Optimization of intercalation dye concentration for short tandem repeat allele genotyping using capillary electrophoresis with laser-induced fluorescence detection

DNA analysis using capillary electrophoresis (CE) with laser-induced fluore scence (LIF) detection requires that polymerase chain reaction products eit her be prepared using primers with fluorescent molecules covalently bonded to them or stained with a fluorescent intercalation dye following amplifica tion. The intercalation technique has the advantage of allowing fluorescenc e detection of any double-stranded DNA (dsDNA) product regardless of the am plification primers used. The increased sensitivity of LIF detection is som etimes compromised by the intercalation dye changing the mass to charge rat io of the DNA. The purpose of this study was to evaluate the changes of mig ration rate, resolution and fluorescent intensity of dye-DNA complexes duri ng electrophoretic separations, and to establish the optimal parameters for short tandem repeats alleles profiling. The alleles of three STR loci THO1 , F13A01 and vWFA31 were intercalated with the monomeric dyes TOPRO-1 and Y OPRO-1, and their corresponding dimers, TOTO-1 and YOYO-1 (Molecular Probes , Eugene, OR, USA). Alleles intercalated before injection onto the CE colum n resulted in loss of resolution and sensitivity when compared to the on-co lumn labeling technique. The results of this experimentation were then appl ied to a STR typing assay using a commercially available polymer and buffer matrix. This assay included development of a unique internal standard used for migration time normalization assignment of alleles. Consequently, the 9 allele and the 9.3 microvariant of the THO1 locus were separated and type d correctly with a resolution of 0.49 in less than 20 min, and the only sam ple preparation necessary after amplification was a dilution step. (C) 1999 Elsevier Science B.V. All rights reserved.