Figure 1.

Schematic diagram depicting fluorescent labeling and fluorescence detection of single nucleic acid molecules using a flow cytometer. In this example, ~22-mer microRNA is extended by rolling circle amplification using a circular DNA template and DNA polymerase to generate large molecules with repeating sequence motifs. The products are then labeled with a mixture of multicolor intercalating dyes and dye-DNA molecules that bind through hybridization. The products are then analyzed as individual events by multispectral fluorescence and scattering in a flow cytometer. The schematics illustrate molecular mechanisms but are not drawn to scale. The specific dye-DNA probes applied determine the density of labeling and the fraction of amplicon that is double-stranded.