Paper detail

Polymorphic gene conferring susceptibility to insulin-dependent diabetes mellitus typed by ps-resolved FRET on nonamplified genomic DNA

This work concerns the identification of the allelic sequences of the DQB1 gene of the human leukocyte antigen system conferring susceptibility to the development of insulin-dependent diabetes mellitus (IDDM) in DNA samples with no need of PCR amplification. Our method is based on the time-resolved analysis of a Förster energy-transfer mechanism that occurs in a dual-labeled fluorescent probe specific for the base sequence of the allelic variant of interest. Such an oligonucleotide probe is labeled, at the two ends, by a pair of chromophores that operate as donor and acceptor in a Förster resonant energy-transfer. The donor fluorescence is quenched with an efficiency that is strongly dependent on the donor-to-acceptor distance, hence on the configuration of the probe after hybridization with the DNA containing or not the selected allelic sequence. By time-correlated single-photon counting, performed with an excitation/detection system endowed with 30-ps resolution, we measure the time-resolved fluorescence decay of the donor and discriminate, by means of the decay time value, the DNA bearing the allele conferring susceptibility to IDDM from the DNAs bearing any other sequence in the same region of the DQB1 gene.