A Quick Method to Synthesize Extrachromosomal Circular DNA In Vitro

作者：Shanru Zuo, Xueguang Li, Yide Yang, Junhua Zhou, Quanyuan He · 发表于：Molecules · 年份：2023 · DOI：10.3390/molecules28104236 · 被引用次数：9 · 研究领域：Cancer Genomics and Diagnostics、RNA modifications and cancer、CRISPR and Genetic Engineering

Extrachromosomal circular DNA (eccDNA) is a special class of circular DNA in eukaryotes. Recent studies have suggested that eccDNA is the product of genomic instability and has important biological functions to regulate many downstream biological processes. While NGS (Next-Generation Sequencing)-based eccDNA sequencing has led to the identification of many eccDNAs in both healthy and diseased tissues, the specific biological functions of individual eccDNAs have yet to be clearly elucidated. Synthesizing eccDNAs longer than 1 kb with specific sequences remains a major challenge in the field, which has hindered our ability to fully understand their functions. Current methods for synthesizing eccDNAs primarily rely on chemical oligo synthesis, ligation, or the use of a specific gene editing and recombination systems. Therefore, these methods are often limited by the length of eccDNAs and are complex, expensive, as well as time-consuming. In this study, we introduce a novel method named QuickLAMA (Ligase-Assisted Minicircle Accumulation) for rapidly synthesizing eccDNAs up to 2.6 kb using a simple PCR and ligation approach. To validate the efficacy of our method, we synthesized three eccDNAs of varying lengths from cancer tissue and PC3 cells and confirmed successful circularization through sequencing and restriction enzyme digestion. Additional analyses have demonstrated that this method is highly efficient, cost-effective, and time-efficient, with good reproducibility. Using th...