两核苷酸合成测序及其在PCR产物分析中的应用研究

DNA sequencing technology has played important roles in modern biological research and it is a frontier research of interdisciplinary studies.The project is to develop a novel real-time sequencing system which is compatible with the existing commercialized sequencing systems. The system is based on the principle that the same kind of molecules is produced even when different types of nucleotides are incorporated into the sequencing primer. Several important issues are explored here. Firstly, the sequencing reactions are optimized, and to increase the sequencing read length and accuracy. It will provide a potential strategy for high-throughput DNA sequencings. Secondly, in this system queried templates are determined without directly measuring base sequences but the code information that give a specific pattern obtained in a single run. And the signal intensities and the read length of each step will be enhanced due to the simultaneous addition of dual mononucleotide into the reaction. These advantages enables the system to be applied to analyze PCR products, and to determinate the molecular markers, providing an alternative way to accurately analyze viruses and microorganisms in the areas of food security and public health. The investigatin of the real-time sequencing strategy based on dual mononucleotide addition has important application value and scientific significance for developing new sequencing methods and related theory.

DNA测序技术是现代生命科学研究中重要的手段之一,是多学科交叉的前沿研究。本项目针对实时合成测序方法的不足，基于不同核苷酸合成反应产生完全相同的检测分子的原理，提出适合第一代、二代和三代实时合成测序平台的两核苷酸合成测序技术并进行系统研究。1)通过优化两核苷酸的循环合成测序的反应条件，大幅度增加测序技术的的阅读长度和准确性，为现有高通量DNA实时测序平台提供一种新策略；2)利用两核苷酸合成测序长、具有信号放大，以及一次合成测序得到的信息由两类编码组成等特点，将这种基于具有唯一性的模式识别而非序列识别的合成测序技术应用到PCR产物的传统焦测序检测中，为疾病核酸分子标志物，以及食品安全、公共卫生等领域的病毒和微生物的分析提供新途径。开展两核苷酸合成测序及其应用研究，对于发展新测序方法及其相关理论具有非常主要的应用价值和科学意义。

在本自然科学基金的资助下，本项目主要开展了以下研究工作：1）研究了两核苷酸合成高通量DNA测序的可行性、特征及其不足的应对策略，为两核苷酸合成高通量DNA测序新策略提供了理论上支撑。2）研究了两核苷酸合成在第一代焦测序平台上的应用，建立了比传统单核苷酸焦定性分析测序长度更长、定量分析检测限更低的两核苷酸合成焦测序技术，可用于核酸遗传分析。3）发展了基于两核苷酸测序的新分析方法、拓广了焦测序应用范围：a）提出一种单倍型定量分析方法；b) 提出一种单倍型定性分析方法；c) 提出一种用于样本与对照序列差异谱分析方法；d）提出一种基于样本组合分析多模板核酸序列的检测方法。4）开展了“探索两核苷酸合成测序新方法、及其应用新途径”研究：a）提出“新型两核苷酸合成测序”策略、有望成为最准确的测序技术。b）提出一种DNA存储的寡核苷酸可变编码方法，可对二进制文件编码成满足要求的寡核苷酸序列。c）研究“基于分析PCR副产物焦磷酸的特异PCR产物快速检测方法“，核酸检测限达到单个拷贝。.本项目共发表SCI收录期刊论文7篇、会议论文7篇；获授权发明专利9项、申请5项；软件著作权1件；培养硕士毕业研究生3人。