Efficient Separation of Single-Stranded DNA of Various Lengths for Applications in Bioimaging, Gene Editing, and Nanostructures

Single-stranded DNA (ssDNA) has extremely high design flexibility and specific functions. Therefore, ssDNA is used in crucial practical applications in many fields, such as molecular detection, gene editing, and nanotechnology. However, the existing methods for ssDNA preparation often present limitations in terms of yield, purity, and length applicability. In order to overcome these challenges, the present study proposes a ssDNA separation method that utilizes the modification of linear polyacrylamide (LPA) combined with denaturing agarose gel electrophoresis (DAGE). The method is referred to as LPA-DAGE. The underlying principle is to produce LPA-modified double-stranded DNA (dsDNA) through PCR using the LPA primer and then achieve efficient ssDNA separation using denaturing agarose gel electrophoresis based on molecular weight differences. This method can stably recover ssDNA showing significant advantages over the existing methods in terms of purity and recovery rate. The results of this study demonstrate that the proposed ssDNA preparation method enables signal amplification using fluorescence <i>in situ</i> hybridization (FISH), CRISPR/Cas9-mediated homologous recombination repair, and nanostructure construction using DNA origami.