A universal, high-quality, and high-yield DNA purification method for mycobacteria, including

Genomic analysis of mycobacteria has become increasingly crucial for understanding drug-resistance mechanisms, molecular epidemiology, and pathogenesis. However, efficient extraction of high-molecular-weight genomic DNA from these organisms remains challenging because of their thick mycolic acid-rich cell walls. In this study, we report the chloroform-bead method, a universal DNA extraction protocol that combines chemical and mechanical disruptions to overcome these challenges. Multi-laboratory evaluation (16 sites) demonstrated the chloroform-bead method's superiority over conventional methods for IMPORTANCE: Mycobacterial genomics is crucial for understanding pathogenesis and drug resistance; however, DNA extraction remains a significant challenge because of its unique cell wall. Traditional methods rely on enzymatic treatments, resulting in complex and time-consuming protocols with variable results. The chloroform-bead method introduces a paradigm shift by chemically and mechanically disrupting the mycolic acid layer and eliminating the need for enzymatic treatment. This standardized approach ensures consistent, high-quality DNA extraction across diverse mycobacterial species, thereby enhancing research capabilities and clinical applications.