BOXTO as a real-time thermal cycling reporter dye

The unsymmetrical cyanine dyes BOXTO (4-[6-(benzoxazole-2-yl-(3-methyl-)-2,3-dihydro-(benzo-1,3-thiazole)-2methylidene)]-1-methyl-quinolinium chloride) and its positive divalent derivative BOXTO-PRO (4-[(3-methyl-6(benzoxazole-2-yl)-2,3-dihydro-(benzo-1,3-thiazole)-2-methylidene)]-1-(3-trimethylammonium-propyl)-quinolinium dibromide) were studied as real-time PCR reporting fluorescent dyes and compared to SYBR GREEN I (SG) (2-[N-(3-dimethylaminopropyl)-N-propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl- quinolinium). Unmodified BOXTO showed no inhibitory effects on real-time PCR, while BOXTO-PRO showed complete inhibition. Sufficient fluorescent signal was acquired when 0.5-1.0 mu M BOXTO was used with RotorGene and iCycler platforms. Statistical analysis showed that there is no significant difference between the efficiency and dynamic range of BOXTO and SG. BOXTO stock solution (1.5 mM) was stable at -20 degrees C for more than one year and 40 mu M BOXTO solution was more stable than 5x SG when both were stored at 4 degrees C for 45 days.