Time-resolved FRET method for typing polymorphic alleles of the human leukocyte antigen system by using a single DNA probe

By tens-of-picosecond resolved fluorescence detection we study Förster resonance energy transfer between a donor and a black-hole-quencher acceptor bound at the 5′- and 3′-positions of a probe-oligonucleotide matching an allelic region of a class II antigen of the human leukocyte antigen system, namely the region between codons 52 and 57 of the HLA DQB1 0201 allele. This dual-labelled probe is annealed with either the exactly complementary allelic sequence or with polymorphic sequences of the same region. We detect the longest-lived donor fluorescence in the case of the perfect hybridization achieved with the 0201 allele and definitely faster and distinct decays for the other allelic variants, some of which are single-nucleotide polymorphic variants.