Comparison of methods for isolation of Mycobacterium avium complex DNA for use in PCR and RAPD fingerprinting

Different methods for DNA isolation from Mycobacterium avium complex cultures were judged on the basis of their (1) ability to yield DNA from small culture samples, (2) speed of processing, (3) low number of manipulations and low potential for DNA crosscontamination, and (4) ability to provide DNA suitable for use as template in PCR and random amplified polymorphic DNA (RAPD) reactions. The glass-bead disruption methods yielded more amplifiable DNA than chemical or heat-induced lysis methods. The simplest DNA preparation method to give reproducible PCR results required only disrupting mycobacterial cells with glass beads, treating the lysate with proteinase K, and heating to 95°C for 10 min. Using the glass-bead lysis method, 20 culture samples could be prepared for PCR in less than 1 h. Addition of hexadecyltrimethylammonium bromide (CTAB) treatment, chloroform extraction to remove cellular debris, and isopropanol precipitation, yielded DNA suitable for restriction endonuclease digestion, PCR, and RAPD fingerprinting while minimizing the number of manipulations and possibilities of contamination.