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Molecular Diagnostics and Genetics |
1 Institute for Clinical and Experimental Pathology, ARUP Laboratories, Salt Lake City, UT.
2 Department of Pathology, University of Utah School of Medicine, Salt Lake City, UT.
aAddress correspondence to this author at: ARUP Laboratories, 500 Chipeta Way, Salt Lake City, UT 84108.
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 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.
The following articles in journals at HighWire Press have cited this article:
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  J. K. Bickmann, W. Kamin, M. Wiebel, F. Hauser, J. J. Wenzel, C. Neukirch, M. Stuhrmann, K. J. Lackner, and H. Rossmann A Novel Approach to CFTR Mutation Testing by Pyrosequencing-Based Assay Panels Adapted to Ethnicities Clin. Chem., June 1, 2009; 55(6): 1083 - 1091. [Abstract] [Full Text] [PDF]
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C. Costa, J.-M. Costa, J. Martin, B. Boissier, M. Goossens, and E. Girodon Multiplex Allele-Specific Fluorescent PCR for Haplotyping the IVS8 (TG)m(T)n Locus in the CFTR Gene Clin. Chem., September 1, 2008; 54(9): 1564 - 1567. [Abstract] [Full Text] [PDF]
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G. Pont-Kingdon, R. L. Margraf, K. Sumner, A. Millson, E. Lyon, and E. Schutz Design and Application of Noncontinuously Binding Probes Used for Haplotyping and Genotyping Clin. Chem., June 1, 2008; 54(6): 990 - 999. [Abstract] [Full Text] [PDF]
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 C. Bareil, C. Guittard, J.-P. Altieri, C. Templin, M. Claustres, and M. des Georges Comprehensive and Rapid Genotyping of Mutations and Haplotypes in Congenital Bilateral Absence of the Vas Deferens and Other Cystic Fibrosis Transmembrane Conductance Regulator-Related Disorders J. Mol. Diagn., November 1, 2007; 9(5): 582 - 588. [Abstract] [Full Text] [PDF]
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V. Mantovani, P. Garagnani, P. Selva, C. Rossi, S. Ferrari, M. Cenci, N. Calza, V. Cerreta, D. Luiselli, and G. Romeo Simple Method for Haplotyping the Poly(TG) Repeat in Individuals Carrying the IVS8 5T Allele in the CFTR Gene Clin. Chem., March 1, 2007; 53(3): 531 - 533. [Abstract] [Full Text] [PDF]
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 B. A. Konfortov, A. T. Bankier, and P. H. Dear An efficient method for multi-locus molecular haplotyping Nucleic Acids Res., January 12, 2007; 35(1): e6 - e6. [Abstract] [Full Text] [PDF]
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C. Costa, M. Goossens, and E. Girodon Simultaneous Molecular Haplotyping of Both IVS8 (TG)m and (T)n Tracts in the CFTR Gene: Still a Challenge. Clin. Chem., August 1, 2006; 52(8): 1621 - 1622. [Full Text] [PDF]
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A. Millson, G. Pont-Kingdon, and E. Lyon Response to the letter "Simultaneous Molecular Haplotyping of Both IVS8 (TG)m and (T)n Tracts in the CFTR Gene: Still a Challenge" by Costa et al. Clin. Chem., August 1, 2006; 52(8): 1622 - 1622. [Full Text] [PDF]
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