Variation in resolution and sensitivity caused by intercalation dyes in capillary electrophoretic separations of DNA fragments

Capillary electrophoresis (CE) is a high resolution analytical technique which provides an alternative to traditional slab gel methodologies. Analysis time is faster due to the efficient heat dissipation of the capillary, allowing for the use of higher applied voltages. CE also offers many on-column detection modes. DNA can be separated and detected using CE with UV absorption detection at 260 nm, but with limited sensitivity. An alternative detection scheme with increased sensitivity, is laser-induced fluorescence (LIF) detection. This requires that the DNA fragments be PCR-amplified using fluorescent labeled primers, or stained on-column using intercalation dyes. This work describes the analysis of short tandem repeat (STR) loci THO1, F13A01 and vWFA31 alleles intercalated with monomeric and dimeric intercalation dyes and separated using CE with LIF detection. The alleles were purified using high performance liquid chromatography (HPLC) to ensure only the desired fragment was present for each sample analysis. These fragments were used to evaluate the absorbance changes of DNA and the fluorescence changes of the dyes after intercalation as a function of base pair to dye concentration. The electrophoresis of the DNA fragments intercalated before injection onto the CE column resulted in loss of resolution and sensitivity when compared to the on-column labeling technique. The study of dye-DNA complexes during electrophoresis required the development of a fluorescently labeled single stranded DNA standard. The results of these experiments were then applied to an STR typing assay. Using this typing assay, the 9 and 9.3 alleles of the THO1 locus were separated with a resolution of 0.49. This CE analysis, using on-column intercalation with a separation polymer containing $250\times10\sp{-6}$ mM TOTO-1, was complete in less than 20 minutes, and the only sample preparation necessary was a dilution step where the PCR product was diluted one to four thousand prior to CE analysis.