A rapid and efficient technique for the isolation of Bacillus genomic DNA using a cocktail of peptidoglycan hydrolases of different type.

ABSTRACT

The paper suggests a rapid and efficient technique for isolation of genomic DNA from the bacteria of the genus Bacillus, which is based on the hydrolysis of cell wall peptidoglycan by a cocktail of peptidoglycan hydrolases of different type (L,D-peptidase and N-acetylmuramidase). The comparing of conventional techniques for the isolation of genomic DNA using a microwave treatment; a treatment with ionic detergents (SDS, CTAB) or a chaotropic agent (GuSCN); and enzymatic hydrolysis (nonspecific, with proteinase K, or specific, with peptidoglycan hydrolases) conducted on Bacillus megaterium, B. subtilis, B. licheniformis, B. cereus showed that the most effective ones were techniques based on the specific hydrolysis of cell wall peptidoglycan. The highest efficiency of hydrolysis was obtained with an enzyme cocktail consisted of hen egg muramidase (HEWL) and highly active phage-specific L,D-peptidase EndoRB49 revealed a pronounced synergism between the peptidase and the muramidase. The cocktail treatment of Bacillus cells could be reduced to 10 min without affecting the yield of nucleic acids. The quality of DNA preparations was assessed using the restriction and PCR assays, as well as agarose gel electrophoresis. Using peptidoglycan hydrolases of different type, which have a good synergy, makes the technique very efficient and perspective for the application when rapid and effective disintegration of cell wall is crucial to avoid adverse effects of macromolecular denaturation.