DNA extraction methods from Mycobacterium leprae and M. lepraemurium
To the Editor:
Mycobacterium leprae and M. lepraemurium, respectively, the etiologic agents of human and murine leprosy, are rich in lipids and have thick cell walls. These mycobacteria thus resist DNA extraction. Although some methods have been used to extract DNA from mycobacteria (2,4,10) they lack efficacy and arc time consuming. There is a need for a rapid, effective and simple method for DNA extraction from Al. leprae and M. lepraemurium which could be used for the diagnosis, epidemiological investigation, molecular biology research and identification of these mycobacteria grown in vitro. Studies were carried out to find a suitable method of DNA extraction from in Wvo-grown M. leprae and M. lepraemurium.
The sources of M. leprae were foot pads of nude mice and armadillo liver tissues, infected earlier with human leprosy bacilli. Bacilli from foot pads of nude mice free from host material were purified by the method of Franzblau and Hastings (5). Density separation of M. leprae from nude mouse foot pads was accomplished by 30% Percoll gradient separation (8). M. leprae recovered from infected armadillo liver were purified by the method described by Clark-Curtiss, et al. (3). M. lepraemurium (Hawaiian strain) isolated from C3H mice lepromas were purified by differential ccntrifugation (6), and were further purified by the method of Franzblau and Hastings (5).
During these studies, five different methods were used to extract DNA from M. leprae and M. lepraemurium bacillary preparations. Bacilli (50 mg wet wt, about 5 x 1010 bacilli) were suspended in 1 ml of buffer, consisting of 100 mM NaCl, 10 mM EDTA, and 10 mM Tris-HCl, pH 7.5. These preparations were used for DNA extraction in the following five methods: 1) the intensive enzymic digestion method (M1) used was the same as described by Visuvanathan, et al. (11). 2) 2-min mechanical glass-bead disruption method (M2) was carried out according to Via and Falkinham (10) and Jacobs, et al. (7). 3) thermal shock method (M3). In this method, 50 mg of cells were suspended in 400 µ l of distilled water and subjected to repeated (six times) heat/cold shock; boiling for 5 min at 100ºC and snapfreezing for 5 min at - 196ºC in liquid nitrogen. 4) modified conventional enzymic digestion method (M4). Briefly, to 1 ml of a bacillary suspension containing 50 mg of bacilli, 2 mg of lysozyme was added and incubated at 37ºC for 1 hr, then 0.3 mg each of proteinase K and SDS (to a final concentration of 3%) were added and the reaction mixture was further incubated at 56ºC for 1 hr. 5) manual disruption with modified conventional enzymic digestion method (M5): 50 mg of cells were triturated for 30 min in a mortar containing dry ice and 0.1 g of glass beads (0.11 mm), and the rest of the procedure was essentially the same as described in M4.
Estimation of DNA concentration was performed spectrophotometrically using the standard method of Sambrook, et al. (9). The highest yield of 2.82 µ g DNA/mg wet wt of M. lepraemurium was obtained by M2; this represents a theoretical yield of 78% (1,11). The lowest DNA yield of 0.01 µ g DNA/mg wet wt of Al. lepraemurium was obtained by using M3.
When M. leprae recovered from armadillo liver were used, DNA yields of 1.25, 1.37 and 1.66 µ g DNA/mg wet wt of cells were obtained, respectively, by M2, M4 and M5. Very low yields of DNA were achieved by Ml and M3. In our experience, it was comparatively more difficult to extract the DNA of M. leprae from the foot pads of nude mice than M. leprae from armadillo liver by all of the methods used. Also, M. leprae isolated from nude mice gave comparatively lower DNA yields. For example, when Al. leprae recovered from the foot pads of nude mice were used, yields of 0.66, 0.27 and 1.43 µ g DNA/mg wet wt of cells were obtained, respectively, by the M2, M4 and M5 methods. These results could be attributed to the variations in cultural conditions and strain differences. M3 and M1 were the least effective for the extraction of DNA from M. leprae and M. lepraemurium. Based on the yields of DNA extracted by all of the methods used, both M2 and M5 are the time-saving (less than 4 hr), effective and simple methods for DNA extraction from in vivo -grown M. leprae and M. lepraemurium .
- Zhang-Qing Zhang, Ph.D.
Post-doctoral fellow
- Muhammad Ishaque, Ph.D.
Professor
Applied Microbiology Research Center
Institute Armand-Frappier
University of Quebec
CP. 100
Laval, Quebec, Canada H7N 4Z3
Acknowledgment. This investigation was generously supported by the Military and Hospitaller Order of Saint Lazarus of Jerusalem, Canada.
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