Direct molecular haplotyping of the IVS-8 poly(TG) and polyT repeat tracts in the cystic fibrosis gene by melting curve analysis of hybridization probes

Background: Molecular haplotyping is a developing technology with great potential for use in clinical diagnostics. We describe a haplotyping method that uses PCR combined with hybridization probes. Methods: We designed a LightCycler assay that uses fluorescence resonance energy transfer hybridization probes to haplotype the poly(TG) and polyT (TG-T) tract in the IVS-8 region of the CFTR gene. The reporter probe was designed as a perfect match to the TG12-5T allele. Results: Analysis of 132 samples revealed 9 unique derivative melting temperatures (T(m)s); the lowest was 42.4 degrees C and the highest was 63.6 degrees C. The lowest T(m)s were in the TGn-9T group, the intermediate T(m)s in the TGn-7T group, and the highest T(m)s in the TGn-5T group. Haplotype frequencies were highest (39%) for TG11-7T and lowest (0.4%) for TG13-5T. Conclusions: Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic T(m)s., This property enables genotyping as well as determination of the phase of multiple variants under the probe, a principle we demonstrated by haplotyping the TG-T repeat tract in the IVS-8 region of the CFTR gene. (C) 2005 American Association for Clinical Chemistry.