Spectral measurements of intercalated PCR-amplified short tandem repeat alleles

Short tandem repeat (STR) alleles are popular for use as forensic markers due to their highly polymorphic nature. Commonly they are separated by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and fluorescence of DNA-intercalation dye complexes as a function of base pair (bp)-to-dye ratio. The DNA samples consisted of STR alleles from loci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in each sample. Alleles ranged in size from 151 bp for locus vWFA (allele 17) to 199 bp for the locus F13A01 (allele 8). The adenine and thymine (AT) content varied from 48% for the THO1 locus to 69% for F13A01 and vWFA31 loci. The homozygous alleles of each locus were mixed individually with the bis-intercalators TOTO-1 and YOYO-1 and their corresponding monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. Subtraction of the dye absorbance rendered the DNA absorbance constant at 260 nm. Fluorescence emission increased dramatically upon intercalation of both the monomeric and dimeric dyes into the DNA helix. A plateau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalator TOTO-1 and 5/1 for YOYO-1 for all three loci. The greatest fluorescence intensity response was obtained with the intercalator YOYO-1 using allele 8 of the F13A01 locus, which had the greatest AT concentration.