Because a sequence complementary to the barcode on each probe has also been coupled to a Luminex bead, the amplicons (and hence the gene-specific sequence) can be identified by hybridization to the beads. A volume of 5 μl of PCR amplicon was transferred to a well containing 30 μl of L1000 bead mix (∼ 100 beads/region/well). The plate was sealed and incubated at 95°C for 2 minutes to denature the DNA. Incubation continued at 45°C for 18 hrs. Beads were pelleted, washed, and stained with 20 μl of 10 ng/ul streptavidin R-phycoerythrin conjugate (Molecular Probes) in 1× TMAC buffer (3 mol/l tetramethylammonium chloride, 0.1% N-lauryl sarcosine, 50 mmol/l tris-HCl [pH 8.0], 4 mmol/l EDTA [pH 8.0]) at 45°C for ten minutes.
