Typing of a Polymorphic Human Gene Conferring Susceptibility to Insulin-Dependent Diabetes Mellitus by Picosecond-Resolved FRET on Non-Purified/Non-Amplified Genomic DNA

This work concerns the identification of the alleles of the polymorphic DQB1 gene of the human leukocyte antigen system, conferring susceptibility to the development of insulin-dependent diabetes mellitus (IDDM) in non-PCR amplified DNA samples and, more importantly, in crude cell extracts. Our method is based on the time-resolved analysis of a Förster energy-transfer mechanism that occurs in a dual-labelled fluorescent probe specific for the IDDM-associated DQB1-0201 allele. Such an oligonucleotide probe is labelled, 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 various DQB1 alleles. 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 'susceptible' allele from the DNAs bearing any other sequence in the same region of the DQB1 gene. We could also distinguish the presence of the DQB1-0201 allele in a homozygous versus a heterozygous condition.