PCR was performed

in a 50-μl reaction mixture containing 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM MgCl2, 200 μM of each dNTP, 0.5 μM of each primer, 50 ng of DNA template, and 2.5 U of Taq DNA polymerase (Promega, USA). Sorafenib price The PCR conditions consisted of an initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation at 94°C for 30 sec, annealing at 56-60°C for 1 min and extension at 72°C for 1-2 min depending on the PCR product size (Table 2), and a final extension at 72°C for 7 min. The PCR products were analyzed by agarose gel electrophoresis and purified using the QIAquick PCR Purification Kit (Qiagen, Germany) prior to submission for DNA sequencing. Table 2 Primers used for amplification and sequencing of M.tuberculosis clinical strains Gene Primer name (position*)

Primer sequence (5′→3′) Annealing temp (°C) PCR product size (bp) Purpose Reference rrs F-rrs (-44) 5′-TTCTAAATACCTTTGGCTCCCT-3′ 51 1,680 PCR/Seq [42] R-rrs (1,636) 5′-TGGCCAACTTTGTTGTCATGCA-3′ 53   PCR/Seq [42] F-rrs1 (554) 5′-CTGGGCGTAAAGAGCTCGTA-3′ 54   Seq This study F-rrs2 (1,114) 5′-GTTGCCAGCACGTAATGGTG-3′ ITF2357 purchase 54   Seq This study R-rrs1 (483) 5′-TCCACCTACCGTCAATCCGA-3′ 54   Seq This study R-rrs2 (1,073) 5′-ATCTCACGACACGAGCTGAC-3′ 54   Seq This study eis (Rv2416c) F-Rv2417c (-316) 5′-GCGGTGCATCACGTCGCCGA-3′ 60 1,661 PCR/Seq This study R-eis-Rv2415c (1,345) 5′-GCAACGCGATCCGCGAGTGC-3′ 60   PCR/Seq This study F-eis1 (247) 5′-AGTTTCGTCGCGGTGGCGCC-3′ 60   Seq This study F-eis2 (816) 5′-GGACCCGTTACCCCACCTGC-3′ 60   Seq This study R-eis1 (240) Cyclic nucleotide phosphodiesterase 5′-GGCGGTCGGGAGCACCACTT-3′ 60   Seq This study R-eis2 (769) 5′-TCAGGGCCCGCCACAACGCA-3′ 60   Seq This study tap (Rv1258c) F-Rv1259 (-496) 5′-CAGGCCGGCCCTATGCAGTG-3′ 60 1,847 PCR/Seq This study R-Rv1257c (1,351) 5′-CGGTCTTGCCGGTAGCCGTC-3′ 60   PCR/Seq This study F-tap1 (41) 5′-TCGCAACGCTGATGGCGGCC-3′ 60   Seq This study F-tap2 (641) 5′-AGGGGCTGCGCTTCGTCTGG-3′ 60   Seq This study R-tap1 (210) 5′-CCCGAAGTAGTCGACCGCGG-3′ 60   Seq This study R-tap2 (862) 5′-GACGGGGAACGCGGATAGCC-3′

60   Seq This study whiB7 (Rv3197A) F URT-whiB7 (-451) 5′-GCTGGTTCGCGGTCGGACCT-3′ 60 550 PCR/Seq This study R whiB7 (99) 5′-CGGGGTATCGGCGAACCACA-3′ 58   PCR/Seq This study tlyA (Rv1694) F-tlyA (1) 5′-GTGGCACGACGTGCCCGCGT-3′ 62 807 PCR/Seq This study R-tlyA (807) 5′-CTACGGGCCCTCGCTAATCG-3′ 58   PCR/Seq This study *The first 5′nucleotide position of each primer was counted from the translation start codon of each gene. DNA sequencing analysis Nucleotide sequencing was performed with the Big-Dye™ Terminator Cycle Sequencing Ready Reaction Kit (Perkin Elmer, USA) using an ABI PRISMR 3700 DNA analyzer at First BASE Laboratories (Malaysia). The PCR products were sequenced in both directions. The obtained nucleotide sequences were compared with those of M. tuberculosis H37Rv (Accession no. NC_000962) by pairwise alignment using the ClustalW program [43].