Applying amplification refractory mutation system technique to detecting cell-free fetal DNA for single-gene disorders purpose

• DOI: 10.3389/fgene.2023.1071406
• Publication Date: 2023-04-11T14:43:56Z
• Authors: Yu Tan; Hui Jian; Ranran Zhang; Jing Wang; Cong Zhou; Yuanyuan Xiao; Weibo Liang; Li Wang
• Keywords: Amplicon; Cell-free fetal DNA; Primer (cosmetics); genomic DNA; Multiple displacement amplification; COLD-PCR

Non-invasive prenatal diagnosis for single-gene disorders (NIPD) is still in development and deserves further study. The advent of next-generation sequencing technology significantly improved the detection multiple mutations non-invasive disorder purposes. However, bespoke amplicon-based NGS assays are costly. In this study, we developed a new strategy screening based on capillary electrophoresis (CE) platform using an amplification refractory mutation system (ARMS)-PCR technique. Allele-specific primers several disease-correlated were designed, subsequently, sensitivity specificity conducted. Assays simulated two-person DNA mixtures showed that three targeting mutant allele could detect minor components 1:500 mixtures. All positive results at 0.01 ng template DNA. Cell-free fetal was extracted from pregnant woman's peripheral blood paternally inherited mutations. Our one primer successfully amplified maternal plasma, which confirmed by genotyping genomic amniotic fluid. This study suggested ARMS-PCR technique, fast cost-effective method, might be promising method used to target de novo or pathogenic plasma.