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

Abstract

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 (Tms); the lowest was 42.4 °C and the highest was 63.6 °C. The lowest Tms were in the TGn-9T group, the intermediate Tms in the TGn-7T group, and the highest Tms 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 ms. 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. © 2005 American Association for Clinical Chemistry.