How Real-Time and End-Point PCR Works in the BAX® System

The PCR tablets in BAX® System real-time assays contain mulitiple, target-specific dye-labeled probes for fluorescent detection. Probes are short DNA sequences with quencher dye at one end that greatly reduces signal from the fluorescent dye at the opposite end. Probes bind to a specific area within the targeted DNA fragment. During PCR the fluorophor separates from the quencher, allowing for increased fluorescent signal. The BAX® System instrument uses dye-specific filters to measure fluorescent signal at the end of each cycle, plotting any amplification and displaying clear positive or negative results at the end of the 50-75 minute program. In standard assays, BAX® System PCR tablets contain intercalating dye, which binds with double-stranded DNA and emits fluorescent signal in response to light. After the amplification phase is complete, the temperature of the samples is raised to the point where the double-stranded DNA begins to denature. This releases the intercalating dye, which lowers the fluorescent signal. The change in signal can be plotted against temperature to generate a melting curve, which is interpreted by the BAX® System software as positive or negative results.