Single strand conformation polymorphism profiles of biotinylated PCR products to detect mutations in rpoB gene of M. tuberculosis - Research Communications.
Selvakumar, N.; Wilson Stuart, M.; Ruth McNerney; Narayanan, P.R.
Current Science; 1997; 73; 773-774
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 tuberculosis by the polymerse chain reaction (PCR). One of the primers, employed in PCR, was biotinylated. The biotinylated strand of the PCR product was separated from the unbiotinylated strand using streptavidin magnetic beads. Both the strands were subjected to single strand conformation 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 indicative of resistance. This strategy was found to case the visualization of the shift in the migration of the strands in 17 of 21 samples and thereby defection of mutations.
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