摘要
目的:大量研究证实线粒体DNA(mtDNA)突变与肿瘤发生及进展密切相关,但使用传统测序方法难以高通量、高精确度的检测mtDNA突变,为此本研究建立了基于新一代测序技术的mtDNA突变检测方法。方法:提取肝癌患者癌、癌旁组织以及外周血细胞总DNA,利用PCR技术对线粒体基因组进行富集并对PCR产物进行平末端、粘性末端连接或对PCR引物进行氨基修饰,构建mtDNA测序文库。经Illumina HiSeq 2000平台测序后利用生物信息学方法与人类mtDNA参考序列进行比对,并进行测序数据分析。结果:通过对不同质量基因组DNA进行评估后,发现三对引物法适用于大部分DNA样本的mtDNA富集。进一步我们发现PCR引物的氨基修饰可显著提高测序数据覆盖均一性,降低测序成本。结论:本研究利用新一代测序技术通过对线粒体DNA富集方法以及测序覆盖度均一性进行优化,建立了一套灵敏、特异、高通量的mtDNA突变检测策略,为mtDNA突变与疾病研究提供了新方法。
Objective： Previous studies proved that somatic mutations of mitochondrial DNA（mtDNA） are strongly associated with tumorigenesis and tumor progression.However,conventional sequencing methods are not able to detect mtDNA mutations with high-throughput and high accuracy.In this study,we established a new approach for detecting mutations in mtDNA based on next-generation sequencing technology.Methods： mtDNA were amplified by PCR using genome DNA samples obtained from tumor tissue,paired normal tissue or peripheral blood.The PCR products went through blunt end ligation,sticky end ligation,or amino-modification before sequencing library preparation.The mtDNA libraries were sequenced utilizing Illumina HiSeq 2000 platform.The sequences were then compared with human mtDNA referencing sequences,and further analysis using bioinformatical methods.Results： After assessing the genomic DNA samples with different qualities,we found that using three pairs of PCR primers is applicable to most mtDNA enrichment reactions.We also found the amino-modified PCR primers could significantly improve the coverage uniformity,and reduce the sequencing costs.Conclusion： A method to optimize mtDNA enching and the coverage uniformity has been built up in this study based on next-generation sequencing technology.Further a sensitive mtDNA mutation detecting system with high specificity and high-throughput was aslo established.This system may provide a new method for research in mtDNA mutation detection and disease association studies.
出处
《现代生物医学进展》
CAS
2013年第13期2433-2436,共4页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81171966)
关键词
线粒体DNA
突变
新一代测序技术
Mitochondrial DNA
Mutation
Next-generation sequencing
