Color multiplexing hybridization probes using the apolipoprotein E locus as a model system for genotyping.

Fluorescent hybridization probes were multiplexed for color genotyping of the apolipoprotein E locus using model oligonucleotide targets. Fluorescence resonance energy transfer was observed during adjacent hybridization of 3'-fluorescein-labeled "donor" probes paired with 5'-labeled "acceptor" probes with different emission spectra reporting at codons 112 and 158. The acceptor dyes emitted at either 640 nm (LightCycler Red 640) or 705 nm (LightCycler Red 705) and were monitored with a LightCycler, a thermal cycler with an integrated fluorimeter. The color of the acceptor dye identified each site and the characteristic melting temperatures of the fluorescein-labeled probes identified single base changes within each codon. Color compensation of temperature-dependent spectral overlap was applied to completely separate each channel. Competition between the probes and the complementary strand for the target sequence decreased resonance energy transfer, indicating an advantage of single-stranded target. Hybridization probes of the same length, but different GC content are T(m) shifted by the same amount during A:C mismatch duplex melting. Genotyping was optimal at both sites if melting curve analysis was preceded by a slow (1 degrees C/s) annealing phase. Although each site preferred different concentrations of Mg(2+) and target strand for optimal genotyping, conditions for multiplexing were found. This method, along with an appropriate amplification technique, should allow real-time multiplex genotyping from genomic DNA.