Question

Rifampicin is an important antibiotic for the treatment of tuberculosis. Some strains of Mycobaterium tuberculosis, the causative agent of tuberculosis, are resistant to rifampin. These strains become resistant through the acquisition of mutations that they alter the rpoB gene, which codes for the RNA polymerase subunit. In these mutants, rifampin does not bind to mutant polymerase and is therefore unable to inhibit start of transcription. The DNA sequences of a large number of M strains. rifampicin-resistant tuberculosis, various strains have been shown to have mutations specific in a 68 bp region of rpoB. One of these resistant strains very well characterized, it has only a single base pair alteration in rpoB, resulting in That a residue of Su is replaced by an Asp in subunit B. Given an infection by M. tuberculosis how would you determine if it is a strain containing the mutation before mentioned?
a) Indicate some method at the protein level b) Indicate some method at the level of ribonucleic acid
c) Indicate some method at the level of deoxyribonucleic acid

Answer 1

a) The goal is to detect the protein that has been mutated. To detect a protein, an antibody specific for this protein can be used. The proteins from the cell can be isolated and the isolate be probed with the antibody. The antibody itself can then be detected using a secondary antibody that has a colorimetric readout.

b) Ribonucleic acid will have a change in the nucleotide or a couple of nucleotides. To detect this change, an RNA probe that is either radioactive or attached to a fluorophore, and is complementary to the target sequence can be used.

c) For a deoxyribonucleic acid, again an RNA probe or a DNA probe that is attached to a fluorophore or is hot can be used to detect the complementary target sequence.