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Molecular Diagnostics and Genetics |
1 Division of Human Genetics, Children’s University Hospital, Inselspital, Bern, Switzerland.
aAddress correspondence to this author at: Head of Division of Human Genetics, Children’s University Hospital, Inselspital, 3010 Bern, Switzerland. Fax: 41-31-632-94-84; e-mail: sabina.gallati{at}insel.ch.
Background: Cystic fibrosis (CF) is associated with at least 1 pathogen point sequence variant on each CFTR allele. Some symptomatic patients, however, have only 1 detectable pathogen sequence variant and carry, on the other allele, a large deletion that is not detected by conventional screening methods.
Methods: For relative quantitative real-time PCR detection of large deletions in the CFTR gene, we designed DNA-specific primers for each exon of the gene and primers for a reference gene (ß2-microglobulin). For PCR we used a LightCycler system (Roche) and calculated the gene-dosage ratio of CFTR to ß2-microglobulin. We tested the method by screening all 27 exons in 3 healthy individuals and 2 patients with only 1 pathogen sequence variant. We then performed specific deletion screenings in 10 CF patients with known large deletions and a blinded analysis in which we screened 24 individuals for large deletions by testing 8 of 27 exons.
Results: None of the ratios for control samples were false positive (for deletions or duplications); moreover, for all samples from patients with known large deletions, the calculated ratios for deleted exons were close to 0.5. In addition, the results from the blinded analysis demonstrated that our method can also be used for the screening of single individuals.
Conclusions: The LightCycler assay allows reliable and rapid screening for large deletions in the CFTR gene and detects the copy number of all 27 exons.
The following articles in journals at HighWire Press have cited this article:
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  F. M. Hantash, A. Rebuyon, M. Peng, J. B. Redman, W. Sun, and C. M. Strom Apparent Homozygosity of a Novel Frame Shift Mutation in the CFTR Gene Because of a Large Deletion J. Mol. Diagn., May 1, 2009; 11(3): 253 - 256. [Abstract] [Full Text] [PDF]
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E. Lyon and C. T. Wittwer LightCycler Technology in Molecular Diagnostics J. Mol. Diagn., March 1, 2009; 11(2): 93 - 101. [Abstract] [Full Text] [PDF]
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I. Schrijver, K. Rappahahn, L. Pique, M. Kharrazi, and L.-J. Wong Multiplex Ligation-Dependent Probe Amplification Identification of Whole Exon and Single Nucleotide Deletions in the CFTR Gene of Hispanic Individuals with Cystic Fibrosis J. Mol. Diagn., July 1, 2008; 10(4): 368 - 375. [Abstract] [Full Text] [PDF]
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C. P. Vaughn, E. Lyon, and W. S. Samowitz Confirmation of Single Exon Deletions in MLH1 and MSH2 Using Quantitative Polymerase Chain Reaction J. Mol. Diagn., July 1, 2008; 10(4): 355 - 360. [Abstract] [Full Text] [PDF]
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 E. Castellsague, S. Gonzalez, M. Nadal, O. Campos, E. Guino, M. Urioste, I. Blanco, T. Frebourg, and G. Capella Detection of APC Gene Deletions Using Quantitative Multiplex PCR of Short Fluorescent Fragments Clin. Chem., July 1, 2008; 54(7): 1132 - 1140. [Abstract] [Full Text] [PDF]
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J. Montgomery, C. T. Wittwer, J. O. Kent, and L. Zhou Scanning the Cystic Fibrosis Transmembrane Conductance Regulator Gene Using High-Resolution DNA Melting Analysis Clin. Chem., November 1, 2007; 53(11): 1891 - 1898. [Abstract] [Full Text] [PDF]
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T. von Kanel, F. Adolf, M. Schneider, J. Sanz, and S. Gallati Sample Number and Denaturation Time Are Crucial for the Accuracy of Capillary-Based LightCyclers Clin. Chem., July 1, 2007; 53(7): 1392 - 1394. [Full Text] [PDF]
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