Direct genotyping of C3435T single nucleotide polymorphism in unamplified human MDR1 gene using a surface plasmon resonance imaging DNA sensor.

Abstract

Optical genotyping of C3435T single nucleotide polymorphisms (SNPs) in unamplified human multidrug resistance (MDR1) gene was here performed by a surface plasmon resonance imaging (SPRi) dual-targeting DNA assay, allowing its selective detection down to 0.18 fM of the whole genomic DNA. The result was achieved by the combination of the rational selection of the DNA probe and an optimized sample pretreatment (i.e., ultrasound fragmentation and thermal denaturation). Some assay developments and tunings were reported in a previously published research, but here, for the first time, the biosensor reliability and its analytical performance were directly tested on the unamplified human DNA extracted from lymphocytes. The assay resulted to be able to differentiate among all the possible genotypes of C3435T (homozygote and heterozygote) in the diluted genomic samples using a label-free approach and by bypassing the classical PCR amplification of the target sequences. Moreover, the reusability of the DNA-based chip allowed up to 40 subsequent measuring cycles, opening new horizons in multi-SNP genotyping based on cheap and daily routine clinical monitoring by optical biosensing.