Design of an optimal allele-specific oligonucleotide probe for the efficient discrimination of a thermodynamically stable (G·T) mismatch

A general approach that allows reliable detection of a single base-pair mismatch is presented. Specifically, how important a careful design of the allele-specific oligonucleotide (ASO) probe is, was demonstrated for a G·T mispair highly stable from the thermodynamic point of view. This study involved comparison of six ASO probes of variable length (25 to 9-mer), with a similar GC composition and designed to hybridise the target in a way that the mutation centrally occurred with respect to the duplex region.All hybridisation experiments were performed on the surface of probe-modified screen-printed gold electrodes. Transduction of the interfacial biorecognition events was then accomplished by means of electrochemical measurements, using a streptavidin-conjugated alkaline phosphatase label and a suitable substrate.While the longest ASO probes (25 and 15-mer) did not allow significant differentiation of the mutant from the wild-type sequence and the shortest probe (9-mer) was incapable of forming a stable hybrid in the given hybridisation conditions, increasing levels of selectivity were observed employing the 13, 12 and 11-mer probes, respectively. In particular, the 11-mer ASO probe allowed resolving matched and mismatched duplexes with relative selectivity as high as 50:1 even operating in non-stringent hybridisation conditions, using buffers with a relatively high ionic strength, at room temperature and without the need for including chemicals such as formamide into the hybridisation medium.The results shown in this paper may serve to further improve the reliability of the hybridisation-based mutation analysis.