Precise Differentiation of Wobble-Type Allele via Ratiometric Design of a Ligase Chain Reaction-Based Electrochemical Biosensor for CYP2C19*2 Genotyping of Clinical Samples

Due to the comparable stability between the perfect-base pair and the wobble-base pair, a precise differentiation of the wobble-type allele has remained a challenge, often leading to false results. Herein, we proposed a ligase chain reaction (LCR)-based ratiometric electrochemical DNA sensor, namely, R-eLCR, for a precise typing of the wobble-type allele, in which the traditionally recognized negative signal of wobble-base pair-mediated amplification was fully utilized as a positive one and a ratiometric readout mode was employed to ameliorated the underlying potential external influence and improved its detection accuracy in the typing of the wobble-type allele. The results showed that the ratio between current of methylene blue (I (MB)) and current of ferrocene (I (Fc)) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: I- MB/I- Fc > 2; GG homozygote: I MB/I Fc < 1; GA heterozygote: 1 < I- MB/I- Fc< 2); the proposed R-eLCR successfully discriminated the three types of CYP2C19*2 allele in nine cases of human whole blood samples, which was consistent with those of the sequencing method. These results evidence that the proposed R-eLCR can serve as an accurate and robust alternative for the identification of wobble-type allele, which lays a solid foundation and holds great potential for precision medicine.

Automated summary

A ratiometric electrochemical DNA sensor was proposed for precise typing of wobble-type allele. By utilizing the traditionally recognized negative signal of wobble-base pair-mediated amplification as a positive one and employing a ratiometric readout mode, the detection accuracy of the wobble-type allele was improved.