Examination of cyanine intercalation dyes for rapid and sensitive detection of DNA fragments by capillary electrophoresis.
Recently, a series of cyanine dyes has been developed that are reported to offer superior fluorescence upon intercalation relative to ethidium bromide (EB). The dimeric cyanine dyes, such as POPO-3 (Molecular Probes, Eugene, OR, U.S.A.), are reported to offer the highest fluorescence enhancement of the cyanine dyes. Although POPO-3 does offer an improvement in sensitivity, achieving reproducible, high-efficiency results by employing pre-column, on-column, and postcolumn labeling strategies proved to be problematic. Evaluation of the labeling rate of these dimeric dyes indicates that perhaps the most limiting factor to using this dye for rapid CE analysis of DNA fragments is the apparent slow rate at which the dye fully intercalates into the DNA structure. Use of the monomeric analog PO-PRO-3 (Molecular Probes) resulted in much better separations and low limits of defection when employed for on-column intercalation. However, we find that the kinetics are too slow to employ this dye in a sheath flow cell for postcolumn derivatization.
D001583:Benzoxazoles; D002021:Buffers; D002232:Carbocyanines; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D007364:Intercalating Agents; D015394:Molecular Structure; D012680:Sensitivity and Specificity; D013997:Time Factors