Improved Isolation of Ultra-High-Molecular-Weight Genomic DNA Suitable for Third-Generation Sequencing

Improved Isolation of Ultra-High-Molecular-Weight Genomic DNA Suitable for Third-Generation Sequencing

Blog Article

Although a variety of protocols to isolate high-molecular-weight genomic DNA exist, the isolation and preservation of ultra-high-molecular-weight genomic DNA of sufficient quality and length for error-free third-generation sequencing remains challenging.Inspired by the isolation of high-molecular-weight DNA in agarose plugs suitable to be separated by pulsed-field gel electrophoresis, we report the construction of an incubation chamber for DNA isolation.The chamber is flanked by semi-permeable polycarbonate membranes of variable Vitamin pore size for the selective diffusion of compounds and components, which allows efficient cell lysis and the subsequent isolation of ultra-high-molecular-weight genomic DNA without shearing.

The designed experimental approach is simple, but effective in isolating high-quality ultra-long genomic DNA that is suitable for third-generation sequencing by Oxford Nanopore Technology from challenging bacterial samples.We envisage that genomic DNA isolation using the incubation chamber, Queen Sofa Bed or variations thereof, will facilitate the regular sequencing of megabasepair-long DNA fragments, with a multitude of applications in microbiology, immunology, oncology, paleontology and forensic science.