The Improved Method and Device for Nucleic Acid Isolation Using a High-Salt Gel Electroelution Trap

The success of DNA analytical methods, including long-read sequencing, depends on the availability of high-quality, purified DNA. Previously, we developed a method and device for isolating high-molecular-weight (HMW) DNA for long-read sequencing using a high-salt gel electroelution trap. Here, we present an improved version of this method for purifying nucleic acids with higher yield and purity from even the most difficult biological samples. The proposed method is a significant improvement over the previously published procedure, offering a simple, fast, and efficient solution for isolating not only HMW DNA but also smaller DNA and RNA molecules. The method utilizes vertical gel electrophoresis in two nested, partially overlapping electrophoretic columns. The upper, smaller-diameter column has a thin layer of agarose gel at the bottom and electrophoresis buffer on top and serves to separate nucleic acids from impurities. After the target nucleic acid has been gel-purified on the upper column, a larger-diameter column with a layer of high-salt gel overlaid with electrophoresis buffer is inserted from below. The purified nucleic acid is then electroeluted into the buffer-filled gap between the separating gel and the high-salt gel, where excess counterions from the high-salt gel slow its migration and cause it to accumulate. The proposed vertical purification system outperforms the previously described horizontal system in terms of nucleic acid yield and purity, while also being more user-friendly, scalable, and adaptable to high-throughput workflows. Furthermore, the vertical system allows for the sequential purification of multiple nucleic acid species from the same sample using interchangeable salt-gel columns.