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 (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.


CONCLUSIONS
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.
Millson
Alison
A
Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT 84108, USA.
Pont-Kingdon
Genevieve
G
Page
Sam
S
Lyon
Elaine
E
D003550:Cystic Fibrosis / Q000235:genetics*; D019005:Cystic Fibrosis Transmembrane Conductance Regulator / Q000235:genetics*; D031541:Fluorescence Resonance Energy Transfer; D006239:Haplotypes; D006801:Humans; D007438:Introns; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D011068:Poly G / Q000235:genetics*; D011071:Poly T / Q000235:genetics*
C505032:CFTR protein, human; D019005:Cystic Fibrosis Transmembrane Conductance Regulator; D011071:Poly T; D011068:Poly G