Increased demands for plasmid DNA (pDNA) for emerging nucleic acid-based vaccines and therapies exacerbate the need to remove bottlenecks from pDNA production. For industry, the problem has been that as pDNA is synthesized and purified, the plasmids sometimes come out of their supercoiled isoform. In therapeutics, the resulting heterogeneous sequence mixtures and uncontrolled transcripts may contribute to dangerous side effects.
Scientists led by Anubhav Tripathi, PhD, professor of biology and medical sciences, Brown University, developed a way to detect supercoiled, linear, and open circular pDNA isoforms using high-throughput microfluidic electrophoresis. The method developed by Tripathi and colleagues detects isoforms as small as 0.1 ng/μL for supercoiled and linear isoforms, and 0.
5 ng/μL for the circular isoform. With a sample volume of 10 μL and a turnaround time of one minute per sample, this method, they say, can help optimize processes and analyze batches. As Tripathi tells , “Where plasmids are part of the final product, it has been shown that supercoiled plasmids have a higher efficiency.
Therefore, our method can be used to ensure no undesired single or double-stranded breaks in the plasmids have been induced.” That is true regardless of whether the plasmids are used as precursors, for transcription, or in the final product. To achieve this, the scientists developed a microfluidic supercoiled pDNA assay, optimized the separation of supercoiled and linear is.