Agarose gel electrophoresis is a classic method of detection. During electrophoresis, the migration of the DNA molecules is almost entirely dependent upon their size. Smaller molecules will migrate faster than larger molecules. Following electrophoresis, some method of DNA staining or probing is required. Ethidium bromide is the most commonly employed stain utilized to visualize DNA products. The patterns generated by test samples are typically compared to those of known controls. Pulse Field Gel Electrophoresis (PFGE) using gel plus electrical current is useful.
Biotin labeling in microwell-based formats (reminiscent of some ELISA assays) is another method utilized in some of the first commercial formats. Using the Roche COBAS® AMPLICOR assays as an example, the master mix contains biotinylated probes for the target sequence. Biotin-labeled amplicons are generated during the extension sequence of the PCR process. During the detection phase, biotin-labeled amplicons bind to a capture probe attached to a magnetic particle. Avidin-horseradish peroxidase conjugate is added, which binds to the biotin-labeled amplicons. After washing, the substrate is added; bound conjugate acts on the substrate to produce a color reaction, read by the analyzer.
In earlier developed PCR methods, amplification and detection/characterization of the amplicon are carried out as independent steps. When detection occurs as a separate process, the analytical time is increased significantly—from 2 to 24 additional hours. In addition, some methods require more extensive manipulation of the nucleic acid amplification test (NAAT) product, creating risks of crossover contamination and requiring greater expertise to perform the assays successfully.
