Healthy Longevity Translational Research Programme, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Department of Microbiology & Immunology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, Singapore.
Methods Mol Biol. 2022;2521:329-338. doi: 10.1007/978-1-0716-2441-8_18.
Nonviral delivery vectors are highly sought after for gene therapeutic applications and genetic vaccination. Dumbbell-shaped DNA minimal vectors have important advantages as compared with plasmids and minicircle DNA. Here, we describe the rapid, cheap, and efficient production of superior dumbbell vectors at high purity using a process termed 1-2-3 gap-primer PCR. This process represents a 1-tube, 2-enzyme, 3 h procedure that comprises a PCR followed by a ligation. The resulting dumbbells harbor mismatches close to the loop structures, which facilitate nuclear diffusion and result in enhanced gene expression.
非病毒递送载体在基因治疗应用和基因疫苗接种中受到广泛关注。哑铃形 DNA 最小载体与质粒和微环 DNA 相比具有重要优势。在这里，我们描述了使用称为 1-2-3 缺口引物 PCR 的过程快速、廉价且高效地以高纯度生产优越的哑铃载体。该过程代表了一个 1 管、2 酶、3 小时的程序，包括 PCR 后连接。所得的哑铃载体具有靠近环结构的错配，这有利于核扩散并导致增强的基因表达。
