DNA Sequence Analysis
Peripheral blood samples were collected for DNA sequencing to determine CFTR mutations in siblings. Genomic DNA was isolated by the salting-out method. CFTR coding regions, exon-intron boundaries, promoter and 3’UTR were amplified in Gene Amplification PCR System 9700 (Applied Biosystem, USA). Standard PCR reaction was performed using the with 5U/µL Taq DNA polymerase (Ampliqon, Denmark), 12,5 mM each dNTPs (Fermantas, USA), 100 nM forward and reverse primer, 10X ammonium buffer (Ampliqon, Denmark) and 100 ng/µl input genomic DNA per reaction. PCR conditions were, 35 cycle: 94°C/30 seconds; denaturation, 57°C/45 seconds; annealing, 72°C/30 seconds; extension. The PCR products were checked in 3% agarose gel electrophoresis containing ethidium bromide (Sigma-Aldrich, USA). After genomic DNA amplification, Sanger DNA sequencing was carried out in ABI 3030 (Thermo-Fisher Scientific, USA).