Abstract
Genotyping of genetic polymorphisms is widely used in clinical molecular laboratories to confirm or predict diseases due to single locus mutations. In contrast, very few molecular methods determine the phase or haplotype of two or more mutations that are kilobases apart. In this report, we describe a new method for haplotyping based on long-range allele-specific PCR. Reaction conditions were established to circumvent the incompatibility of using allele-specific primers and a polymerase with proofreading activity. Haplotypes are determined by post-PCR analysis using different detection methods. The clinical application presented here directly determines the phase of two mutations separated by 17.7 kilobases in the cystic fibrosis transmembrane conductance regulator gene. Each mutation, the missense mutation R117H in exon 4 and the 5T polymorphism in intron 8 (IVS-8), have mild phenotypic effect unless they are present on the same chromosome (in cis). If an individual is heterozygous for both R117H and the IVS-8 5T variant, cis/trans testing is required to completely interpret results. The molecular method presented here bypasses the need to perform family studies to establish haplotypes. We propose use of this assay as a reflex clinical test for R117H- 5T-positive samples.