Step-wise isolation of RNA and DNA from mycobacteria.

Mycobacterium phlei and M. smegmatis were lysed at -15 degrees C in the presence of guanidinium salts and poly(A)+-RNA. Nonpolyadenylated RNA (mainly rRNA) and DNA were isolated in successive steps from the same lysate. DNA isolated by this procedure was sufficient to yield distinct bands on treatment with restriction endonucleases as shown by hybridization to 125I-labeled rRNA. The successive isolation of poly(A)+-RNA, nonpolyadenylated RNA, and high molecular weight DNA from the same lysate by this procedure, which is being reported for the first time, provides a very economical approach for isolation and purification of nucleic acids from mycobacteria and other organisms. This could be of special value for various genetic recombination studies, particularly in the case of mycobacteria which are available in very limited quantities, e.g., noncultivable or difficult-to-grow mycobacteria, especially M. leprae.