Warfarin Pharmacogenomics: Designing Electrochemical DNA-Based Sensors to Detect *2 Gene Variation.

BACKGROUND/OBJECTIVES: The CYP2C9 enzyme is involved in the metabolism of warfarin. The gene harbors several single-nucleotide polymorphisms (SNPs), including (rs1799853), which is known to affect warfarin's therapeutic response. So, it is important to develop analytical tools capable of genotyping these SNPs to adjust warfarin's therapeutic outcomes. In this work, an electrochemical DNA-based sensor was constructed and optimized for the detection of the *2 polymorphism.

METHODS

Using bioinformatic database platforms, two 71 base pair DNA target probes with the polymorphic variants A and G were chosen and designed. A DNA-based sensor was composed by mercaptohexanol and the *2 DNA capture probe in a self-assembled monolayer connected to screen-printed gold electrodes. Two independent hybridization events of the *2 allele were designed using complementary fluorescein-labeled DNA signaling to improve selectivity and avoid secondary structures. Three human samples with the homozygous variant (G/G) and non-variant (A/A) and heterozygous (G/A) genotypes were amplified by PCR and then applied to the developed genosensor.

RESULTS

Chronoamperometry measurements were performed for both polymorphic probes. A calibration curve in the 0.25 to 2.50 nM (LOD of 13 pM) and another in the 0.15 to 5.00 nM range (LOD of 22.6 pM) were obtained for the homozygous non-variant and variant probes, respectively. This innovative tool was capable of identifying the hybridization reaction between two complementary strands of immobilized DNA, representing a genotyping alternative to the classical PCR methodology.

CONCLUSIONS

The developed electrochemical DNA-based sensor was able to discriminate two synthetic SNP target sequences (Target-A and Target-G) and detect, with specificity, the three patients' genotypes (G/G, G/A, and A/A). This tool is therefore a promising, sensitive, and cost-effective analytical way to determine and discriminate an individual's genotype and predict the appropriate warfarin dose.