Context
Genomic and proteomic research on mycobacterial diseases requires high quality and quantity preparation of DNA or proteins. Effective lysis of mycobacterial cells for DNA or protein extraction is demanding due to the mycobacteria’s robust and waxy cell wall features. Also, many mycobacteria are slow growers, often resulting in small amounts of starting material from a culture. Commercially available kits are not usually applicable to research methods on mycobacterial genomics and proteomics [1] [2].
[1] Kaser, et al. 2009. Optimized method for preparation of DNA from pathogenic and environmental mycobacteria.Appl.Environ.Microbiol. 75, no. 2 (January): 414-418.[2] Kaser, et al. 2010. Optimized DNAPreparation from Mycobacteria. Cold Spring Harb Protoc 2010, no. 4 (April) :pdb.prot5408.doi:10.1101/pdb.prot5408.
Results
Several elements of published DNA extraction protocols were combined and tested to improve DNA yield. Mechanical disruption of the mycobacterial cell wall, after incubation with 4% SDS (final concentration), was found to be crucial for sufficient and satisfactory yields. DNA yield achieved when testing both combinations of DNA extraction procedures (chemical lysis only vs chemical and mechanical disruption) are shown in the Figure 1.
Figure 1. Twenty-milligram (wet weight) pellets of M. ulcerans strain IFIK1066089 were used. (Superscript 1) SDS was applied to a final concentration of 4%. (Superscript 2) Mechanical disruption performed with Precellys 24.
