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

Selvakumar, N and Ding, B C and McNerney, R and Wison, S M and Narayanan, P R (1998) Use of streptavidin magnetic beads in single strand conformation polymorphism profiles to detect mutations in rpoB gene of M.tuberculosis. In: Workshop on Tuberculosis Research : in to the 21st century, 1998.

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Abstract

Single strand conformation polymorphism (SSCP) is one of the promising techniques to identify mutations in short pieces of DNA (Orita et al. 1989). In this technique, DNA of interest is often amplified by the polymerase chain reaction (PCR) and then denatured by heat or alkali treatment before electrophoresis on a non denaturing polyacrylamide gel. Differences in mobility of either of the single strands compared to the control DNA indicate mutations which affect the secondary structure and alter the mobility of the DNA. We applied PCR-SSCP for the detection of mutations in the rifampicin resistance determining region (RRDR) of the rpoB gene of M. tuberculosis (Telenti et al. 1993a; 1993b). A nested PCR was used to amplify the RRDR. In the first PCR, 293-bp product was amplified and in the second PCR a 103- bp of the first PCR product was amplified. However, in our experience using denaturation by alkali or heating, the denatured PCR product most often reannealed to form a large proportion of double stranded DNA during the electrophoresis (Selvakumar et al. 1997a). After visualisation by staining with ethidium bromide or silver staining, most of the DNA was in the double stranded form, with very little or no single stranded DNA. The single strands that could be observed often ran close together, making analysis of any difference in mobility difficult. Therefore an attempt was made to generate biotinylated PCR product using a biotinylated forward primer and later the biotinylated strand was separated using sterptavidin magnetic beads. The separated strands eliminated the problem of strand reannealing during SSCP and were silver stained to detect the shift in the mobility. Since the nested PCR requires more time and is more expensive. a biotinylated PCR product was generated in a single PCR using a biotinylated forward primer and an unbiotinylated reverse primer. This simplified protocol was applied to clinical isolates in an attempt to detect rifampicin resistance.

Item Type:	Conference or Workshop Item (Paper)
Additional Information:	RP199818
Subjects:	Tuberculosis > Laboratory Research > Bacteriological
Divisions:	Basic Science Research > Bacteriology
Depositing User:	Dr. Rathinasabapati R
Date Deposited:	30 Oct 2013 06:10
Last Modified:	14 Mar 2016 04:33
URI:	http://eprints.nirt.res.in/id/eprint/473

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