直接测序与克隆测序检测不育男性H19基因甲基化印记的比较

目的建立亚硫酸氢盐修饰后测序技术,比较直接测序与克隆测序在不育男性精子印记基因DNA甲基化状态检测中的差别。方法对样本进行精液分析和精子形态学分析,密度梯度离心法制备精液。提取精液基因组DNA并行亚硫酸氢盐处理,行半巢式PCR,将纯化后PCR产物与pCR?2.1-TOPO?载体连接及转化,分别对PCR产物及阳性克隆菌液进行测序。结果 PCR产物直接测序,前半部分序列丢失约40bp,1号CpG位点甲基化状态信息丢失。每例标本只有一个测序结果,会出现CpG位点的部分甲基化状态。挑取15个克隆进行测序,序列无丢失,18个CpG位点甲基化状态信息均完整。15个克隆CpG位点甲基化状态有所不同,显示出此样本的平均甲基化状态。结论克隆测序不会造成序列丢失,CpG位点甲基化状态信息完整,克隆测序能较好地反应样本的平均甲基化状态。

A Comparison between direct sequencing and clone sequencing in detection for methylation imprinting of H19 gene in infertile males

Objective To establish bisulfite sodium sequencing technology. Compare the direct sequencing and clone sequencing in DNA methylation detection of imprinted genes in infertile male. Methods Routine semen analysis and morphological analysis were performed, sperm were purified by density gradient centrifugation and DNA was extracted and treated with bisulfite. Then a hemi-nest PCR was performed. The purified PCR products were cloned into pCR?2.1-TOPO? vector and proceed to Chemical Transformation. For each sample, PCR products and positive clones were selected for sequencing analysis. Results In direct sequencing of PCR products, the front of the sequence was lost about 40 bp and methylation information of CpG 1 was lost. There is only one sequence result in each sample, and there will present partial methylation in CpG sites. In Clone sequencing, there is no loss of sequence and methylation information of all CpG sites was complete. There is different methylation in 15 clones, which could show the average methylation of this sample. Conclusion Clone sequencing did not lose any sequence, the methylation information of CpG sites was complete. 15 clones selected from each sample for sequencing could show the average methylation of the sample.