We presented a low-abundance mutation detection method with lambda exonuclease and DNA three-way junction structure. The assistant strand in the DNA three-way junction structure could regulate the reaction system from the kinetics and thermodynamics aspects. The optimization of the assistant strand helps to improve the selectivity of the mutant-type DNA to the wild-type DNA about 35 times. Moreover, the cost of the optimization process could be saved by about 90%. The method was applied to the detection of a human ovarian cancer-related gene mutation BRCA1 (rs1799949, c.2082C>T). The limit of detection to the mutation abundance in the DNA three-way junction structure system (0.2%) was one order lower compared with that in the double-stranded DNA structure system (2%). The mutation abundance in different standard samples was quantitively measured, and the results were consistent with the initial abundance in the standard samples.

我们提出了一种带有λ核酸外切酶和DNA三向连接结构的低丰度突变检测方法。DNA三向连接结构中的辅助链可以从动力学和热力学方面调节反应系统。辅助链的优化有助于将突变型DNA对野生型DNA的选择性提高约35倍。此外，优化过程的成本可以节省约90％。该方法用于检测人类卵巢癌相关基因突变BRCA1（rs1799949，c.2082C> T）。与三链DNA结构系统（2％）相比，DNA三元连接结构系统（0.2％）的突变丰度检测限低一级。定量测量了不同标准样品中的突变丰度，其结果与标准样品中的初始丰度一致。