LightCycler® 480 Real-Time PCR System in Human Genetics
Featured project: High-Resolution Melting for CFTR Mutation Scanning
Stefanie Beck-Wödl and Peter Bauer
Molecular Pathology Research and Development Laboratory, Department of Pathology, University of Tübingen, Germany
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Cystic fibrosis (CF) is the most common autosomal recessive monogenic disease (1:2500, with a carrier frequency of 1 in 25). More than 1,000 mutations are described in the CFTR (cystic fibrosis transmembrane conductance regulator) gene. In Germany, 86% of all CFTR alleles are defined by 19 different mutations, with F508del being the most prevalent (73%). These alleles are located in nine different PCR fragments. Complete gene analysis by scanning and/or sequencing is seldom performed because of the cost, time, and labor involved. High-resolution melting analysis (HRM) is a rapid, closed-tube alternative.
To assess the feasibility of using HRM in CFTR mutation screening for research purposes, we set up PCR protocols for CFTR exons 4, 7, 10, 11, 14b, 19, 20, and 21, enabling the analysis of the most prevalent CFTR mutations in the German population. In parallel, unlabeled probes were tested to directly discern known CFTR mutations in these exons.
Materials and Methods
Several CFTR-mutation-positive samples were selected for this study, including samples for F508del, I507del, R117H, R347P, G542X, G551D, R553X, 1717-1G>A, 2789+5G>A, S1255X, W1272X, and N1303K. CFTR genotypes had been previously confirmed for all samples, either by direct sequencing or RFLP-analysis. Unlabeled probes were designed complementary to the wild-type sequences. PCR fragments usually had amplicon lengths of less than 400 bp. Amplification was carried out for 45 cycles followed by a melting curve from +50 to +85°C. Ten picomoles of each primer and 3 pmol of each unlabeled probe were used for detection.
Results and Discussion
So far, we have been able to establish an HRM analysis panel with 18 CFTR mutations amplified in one PCR assay. This panel covers approximately 85% of the German population. All mutations in this panel were determined to be heterozygous, as indicated by aberrant melting curves. F508del and compound heterozygote alleles in exon 11 (G551D/R553X) where clearly discernible by analysis of the melting curves of the unlabeled probes.
Figure 1: Genetic variation in the human CFTR gene analyzed by high-resolution melting. A 198 bp fragment of the human CFTR gene was amplified using the LightCycler® 480 High Resolution Melting Master and subjected to amplicon melting at high resolution. Difference plot analysis revealed three different groups of heterozygotes (blue, red, and green) in addition to the homozygous samples (black).
Figure 2: Genotype differentiation using high-resolution melting analysis in the presence of unlabeled probes. Samples not differentiated by amplicon melting alone (green in Figure 1) could be separated based on the Tm of an unlabeled probe covering both mutated sites (see insert).
High-resolution melting analysis with unlabeled probes enables accurate genotyping and mutation screening in the CFTR gene. The sensitivity obtained in this experiment was almost 100%, and specificity was guaranteed by mutation-specific melting peaks.
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