To ligate juxtaposed probe pairs, 5 μl mix containing 2.5 units Taq DNA ligase in ligase buffer was added, plates were sealed, and incubation proceeded at 45°C for 1 hour followed by 65°C for 10 minutes. The plate wells were emptied as described above, and the resulting amplification templates were subject to PCR using T3 and 5′-biotinylated T7 universal primers. PCR was initiated by adding 15 μl master mix, containing 1.5 umole of each primer, 2.4 nmol of each dNTP, and 4.8e-4 units of HotStarTaq in reaction buffer. Plates were sealed and loaded into a Thermo Electron MBS 384 Satellite Thermal Cycler. Initial denaturation was performed at 95°C for 15 minutes, and then the plates were subjected to 29 cycles as follows, one minute per step: 92°C(denature), 60°C (anneal), 72°C (elongation). The resulting amplicons were gene-specific, barcoded, and biotinylated.
