Modification of indium tin oxide electrodes with repeat polynucleotides: Electrochemical detection of trinucleotide repeat expansion

Genomic expansion of the triplet repeat sequences 5'-(CTG)(n) and 5'-(CGG)( n) leads to myotonic dystrophy and fragile X syndrome, respectively. Method s for determining the number of repeats in unprocessed nucleic acids would be useful in diagnosing diseases based on triplet repeat expansion. Electro chemical reactions based on the oxidation of guanine were expected to give larger signals per strand for expansion of repeats containing guanine. A no vel PCR reaction was used to generate fragments containing 150, 230, 400, a nd 830 repeats of (CTG)(n), which codes for myotonic dystrophy, and 130 and 600 repeats of (CGG)(n), which codes for fragile X syndrome. These PCR fra gments were immobilized to indium tin oxide electrodes, and oxidation of gu anine in the fragments was realized using electrocatalysis by Ru(bPY)(3)(2) (bpy = 2,2'-bipyridine). The catalytic currents due to oxidation of the i mmobilized guanines by Ru(bPY)(3)(3+) increased with the number of repeats and were a linear function of the repeat number when normalized to the numb er of strands immobilized. These results suggest a sensing strategy for rep eat length based on the combination of the electrocatalytic strategy for de termining the repeat length combined with existing methods for determining the number of strands.