Split hybridisation probes for electrochemical typing of single-nucleotide polymorphisms

This paper describes the development of a highly selective single-nucleotide polymorphisms (SNPs) typing method based on the use of split hybridisation probes and demonstrates the concept through the electrochemical analysis of single-base mutations in actual patient samples. The requirement that two probes hybridised adjacent to one another to allow for stabilisation (via base-stacking) and binding of the allele-specific oligonucleotide (ASO), imparted highly stringent selectivity criteria to the assay. Simple rules for tuning the characteristics of such stacking/ASO probe pairs and achieve full mismatch discrimination at ambient conditions (with no need to strictly control the temperature) are provided. All genotyping experiments were indeed performed at room temperature, using the planar surface of disposable probe-modified gold electrodes as the genosensing platform. The ability to detect nanomolar amounts of a synthetic target even within a vast excess of single-base substituted sequences gave strong evidence of the specificity of the split probes assay. Proving the general validity of this genotyping approach, application of the analytical pathway was further demonstrated for clinical targets (amplified from the human TP53gene) whose mutational site was poorly accessible, being part of a thermodynamically stable hairpin. In combination with use of auxiliary oligonucleotides (which restored the availability of each pre-defined hybridisation site), the assay demonstrated the ability to fully discriminate single-base mutations with detection limits in the high picomolar range (total analysis time: 60 min). Our specific probe design, hybridisation and signal transduction paths make the analytical process remarkably simple, relatively low cost and, thus, well suited for low throughput analysis of clinically relevant samples.