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).