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

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...

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Bibliographic Details
Published in:Clinical chemistry (Baltimore, Md.) Md.), 2005-09, Vol.51 (9), p.1619-1623
Main Authors: Millson, Alison, Pont-Kingdon, Genevieve, Page, Sam, Lyon, Elaine
Format: Article
Language:English
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
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Summary: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.
ISSN:0009-9147
1530-8561
DOI:10.1373/clinchem.2005.052159