Sargent describes a new method for isolating high-quality DNA from Bacillus subtilis 168 spores that overcomes previous difficulties with low yields and poor DNA activity. The procedure uses a multi-step approach: first treating spores with a specialized solution containing urea, SDS, and reducing agents at pH 9.8 to disrupt the spore coat; removing SDS completely; treating with lysozyme to digest the peptidoglycan layer; then lysing with freeze-thaw cycles, protease, and detergent. The method recovers over 80% of DNA with minimal loss to spore residue. Critically, the isolated DNA demonstrates transformation efficiency comparable to vegetative cell DNA and produces restriction patterns similar to vegetative DNA when treated with restriction endonucleases. The key innovation is avoiding extreme pH conditions during extraction while ensuring complete removal of SDS before lysozyme treatment and maintaining reduced thiol groups throughout. The procedure successfully isolates DNA from multiple B. subtilis 168 strains and enables investigation of chromosomal configuration in spores and DNA replication during sporulation.

Key findings

• A new DNA extraction procedure recovers >80% pure DNA from B. subtilis spores with minimal degradation
• Isolated spore DNA shows transformation efficiency (2-5×10⁵ transformants per 10⁸ cells) comparable to vegetative cell DNA
• The DNA is susceptible to restriction endonucleases and produces restriction patterns similar to vegetative DNA
• Complete SDS removal before lysozyme treatment and maintenance of reduced thiol groups are critical for successful spore lysis
• The method avoids extreme pH conditions that can damage DNA, preventing undesirable protein modifications and cross-linking