二代测序应用于检测GT1-7细胞中KISS1和GnRH启动子的DNA甲基化状态的研究

目的:利用二代测序技术检测GT1-7细胞中KISS1和GnRH基因启动子范围内的甲基化状态,并用金标准的亚硫酸氢盐修饰后的克隆测序作为对照,比较二代测序与金标准克隆测序在研究DNA甲基化检测中的差别。方法:提取GT1-7细胞基因组DNA并进行亚硫酸氢盐处理。进行巢式PCR,将PCR产物进行二代测序。同时采用金标准的亚硫酸氢盐修饰后克隆测序的方法作为对照,对相同批次的PCR产物进行克隆测序。结果:PCR产物二代测序结果表明KISS1和GnRH两个基因的27个CpG甲基化位点信息完整,结果准确。挑取10个克隆进行一代测序结果表明序列无丢失,KISS1和GnRH两个基因的27个CpG甲基化位点信息完整。结论:利用高通量的二代测序技术能够有效的对DNA甲基化的PCR产物进行检测,二代测序和克隆测序都是研究DNA甲基化的有效方法,但前者与克隆测序相比每一个读取序列(reads)都相当于一个单克隆,且二代测序每个区段得到成百上千个reads,因此二代测序结果更加精确。

Research on the Application of Next Generation Sequencing for the Detection of Promoter Methylation Status of KISS1 and GnRH in GT1-7 Cell

ABSTRACT Objective: The next generation sequencing(NGS) technology was used to detect the methylation status of the promoter region of KISS1 and GnRH gene in GT1-7 cell, and the clone sequencing was used as a control.Compare the diffenence between the next generation sequencing and clone sequencing in DNA methylation detection. Methods: Genome DNA was extracted from GT1-7 cells and treated with heavy hydrogen and hydrogen sulfate. Then the Nested PCR were performed and the PCR products were used for the next generation sequencing. At the same time, the same batch of purified PCR products were cloned into pGM-T Fast vector and proceed to Chemical Transformation. Positive clones were selected for Sanger sequencing analysis. Results: In the next generation sequencing of PCR production, the information of 27 CpG methylation sites was complete and the results were accurate. In clone sequencing, there is no loss of sequence and methylation information of all CpG sites was complete. Conclusion: The next generation sequencing and clone sequencing are effective methods for the research of DNA methylation, compared with clone sequencing, the next generation sequencing each reads is equivalent to a monoclonal and the next generation sequencing get thousands of reads, so the next generation sequencing re- sults are more accurate.