Hairpin amplicon cleavage PCR (HAC-PCR): A novel, highly sensitive and cost-effective approach for detection of low-frequency DNA mutations

放大器 分子生物学 多重聚合酶链反应 生物 多重位移放大 突变体 聚合酶链反应 数字聚合酶链反应 多路复用 DNA 基因 DNA提取 遗传学
作者
Rui Zhang,Yuxuan Zhang,Ji Li,Lingli Gong,Yong Ji,Fengsheng Zhou,Ying Yin,Koukou Li,Ping Sun,Zhening Pu,Zhaocheng Liu,Jian Zou
出处
期刊:Microchemical Journal [Elsevier BV]
卷期号:196: 109654-109654 被引量:1
标识
DOI:10.1016/j.microc.2023.109654
摘要

Highly sensitive detection of gene variants holds immense significance for cancer diagnosis and treatment. However, the presence of highly redundant wild DNA poses a substantial challenge when identifying low-frequency mutant circulating tumor DNA (ctDNA) in cancer cases. Here, a novel technique called hairpin amplicon cleavage PCR (HAC-PCR) for detecting low-level DNA mutations with exceptional sensitivity was presented. What sets HAC-PCR apart is its ability to achieve high sensitivity independently of strand-displacement, base modification, or the use of additional reagents. The discrimination between wild-type targets (WT) and mutant targets (MT) is significantly improved by introducing uniquely designed hairpin structures. These hairpins hinder the amplification of WT and are cleaved during the elongation of MT. For the first time, the unique mechanism of hairpin amplicon cleavage by Taq polymerase was exploited for mutation identification. The optimized HAC-PCR consistently demonstrates the capability to discriminate mutant DNA at levels as low as 0.01% variant allele fraction (VAF) for five mutation types. Furthermore, this impressive sensitivity of 0.01% VAF has also been achieved in a multiplex assay. When applied to the testing of tissue DNA from 24 patients with thyroid cancers and plasma ctDNA from 12 patients with non-small cell lung cancers (NSCLCs), HAC-PCR exhibited a 100% concordance rate with the existing commercial kit and droplet digital PCR (ddPCR). Therefore, HAC-PCR stands as a cost-effective and highly effective tool for variant identification in clinical testing.
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