索引
分子生物学
等位基因
数字聚合酶链反应
塔克曼
寡核苷酸
化学
遗传学
DNA
聚合酶链反应
单核苷酸多态性
基因
生物
基因型
作者
Kerou Zhang,L. Raposo Rodríguez,Lauren Y. Cheng,Michael Wang,David Y. Zhang
标识
DOI:10.1021/acs.analchem.1c03716
摘要
Clinically and biologically, rare DNA sequence variants are significant and informative. However, existing common detection technologies are either complex and time-consuming in workflow, or restricted in the limit of detection (LoD), or do not allow for multiplexing. Blocker displacement amplification (BDA) method can stably and effectively detect and enrich multiple rare variants with LoD around 0.1% variant allele fraction (VAF). Nonetheless, the detailed mutation information has to be identified by additional sequencing technologies. Here, we present allele-specific BDA (As-BDA), a method combining BDA with allele-specific TaqMan (As-TaqMan) probes for effective variant enrichment and simultaneous single nucleotide variant or small insertions and deletions (INDELs) profiling. We demonstrated that As-BDA could detect mutations down to 0.01% VAF. Further, As-BDA could detect up to four mutations with low to 0.1% VAF per reaction using only 15 ng DNA input. The median error of As-BDA in VAF determination is approximately 9.1%. Comparison experiments using As-BDA and droplet digital PCR on peripheral blood mononuclear cell clinical samples showed 100% concordance for samples with mutations at ≥ 0.1% VAF. Hence, we have shown that As-BDA can achieve simultaneous enrichment and identification of multiple targeted mutations within the same reaction with high clinical sensitivity and specificity, thus helpful for clinical diagnosis.
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