Chemical cleavage of mismatches in heteroduplexes of the rpoB gene for detection of mutations associated with resistance of Mycobacterium tuberculosis to rifampin

Abstract

In order to detect mutations in the core region of the RNA polymerase B (rpoB) subunit gene of Mycobacterium tuberculosis that are known to be associated with resistance to rifampin, we applied rapid chemical cleavage of mismatches (CCM) to heteroduplexes formed between the DNA of M. tuberculosis H37Rv and strains resistant to rifampin. DNA fragments amplified from normal and mutant rpoB genes by polymerase chain reaction were mixed, denatured and re-annealed to create heteroduplexes containing mispaired bases reactive to modification by hydroxylamine (cytosine mismatches) or osmium tetroxide (thymine mismatches) and cleavage of DNA by piperidine at the position of modified base. The cleaved products and the heteroduplexes were separated by polyacrylamide-urea gel electrophoresis and detected by autoradiography. The position of mutations was confirmed by DNA sequencing of the amplified DNA fragments. The results suggest further applicability of the CCM method as a means to screen M. tuberculosis isolates for mutations associated with drug resistance.