Modification of Indium Tin Oxide Electrodes with Repeat Polynucleotides: Electrochemical Detection of Trinucleotide Repeat Expansion

Genomic expansion of the triplet repeat sequences 5ʹ-(CTG)n and 5ʹ-(CGG)n leads to myotonic dystrophy and fragile X syndrome, respectively. Methods for determining the number of repeats in unprocessed nucleic acids would be useful in diagnosing diseases based on triplet repeat expansion. Electrochemical reactions based on the oxidation of guanine were expected to give larger signals per strand for expansion of repeats containing guanine. A novel PCR reaction was used to generate fragments containing 150, 230, 400, and 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 fragments were immobilized to indium tin oxide electrodes, and oxidation of guanine in the fragments was realized using electrocatalysis by Ru (bpy)32+ (bpy = 2,2ʹ-bipyridine). The catalytic currents due to oxidation of the immobilized guanines by Ru(bpy)33+ increased with the number of repeats and were a linear function of the repeat number when normalized to the number of strands immobilized. These results suggest a sensing strategy for repeat length based on the combination of the electrocatalytic strategy for determining the repeat length combined with existing methods for determining the number of strands.