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

Anal Chem. 2023 Oct 3;95(39):14592-14599. doi: 10.1021/acs.analchem.3c01907. Epub 2023 Sep 8.

Abstract

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 (IMB) and current of ferrocene (IFc) was partitioned in three regions and three types of wobble-type allele were thus precisely differentiated (AA homozygote: IMB/IFc > 2; GG homozygote: IMB/IFc < 1; GA heterozygote: 1 < IMB/IFc < 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Biosensing Techniques*
  • Cytochrome P-450 CYP2C19
  • Electrochemical Techniques
  • Genotype
  • Gold
  • Humans
  • Ligase Chain Reaction*
  • Limit of Detection

Substances

  • Cytochrome P-450 CYP2C19
  • Gold
  • CYP2C19 protein, human