基于DNA三向接合结构和Lambda核酸外切酶辅助的低丰度基因突变检测研究

Genetic point mutations are related to many diseases and regarded as important biomarkers for various cancers. Liquid biopsy based on mutation detection in the circulating cell-free DNA in the blood of the cancer patient has advantages of minimally invasive, simple, fast and easy to be used at early stage of the cancer. DNA probe hybridization-based mutation detection methods do not have high selectivity and high sensitivity at the same time so that the limit of detection is not suitable for the requirement of cancer-related mutation detection in the cell-free DNA which can be as low as 0.001%. In the previous work, the applicant has developed a low-abundance mutation detection method with Lambda exonuclease. The applicant has also found that the modification at the 5’-end of the DNA could inhibit the side activity of Lambda exonuclease. Based on these works, the applicant designed a new low-abundance mutation detection method in this project. In the method, Lambda exonuclease will work as the bridge to link the mutation detection and exponential amplification. Together with the design of DNA three-way junction structure, this method will solve the problem about the linkage among mutation recognition by DNA strands structure, mutation recognition by the enzyme and the exponential signal amplification. In this way, mutation abundance at 0.001% could be detected. In the project, the applicant plans to detect cancer-related mutations from 50 to 100 endometrial cancer patients and provide a powerful tool for diagnosis and therapy for cancers at the early stage and for individual precise medicine.

基因突变与疾病密切相关，是众多癌症的重要生物标志物。基于癌症病人血液中循环游离DNA检测的液体活检技术具有微创性、简单快速、可在发病早期检测等优点。然而，目前基于DNA探针杂交的突变检测方法难以兼具高选择性和高灵敏度，无法满足循环游离DNA中癌症相关基因突变（突变链比例低至0.001%）的检测需求。申请人在前期研究和预实验中开发了Lambda核酸外切酶辅助的低丰度基因突变检测方法，发现了通过DNA的5’末端修饰抑制Lambda核酸外切酶副活性的性质。本项目以此为基础开发了低丰度基因突变检测新方法，方法以Lambda核酸酶为桥梁连接突变识别和指数放大，辅以DNA三向接合结构设计，解决DNA链结构识别突变、酶识别突变和指数放大的衔接难题，有望使突变丰度检测限低至0.001%。本项目将检测50至100例子宫内膜癌患者的癌症相关基因突变，为癌症的早期诊断治疗和个体化的精准医疗提供强有力的工具。

基因突变检测有助于疾病的早期诊断和治疗。基于DNA探针杂交的突变检测方法设计较简单、成本较低，有望用于即时常规检测。DNA探针杂交中，常引入额外核酸酶的辅助以提升方法的选择性和灵敏度。然而，链结构识别和核酸酶识别不易优化，存在背景信号干扰，使得基于DNA探针杂交的突变检测方法难以兼具高选择性和高灵敏度。本项目详细探究了Lambda核酸外切酶的性质，发现Lambda核酸外切酶可以水解含有5’-PO4末端的单链，且对不同的5’末端底物具有不同的活性，其水解速率具有5’-PO4 > 5’-FAM > 5’-OH的关系等。利用Lambda核酸外切酶的特性，可以控制Lambda核酸外切酶的水解活性，更好地设置底物结构，降低背景信号。本项目开发了基于催化发夹自组装、末端脱氧核糖核酸转移酶和核酸内切酶IV等构建的多种二次放大和指数放大方法。这些方法的构造较为简单，可以检测pM级浓度的目标链，且具有碱基错配区分的能力，可以用于生物传感和基因突变检测。本项目建立了 DNA 三向接合结构识别突变的理论模型，从理论上阐述 DNA 三向接合结构相较于传统双链结构在条件优化、选择性提升上的优势。根据理论预测，固定目标链和探针链，在一定范围内优化辅助链，利用 Lambda核酸外切酶辅助水解探针链检测荧光信号得到反应体系的最佳区分度，完成突变检测。由于辅助链没有昂贵的标记，它比探针便宜了约90％，这大大节省了优化成本。项目还探究了辅助链对Endo IV辅助的基因突变检测影响，开发了脱氧核酶马达作为信号产生的平台，拓展了基因突变检测的工具。本项目开发基因突变检测成功用于BRCA1 c.2082C> T、EGFR T790M、EGFR L858R等与癌症密切相关的突变检测中，并在临床样品中进行了验证，突变丰度检测限可达0.005%。