Labelless electrochemical melting curve analysis for rapid mutation detection

• In the post-genome era there is an increasing demand for cost effective and rapid methods for thedetection of specific mutations and single nucleotide polymorphisms. Here we describe a method forthe rapid detection of mutations exploiting labelless electrochemical melting curve analysis, using thedetection 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 theDF508 mutation lies, was immobilised on a gold electrode and hybridised to a synthetic analogue ofsingle 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 electroactiveindicator, methylene blue. Upon hybridisation of the immobilised probe to the target, the number ofguanine bases present in close proximity with the sensor surface increased from 3 to 14, resulting ina significant increase in signal. Ramping of the temperature caused denaturation of the on-surfaceimmobilised duplex and a concomitant reduction in signal. From the first derivative of the meltingcurves a clear differentiation between the mutant and wild-type target could be observed. The proposedapproach can be extended to array based melting curve analysis, allowing the simultaneous detection ofmultiple mutations and SNPs, and moreover the melting properties observed can also be used to designgenosensors for single target detection.

Ämnesord

NATURVETENSKAP  -- Kemi -- Analytisk kemi (hsv//swe)

NATURAL SCIENCES  -- Chemical Sciences -- Analytical Chemistry (hsv//eng)

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