Use of streptavidin magnetic beads in single strand conformation polymorphism profiles to detect mutations in rpoB gene of M.tuberculosis

摘要

Single strand conformation polymorphism (SSCP) is one of the promisingudtechniques to identify mutations in short pieces of DNA (Orita et al. 1989). In thisudtechnique, DNA of interest is often amplified by the polymerase chain reaction (PCR)udand then denatured by heat or alkali treatment before electrophoresis on a nonuddenaturing polyacrylamide gel. Differences in mobility of either of the single strandsudcompared to the control DNA indicate mutations which affect the secondary structureudand alter the mobility of the DNA. We applied PCR-SSCP for the detection ofudmutations in the rifampicin resistance determining region (RRDR) of the rpoB gene ofudM. tuberculosis (Telenti et al. 1993a; 1993b). A nested PCR was used to amplify theudRRDR. In the first PCR, 293-bp product was amplified and in the second PCR a 103-udbp of the first PCR product was amplified. However, in our experience usinguddenaturation by alkali or heating, the denatured PCR product most often reannealed toudform a large proportion of double stranded DNA during the electrophoresisud(Selvakumar et al. 1997a). After visualisation by staining with ethidium bromide orudsilver staining, most of the DNA was in the double stranded form, with very little orudno single stranded DNA. The single strands that could be observed often ran closeudtogether, making analysis of any difference in mobility difficult. Therefore an attemptudwas made to generate biotinylated PCR product using a biotinylated forward primerudand later the biotinylated strand was separated using sterptavidin magnetic beads. Theudseparated strands eliminated the problem of strand reannealing during SSCP and wereudsilver stained to detect the shift in the mobility. Since the nested PCR requires moreudtime and is more expensive. a biotinylated PCR product was generated in a single PCRudusing a biotinylated forward primer and an unbiotinylated reverse primer. Thisudsimplified protocol was applied to clinical isolates in an attempt to detect rifampicinudresistance.