Custom pcr master mix

In order to ascertain whether the LRPS assay correctly identifies MTBC lineage we compared the LRPS results with LSP-PCR data using genomic DNA extracted from 70 MTBC stored isolates. LSP-PCR was performed using RD 724 deletion primers (specific for MTB Uganda family) and RD750 deletion primers (Specific for MTB lineage 3) as described by Gagneux et al. [25 (link)] and Tsolaki et al. [26 (link)]. The 10 μl reaction volume PCR was containing 5.5 μl water, 1 μl (10 μM final concentration) forward primer (RD 724 or RD 750) and 1 μl (10 μM final concentration) reverse primer (Reverse RD 724 or RD 750), 1 μl of 10 x Thermo Fischer Scientific Custom PCR Master mix, 1 μl template DNA (at least 50 ng) and 0.5 μl (0.5 unit) DNA polymerase. The reaction was run in a standard thermocycler programmed at 95 °C for 10 min 35 cycles of 95 °C for 1 min, 64 °C for 30 s and 72 °C for 30 s. PCR products were analyzed by gel electrophoresis.

SP-PCR was carried out using flanking primers DM-C and DM-DR as previously described^{18 (link),30 (link)} using Custom PCR Master Mix (Thermo Fisher Scientific [Waltham, MA] #SM-0005) supplemented with 69 mM 2-mercaptoethanol. Taq polymerase (Sigma-Aldrich UK, Gillingham) was used at 1 unit per 10 µL. Where required, reactions were supplemented with 10% DMSO and the annealing temperature reduced to 63.5°C. PCR products were digested with AciI (New England Biolabs UK, Hitchin) in accordance with the manufacturer's instructions. DMSO was removed prior to AciI digestion using the QIAquick (Qiagen, Venlo, the Netherlands) PCR purification kit. DNA fragments were resolved by agarose gel electrophoresis and Southern blot hybridized as described.^{18 (link),30 (link)} Autoradiographic images were scanned and ePAL and modal allele lengths were estimated from the lower boundary^{18 (link)} and the densest part of the expanded allele distribution respectively by comparison against the molecular weight ladder, using CLIQS 1D gel analysis software (TotalLab UK, Newcastle upon Tyne).

Total genomic DNA was extracted from peripheral blood samples, as previously described [8 (link)]. To estimate the length of the expanded mode allele, small-pool PCR (SP-PCR) was carried out with small amounts of input DNA (300 pg), using flanking primers DM-C and DM-DR, as previously described [9 ,10 (link)]. PCR was performed using a Custom PCR Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 69 mM 2-mercaptoethanol, and Taq polymerase (Sigma-Aldrich, Gillingham, UK) at 1 unit per 10 µL. All reactions were supplemented with 5% DMSO and the annealing temperature was 63.5 °C. DNA fragments were resolved by electrophoresis on a 1% agarose gel, and Southern blot hybridized as described in references [9 ,10 (link)]. Autoradiographic images were scanned and the CTG size of the mode was estimated through comparison against the molecular weight ladder using GelAnalyzer 19.1 software (www.gelanalyzer.com, by Istvan Lazar Jr. and Istvan Lazar Sr.).