重叠延伸PCR生成长重复DNA序列及其在纳米孔测序中的应用  

Overlap-Extension PCR Generating Long Repetitive DNA Sequence and Its Application in Nanopore Sequencing

作　　者：宋子婷 程冰晓 胡坤灵 安然[1,2] 梁兴国 Song Ziting;Cheng Bingxiao;Hu Kunling;An Ran;Liang Xingguo(College of Food Science and Engineering,Ocean University of China,Qingdao 266404,China;Laboratory for Marine Drugs and Bioproducts,Qingdao Marine Science and Technology Center,Qingdao 266237,China)

机构地区：[1]中国海洋大学食品科学与工程学院,山东青岛266404 [2]青岛海洋科技中心海洋药物与生物制品功能实验室,山东青岛266237

出　　处：《中国海洋大学学报（自然科学版）》2024年第9期157-164,共8页Periodical of Ocean University of China

基　　金：国家自然科学基金项目(32102064);国家重点研究发展计划项目(2019YFD0901701)资助。

摘　　要：短链DNA的纳米孔测序存在孔道利用率低、数据质量差等问题。因此,亟需建立一种用纳米孔测序技术准确测定短链DNA序列的方法。本研究以1 nmol/L完全互补的序列为引物,以短链DNA为模板,在72℃退火温度下进行PCR,实现了短链DNA的重叠延伸,最终生成长串联重复序列,其产物长度为原始长度的5~20倍。生成的长链DNA可直接测序,仅需对齐单个或少数几个Reads进行简单分析即可获得精确的序列信息。本研究所开发的重叠延伸法将纳米孔One read精确度提高至99.28%,这对提高纳米孔测序的精度具有重要作用。Nanopore sequencing of short DNA fragments suffers from low pore utilization and poor data quality.It is urgent to establish a method of obtaining the sequence of short DNA fragments.In this study,PCR was performed at constant 72℃using 1 nmol/L fully complementary primers and short DNA fragments as the template.The overlapping extension of short DNA fragments ultimately generated long tandem repeats,5~20 times longer than the original.The long DNA constructs can be sequenced directly with the accurate original sequence obtained simply by aligning only a few reads.The developed overlap-extension method improved the one read accuracy of nanopore sequencing to 99.28%,which plays an important role in improving the accuracy of nanopore sequencing.

关 键 词：短链DNA 重复序列 纳米孔测序 重叠延伸 高精度 

分 类 号：Q819[生物学—生物工程]