Concise Analysis of Single-Stranded DNA of Recombinant Adeno-Associated Virus By Automated Electrophoresis System.

Abstract

Recombinant adeno-associated virus (rAAV) is a prominent viral vector currently available for human gene therapy. The diameter of the rAAV capsid is ∼25 nm, and a positive or negative single-stranded DNA is packaged within the vector capsid. In this report, we describe a concise method to examine the extracted rAAV genome using an automated electrophoresis system. The rAAV genome, prepared from vector particles through either heat treatment at 95°C for 10 min or the phenol-chloroform extraction method, was analyzed using an automated electrophoresis system under denaturation conditions. The heat treatment protocol demonstrated a comparable yield with the phenol-chloroform extraction protocol, and the quantified amounts of the rAAV genome obtained using the automated electrophoresis system were consistent with those quantitated by quantitative PCR. Additionally, crude rAAV extractions could also be analyzed by the automated electrophoresis system after DNase I treatment. These results indicated that this simple and quick analysis using automated electrophoresis is highly useful for confirming the purity and integrity of the rAAV genome.