Improved preparation of high molecular weight DNA for pulsed-field gel electrophoresis from mycobacteria.
Molecular typing is now widely used to aid and supplement conventional epidemiological studies of mycobacterial diseases. Pulsed-field gel electrophoresis (PFGE), in which the entire genome can be represented as a distinct pattern of DNA restriction fragments, is a particularly powerful tool in epidemiology for the determination of clonal identity of bacteria providing information for understanding and controlling the spread of disease. Application of PFGE to the study of mycobacterial diseases has been limited because isolation of high-quality genomic DNA from mycobacterial sources has proved problematic. Here we report a simple, highly effective method for the preparation of high molecular weight DNA from a range of mycobacterial species. Cultures are continuously stirred and are homogeneous. This enables accurate quantification. The presence of detergent in buffers keeps the cells in suspension throughout preparation enabling efficient lysis. In addition, it is compatible with heat-inactivation of pathogenic mycobacteria and all of the preparation procedures can be carried out with a category III facility. This standardised method of preparation of DNA from mycobacteria means that PFGE should now be evaluated as a method for typing these organisms and it may be particularly important as a means of typing less well-characterised mycobacteria for which other techniques are not available.
Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH 29 OPZ, UK.
V M
VM
Hughes
K
K
Stevenson
J M
JM
Sharp
D002138:Calibration; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000737:chemistry / Q000302:isolation & purification*; D003720:Densitometry; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry; D016521:Electrophoresis, Gel, Pulsed-Field / Q000379:methods*; D019286:Endopeptidase K / Q000737:chemistry; D009161:Mycobacterium / Q000737:chemistry / Q000235:genetics* / Q000254:growth & development; D011136:Polysorbates / Q000737:chemistry
D004269:DNA, Bacterial; D011136:Polysorbates; D015252:Deoxyribonucleases, Type II Site-Specific; C035594:TTTAAA -specific type II deoxyribonucleases; D019286:Endopeptidase K