SPECTRAL MEASUREMENTS OF INTERCALATED PCR-AMPLIFIED SHORT TANDEM REPEAT ALLELES

Short tandem repeat (STR) alleles are popular for use as forensic mark ers due to their highly polymorphic nature. Commonly they are separate d by gel electrophoresis and visualized using intercalation dyes. The purpose of this study was to determine the changes in absorbance and f luorescence of DNA-intercalation dye complexes as a function of base p air (bp)-to-dye ratio. The DNA samples consisted of STR alleles from l oci THO1, F13A01, and vWFA31. The alleles were PCR amplified and HPLC purified to ensure that only the desired DNA fragment was present in e ach 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 individu ally with the bis-intercalators TOTO-1 and YOYO-1 and their correspond ing monomeric dyes TOPRO-1 and YOPRO-1. The absorbance of the DNA-dye complex at 260 nm increased with addition of each intercalation dye. S ubtraction of the dye absorbance rendered the DNA absorbance constant at 260 mm. Fluorescence emission increased dramatically upon intercala tion of both the monomeric and dimeric dyes into the DNA helix. A plat eau of fluorescence intensity was observed at base pair-to-dye ratios of 10/1 for the bis-intercalators TOTO-1 and 5/1 for YOYO-1 for all th ree loci. The greatest fluorescence intensity response was obtained wi th the intercalator YOYO-1 using allele 8 of the F13A01 locus, which h ad the greatest AT concentration.