基于核酸内切酶IV和分支迁移系统的低丰度单碱基点突变的测定

Determination of low-abundance single-base point mutations based on endonuclease IV and branch migration system.

作者信息

Tang Xiaofeng, Chen Na, Liu Ruijie, Hu Qingyi, Liu Na, Xiao Xianjin

机构信息

Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China.

Institute of Reproductive Health, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, PR China; Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, 430022, PR China.

出版信息

Anal Chim Acta. 2020 Oct 16;1134:28-33. doi: 10.1016/j.aca.2020.08.014. Epub 2020 Aug 20.

DOI:10.1016/j.aca.2020.08.014

PMID:33059863

Abstract

Combining endonuclease IV and branch migration competition systems, we have designed a new single-base mutation detection method which is capable of thermostatic, sensitive, simple, and cost-effective at the same time, while other probe method based on endonuclease IV hardly achieve. Our method has better discrimination factors (6.81-83.10) and a low abundance detection limit of mutant-type DNA (MT) (0.05% mutant-type DNA/total DNA) without complex temperature optimize process. The concentration detection limit of MT is 0.03 nM.The abundance detection limit for EGFR L858R (0.1%) and PTEN R130Q (0.05%) in clinical samples suggested that the method has potential applications in clinical diagnosis.

摘要

结合核酸内切酶IV和分支迁移竞争系统，我们设计了一种新的单碱基突变检测方法，该方法能够同时实现恒温、灵敏、简单且经济高效，而其他基于核酸内切酶IV的探针方法很难做到这一点。我们的方法具有更好的区分因子（6.81 - 83.10），并且在无需复杂温度优化过程的情况下，对突变型DNA（MT）具有较低的丰度检测限（0.05%突变型DNA/总DNA）。MT的浓度检测限为0.03 nM。临床样本中EGFR L858R（0.1%）和PTEN R130Q（0.05%）的丰度检测限表明该方法在临床诊断中具有潜在应用价值。