Abstract：
Detecting low-frequency DNA mutations hotspots cluster is critical for cancer diagnosis but remains challenging. Quantitative PCR (qPCR) is constrained by sensitivity, and allele-specific PCR is restricted by throughput. Here we develop a long blocker displacement amplification (LBDA) coupled with qPCR for ultrasensitive and multiplexed variants detection. By designing long blocker oligos to perfectly match wildtype sequences while mispairing with mutants, long blockers enable 14-44 nt enrichment regions which is 2-fold longer than normal BDA in the experiments. For wild template with a specific nucleotide, LBDA can detect different mutation types down to 0.5 % variant allele frequency (VAF) in one reaction, with median enrichment fold of 1,000 on 21 mutant DNA templates compared to the wild type. We applied LBDA-qPCR to detect KRAS and NRAS mutation hotspots, utilizing a single plex assay capable of covering 81 mutations and tested in synthetic templates and colorectal cancer tissue samples. Moreover, the mutation types were verified through Sanger sequencing, demonstrating concordance with results obtained from next generation sequencing. Overall, LBDA-qPCR provides a simple yet ultrasensitive approach for multiplexed detection of low VAF mutations hotspots, presenting a powerful tool for cancer diagnosis and monitoring.
摘要：
检测低频DNA突变热点簇对于癌症诊断至关重要，但仍然具有挑战性。定量PCR（qPCR）受到灵敏度的限制，和等位基因特异性PCR受到通量的限制。在这里，我们开发了一种长阻断剂置换扩增（LBDA）与qPCR相结合，用于超灵敏和多重变体检测。通过设计长阻断剂寡核苷酸来完美匹配野生型序列，同时与突变体错配，在实验中，长阻断剂使14-44nt富集区域比正常BDA长2倍。对于具有特定核苷酸的野生模板，LBDA可以在一个反应中检测到低至0.5％变异等位基因频率（VAF）的不同突变类型，与野生型相比，在21个突变DNA模板上的中值富集倍数为1,000。我们应用LBDA-qPCR检测KRAS和NRAS突变热点，利用能够覆盖81个突变的单一plex分析，并在合成模板和结直肠癌组织样本中进行测试。此外，通过Sanger测序验证了突变类型，证明与下一代测序获得的结果一致。总的来说,LBDA-qPCR为低VAF突变热点的多重检测提供了一种简单而超灵敏的方法，为癌症诊断和监测提供了强大的工具。
