Identification of transcriptionally active open reading frames within the RD1 genomic segment of Mycobacterium tuberculosis.

OBJECTIVE To identify transcriptionally active open reading frames (ORFs), predicted by bioinformatics, within RD1 genomic segment of Mycobacterium tuberculosis using reverse transcription-polymerase chain reaction (RT-PCR). MATERIALS AND METHODS M. tuberculosis H37Rv was grown in Middlebrook 7H9 medium for 8 weeks and total RNA was isolated using standard procedures. The cDNA was synthesized using first-strand cDNA synthesis kit and general primers provided in the kit [pd (N)6, and/or Not I-d(T)18] as well as forward primers specific for each predicted RD1 ORF. Specific forward and reverse primers in PCR were used to amplify ORF-specific cDNA. The amplified products were identified on the basis of size using agarose gel electrophoresis, and their identity was confirmed by DNA sequencing. RESULTS RT-PCR demonstrated expression of 13 of the 14 bioinformatics-predicted ORFs within RD1 genomic segment of M. tuberculosis. However, cDNA synthesis and PCR amplifications of specific products varied with respect to primer requirement and reaction conditions, respectively. All ORFs of <1.5 kb were amplified in standard RT-PCR, whereas several large-size ORFs (>1.5 kb) required internal primers for amplification in semi-nested RT-PCR. The sequencing of RT-PCR-amplified products of ORFs confirmed their identity. CONCLUSION Bioinformatics analysis of DNA can accurately predict ORFs within M. tuberculosis-specific genomic regions, and RT-PCR is a suitable technique to confirm their expression in bacteria.