化学
核酸
核酸内切酶
基因组DNA
检出限
单核苷酸多态性
分子生物学
核苷酸
DNA
基因
酶
限制性酶
DNA连接酶
生物化学
计算生物学
遗传学
生物
基因型
色谱法
作者
Xuanhao Zhang,Qian Li,Qiqi Chao,Yuxi Zhang,Xufeng Sun,Gao‐Chao Fan,Zhiling Song,Rongmei Kong,Xiliang Luo
标识
DOI:10.1016/j.aca.2023.340811
摘要
To establish protein enzyme-free and simple approach for sensitive detection of single nucleotide polymorphisms (SNPs), the nucleic acid amplification reactions were developed to reduce the dependence on protein enzymes (polymerase, endonuclease, ligase). These methods, while enabling highly amplified analysis for the short sequences, cannot be generalized to long genomic sequences. Herein, we develop a protein enzyme-free and general SNPs assay based on asymmetric MNAzyme probes. The multi-arm probe (MNAzyme-9M-13) with two asymmetric recognition arms, containing a short (9 nt) and a long (13 nt) arm, is designed to detect EGFR T790 M mutation (MT). Owing to the excellent selectivity of short recognition arm, MNAzyme-9M-13 probe can efficiently avoid interferences from wild-type target (WT) and various single-base mutations. Through a one-pot mixing, MNAzyme-9M-13 probe enables the sensitive detection of MT, without protein enzyme or multi-step operation. The calculated detection limit for MT is 0.59 nM and 0.83%. Moreover, this asymmetric MNAzyme strategy can be applied for SNPs detection in long genomic sequences as well as short microRNAs (miRNAs) only by changing the low-cost unlabeled recognition arms. Therefore, along with simple operation, low-cost, protein enzyme-free and strong versatility, our asymmetric MNAzyme strategy provides a novel solution for SNPs detection and genes analysis.
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