2.2. Analysis of Alternating Mycobacterium Positive and Negative Tissue Samples
First the mycobacterium-positive and then negative tissue blocks were loaded into an automated tissue microarrayer (TMA Grandmaster, 3D Histech, Budapest, Hungary). Four punches at 0.6 mm in diameter were taken from the positive sample and transferred to a 0.2 mL PCR tube. The punching tool was mechanically cleaned in an empty paraffin block by insertion and removal multiple times (Figure 2). Then, the negative tissue block was cored similarly and tissue punches placed into the respective tube. Again a mechanical cleaning took place. This sequence was repeated three times such that six different tubes containing alternating positive and negative samples were obtained.
DNA was isolated from tissue punches by overnight digestion with proteinase K following purification using a BioRobot EZ1 (Qiagen, Hilden, Germany). The primer pair used for PCR amplification of a fragment from the 6110 insertion sequence specific for mycobaterium tuberculosis complex was 5'-CCTGCGAGCGTAGGCGTCGG-3' and 5'-GTTTCTCGTCCAGCGCCGCTTCGG-3'. The forward primer was labeled by FAM. Amplification was performed in 40 cycles of 95 °C for 1 min, 62 °C for 1 min and 72 °C for 1.5 min. The PCR product was analyzed by capillary electrophoresis using a Genetic Analyzer 3500 (Life Technologies, Zug, Switzerland). 5 fg of mycobacterium tuberculosis DNA corresponding to 5–10 bacteria was used as a positive control. Human DNA was used as a negative control.
Figure 2  Schematic diagram showing (above) the alternating sequence of mycobacterium-positive and negative tissue blocks in the arrayer; (below) alternating colorectal cancer tissue blocks with known mutational status, followed by mechanical cleaning of the punching device in a paraffin block.

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