Labelless electrochemical melting curve analysis for rapid mutation detection

摘要

In the post-genome era there is an increasing demand for cost effective and rapid methods for the detection of specific mutations and single nucleotide polymorphisms. Here we describe a method for the rapid detection of mutations exploiting labelless electrochemical melting curve analysis, using the detection of the cystic fibrosis associated DF508 mutant as a model. A 21-base long thiolated probe, complementary to the region of Exon 10 of the cystic fibrosis transmembrane regulatory gene where the DF508 mutation lies, was immobilised on a gold electrode and hybridised to a synthetic analogue of single stranded PCR products for each of the mutant (85 bases) and wild type (82 bases) targets. Experimental conditions were optimised to exploit the guanine-specific interaction of the electroactive indicator, methylene blue. Upon hybridisation of the immobilised probe to the target, the number of guanine bases present in close proximity with the sensor surface increased from 3 to 14, resulting in a significant increase in signal. Ramping of the temperature caused denaturation of the on-surface immobilised duplex and a concomitant reduction in signal. From the first derivative of the melting curves a clear differentiation between the mutant and wild-type target could be observed. The proposed approach can be extended to array based melting curve analysis, allowing the simultaneous detection of multiple mutations and SNPs, and moreover the melting properties observed can also be used to design genosensors for single target detection.