FIGURE 8.

Categorization of detection methods. Reactions can be monitored using simpler, but less-specific, sequence-independent methods (e.g., pH changes) or the somewhat more complex but equally more-specific sequence-dependent methods. These, in turn, can either be monitored in real time (e.g., detection of amplification by releasing of quenching [DARQ], intercalating dyes) allowing amplicon formation to be monitored kinetically or as an end-point, stopping/recording the result at a defined time (e.g., quantify and annotate short reads in R [QuasR]). DNA sequencing is the ultimate sequence-dependent endpoint method. When sequence-independent methods are used, false-positive results can be an issue. Most sequence-dependent methods also allow for multiplexing multiple targets in the same reaction. Sequencing of amplicons can allow detection of different variants. Variations on these themes have been described—the location of the icon in the 4-box is purely indicative.