Molecular Biology Today (2002). 3: 43-49.

Qualitative Evaluation of Mycobacterial DNA Extraction Protocols for Polymerase Chain Reaction

Jain Amita, Tiwari Vandana, R.S. Guleria, and R.K. Verma

We have compared the efficacy of various reported protocols of mycobacterial DNA extraction for detection of mycobacterial DNA by PCR assay. Seven DNA extraction protocols were tested for their quantitative as well as qualitative yield of mycobacterial DNA in 15 known positive sputum samples having occasional acid fast bacilli (AFB). DNA samples obtained by various methods were amplified in uniform standard conditions and analysed on 3% agarose gel. Protocol 6 and 7 showed 100% detection sensitivity with strong bands on agarose gel. Protocols 1-5 were found to be unsatisfactory because they yielded either low quantity or poor quality of DNA or were unable to remove inhibitors of DNA amplification. We conclude that a strong physical treatment, use of a detergent and enzyme for lysis, treatment with proteinase K, DNA purification step with or without phenol and DNA precipitation in ethanol or isopropanol are essential steps for extraction of mycobacterial DNA from clinical samples. Protocol 6 is standard in our laboratory and we have found reproducible results with this method. Purification with phenol followed by chloroform treatment was not found to have any inhibitory effect on amplification. A more extensive evaluation of this protocol in samples with lower bacterial load may be necessary.