甲基化提供了新的方法。同时，成年小鼠所有的八个时钟基因均为非甲基化状态。

Promoter methylation analysis of clock genes in mice peripheral tissues

Abstract: Objective To establish a simple, rapid and inexpensive method to determine the methylation status of eight key clock genes, BMAL1, BMAL2, CLOCK, NPAS2, PER1, PER2, CRY1 and CRY2. To examine the methylation of clock promoters in five peripheral tissues with the developed method. Methods Genome DNA was deaminated by Sodium metabisulfite solution and hydroquinone. The two sets of PCR primers for unmenthylated and methylated DNA, respectively, were used to amplify the promoter of clock genes. Polymerase chain reaction (PCR) products were then loaded and electrophoresed on 3% agarose gels. The PCR products for each reaction were sequenced directly. Results Specific amplicons with correct size were amplified. PCR products were also confirmed by sequencing. Conclusion In the present study, a new method of detection of the methylation of clock genes was successfully established; it may apply a new technique for clock genes promoter methylation detection. All of the clock promoters are free from methylation in the adult mouse.