Efficient Generation of Superior Dumbbell-Shaped Nonviral DNA Delivery Vectors Using 1-2-3 Gap-Primer PCR

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.