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

• Published in: Clinical chemistry (Baltimore, Md.) Vol. 51; no. 9; pp. 1619 - 1623
• Main Authors: Millson, Alison, Pont-Kingdon, Genevieve, Page, Sam, Lyon, Elaine
• Format: Journal Article
• Language: English
• Published: Washington, DC Am Assoc Clin Chem 01.09.2005; American Association for Clinical Chemistry; Oxford University Press
• Subjects: Analytical, structural and metabolic biochemistry; Annealing; Biological and medical sciences; Chromosomes; Cystic fibrosis; Cystic Fibrosis - genetics; Cystic Fibrosis Transmembrane Conductance Regulator - genetics; Energy transfer; Fluorescence Resonance Energy Transfer; Fundamental and applied biological sciences. Psychology; Genotype & phenotype; Haplotypes; Humans; Hybridization; Introns; Investigative techniques, diagnostic techniques (general aspects); Medical sciences; Melting; Mutation; Nucleic Acid Amplification Techniques; Poly G - genetics; Poly T - genetics; Probes; Proteins; Temperature; Respiratory disease; Metabolic diseases; Cystic fibrosis; Biochemistry; Melting curve; Hybridization; Probe; Genetic disease; Direct method; Gene; Clinical biology; Digestive diseases; Genetics; Molecular biology; Pancreatic disease
• ISSN: 0009-9147; 1530-8561
• DOI: 10.1373/clinchem.2005.052159

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. 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. Analysis of 132 samples revealed 9 unique derivative melting temperatures (Tms); the lowest was 42.4 degrees C and the highest was 63.6 degrees 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. Different combinations of polymorphisms under the reporter hybridization probe had unique and characteristic Tms. 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.