SINGLE-STRAND CONFORMATION POLYMORPHISM PROFILES WITH BIOTINYLATED PCR PRODUCTS TO DETECT MUTATIONS IN RPOB GENE OF MYCOBACTERIUM-TUBERCULOSIS

A fragment of the rpoB gene, including the region shown to be involved in rifampicin resistance, was amplified from 15 rifampicin-resistant and 6 rifampicin-sensitive clinical isolates of Mycobacterium tubercul osis by the polymerase chain reaction (PCR). One of the primers, emplo yed in PCR, was biotinylated. The biotinylated strand of the PCR produ ct was separated from the unbiotinylated strand using streptavidin mag netic beads. Both the strands were subjected to single strand conforma tion polymorphism in polyacrylamide gel electrophoresis. The DNA bands were silver stained to study their migration pattern. A shift in the migration of either of the strands of the test strain compared to the strands from a control rifampicin-sensitive strain was considered as i ndicative of resistance. This strategy was found to ease the visualiza tion of shift in the migration of the strands in 17 of 21 samples and thereby detection of mutations.