Beijing National Laboratory for Molecular Sciences, MOE Key Laboratory of Bioorganic Chemistry and Molecular Engineering, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871, China.
School of Pharmacy, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, China.
Anal Chem. 2021 May 11;93(18):7086-7093. doi: 10.1021/acs.analchem.1c00564. Epub 2021 Apr 26.
Sensitive detection of low-abundance driver mutations may provide valuable information for precise clinical treatment. Compared to next-generation sequencing and droplet digital PCR methods, fluorescent probes show great flexibility in rapid detection of specific mutations with high sensitivity and easily accessible instruments. However, existing approaches with fluorescent probes need an additional step to convert duplex DNA to single-stranded DNA (ssDNA) before the detection step, which increases the time, cost, and risk of loss of low-input target strands. In this work, we attempt to integrate the ssDNA-generation step with the subsequent detection into a programable one-pot reaction by employing lambda exonuclease (λ exo), a versatile nanopore nuclease which exercises different functions on different substrates. The capability of λ exo in discrimination of mismatched bases in 5'- FAM-ended 2 nt-unpaired DNA duplexes was first demonstrated. Specific fluorescent probes were developed for exon 19 E746-A750del and E545K mutations with discrimination factors as high as 8470 and 884, respectively. By mixing the probes and λ exo with the PCR products of cell-free circulating DNA extracted from plasma samples, the reaction was immediately initiated, which allowed sensitive detection of the two types of mutations at an abundance as low as 0.01% within less than 2 h. Compared to existing approaches, the new method has distinct advantages in simplicity, low cost, and rapidity. It provides a convenient tool for companion diagnostic tests and other routine analysis targeting genetic mutations in clinical samples.
灵敏检测低丰度驱动突变可为精准临床治疗提供有价值的信息。与下一代测序和液滴数字 PCR 方法相比，荧光探针在快速检测特定突变方面具有很大的灵活性，具有高灵敏度和易于获取的仪器。然而，现有的荧光探针方法需要在检测步骤之前额外进行一步，将双链 DNA 转化为单链 DNA（ssDNA），这增加了时间、成本和低输入靶链丢失的风险。在这项工作中，我们尝试通过使用 λ 外切核酸酶（λ exo）将 ssDNA 生成步骤与随后的检测集成到一个可编程的一锅反应中，λ exo 是一种多功能的纳米孔核酸内切酶，在不同的底物上发挥不同的功能。首先证明了 λ exo 在区分 5'- FAM 末端 2 个碱基未配对 DNA 双链中的错配碱基的能力。针对外显子 19 E746-A750del 和 E545K 突变开发了特异性荧光探针，其区分因子分别高达 8470 和 884。通过将探针和 λ exo 与从血浆样本中提取的无细胞循环 DNA 的 PCR 产物混合，反应立即开始，在不到 2 小时的时间内，即可在低至 0.01%的丰度下灵敏地检测到两种类型的突变。与现有的方法相比，新方法在简单性、低成本和快速性方面具有明显的优势。它为针对临床样本中基因突变的伴随诊断测试和其他常规分析提供了一种方便的工具。
