Fig 3

Long-range digital PCR to determine whether p.Arg117His and poly T (5T/7T/9T) in CFTR are in cis or in trans. A, Schematic diagram of the p.Arg117His (R117H) and poly T (5T/7T/9T) allele in CFTR. p.Arg117His is in exon 4. The polyT region is in intron 8. Two primers were designed for the long-range 18-kb PCR fragment, CF-F and CF-R. To increase the specificity, nested primers (CF-117-F-S with CF-117-R-S for p.Arg117His and CF-pT-F-S with CF-117-R-S for the polyT region) were used in Sanger sequencing. B, Example of the sequencing results of the second round PCR after initial digital PCR of wells 13 and 28 for cell line gDNA sample NA13591. Sequencing indicated that well 13 contained a template that had both p.Arg117 (R117) and 9T; well 28 contained a template that had an allele with both p.His117 (H117) and 5T. C, Example of the sequencing results of the second round PCR after initial digital PCR for patient P10. Sequencing indicated that P10 had two alleles, one with p.His117 (H117) and 7T (well 16) and another with p.Arg117 (R117) and 5T (well 32).