Southern and PCR analyses of <i>M. tuberculosis</i> strains.

<p><b>a</b>). Southern analysis of genomic DNA of <i>M. tuberculosis (Mtb)</i> wild type (H37Rv), <i>fbpA</i> mutant (ΔfbpA) and <i>fbpA/sapM</i> double knock out (DKO) strains. Genomic DNA was digested with NdeI and BamHI, separated on 1% agarose gels and transferred to nitrocellulose membranes. Membranes were hybridized with [³²P]dCTP labeled 3.3 kb DNA fragment containing <i>sapM</i> region and signals captured by autoradiography. Arrows indicate the sizes of the signals. <b>b</b>–<b>c</b>). PCR analysis for <i>sapM</i> region in <i>M. tuberculosis</i> H37Rv, ΔfbpA and DKO strains. PCR was performed using standard protocols with genomic DNA from the above strains as templates. Primer pairs RV3310EX1 and Rv3310EX2 (<b>b</b>) and RV3310EX1 and RV3310RT2 (<b>c</b>) were used to amplify DNA. <b>d</b>. RT-PCR analysis for <i>sapM</i> expression in <i>M. tuberculosis</i> H37Rv, ΔfbpA and DKO strains. Total RNA was used to synthesize cDNA from these strains. RT+ and RT- indicate cDNA templates generated in the presence or absence of reverse transcriptase (Superscript II; Invitrogen). The products obtained from both reactions were used as templates in RT-PCR to prove the absence of DNA contamination in total RNAs used for reverse transcriptions. PCR was performed using primers RV3310RT1 and RV3310RT2. MW: molecular weight marker; arrow indicates the size of the band. PCR products were separated on 1% agarose gels.