AUTHOR=Tan Yu , Jian Hui , Zhang Ranran , Wang Jing , Zhou Cong , Xiao Yuanyuan , Liang Weibo , Wang Li 

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

JOURNAL=Frontiers in Genetics

VOLUME=Volume 14 - 2023

YEAR=2023

URL=https://www.frontiersin.org/journals/genetics/articles/10.3389/fgene.2023.1071406

DOI=10.3389/fgene.2023.1071406

ISSN=1664-8021

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