Time-Resolved Förster Resonance Energy Transfer Analysis of Single-Nucleotide Polymorphisms: Towards Molecular Typing of Genes on Non-Purified and Non-PCR-Amplified DNA

Quantitative assessment of the fluorescence resonance energy transfer (FRET) efficiency between chromophores labeling the opposite ends of gene-specific oligonucleotide probes is a powerful tool to detect DNA polymorphisms with single-nucleotide resolution. The FRET efficiency can be most conveniently quantified by applying a time-resolved fluorescence analysis methodology, time-correlated single-photon counting. Recently, we probed by such technique the highly polymorphic DQB1 human gene. Namely, by using a single oligonucleotide probe and acting on non-amplified DNA samples contained in untreated cell extracts, we demonstrated the ability of pursuing unambiguous recognition of subjects bearing the homozygous DQB1-0201 genotype by exploiting the subtle, yet statistically significant, structural differences between the duplex formed by the probe with DQB1-0201 on the one end and duplexes formed with any of the other alleles, on the other end. The relevance of homozygous DQB1-0201 genotype recognition reseeds in the fact that the latter is overexpressed in subjects affected by insulin-dependent diabetes mellitus in north-eastern Italy. In this article we review our preceding achievements and report on additional in-vitro experiments aimed at characterizing the duplexes obtained by annealing of the DQB1 allelic variants with a second oligonucleotide probe, with the final scope to achieve full genotyping of DQB1 on raw DNA samples by means of cross-combination of the FRET responses of both probes.