Fig. 5: Generation and characterization of patient-specific KCNQ1 minigene constructs.

a Structure of the 2078-bp KCNQ1 minigene fragment from exon 13 (E13) to 15 (E15), which includes the c.1686−9 T > C variant in intron 13, that was amplified from the patient genomic DNA and cloned in-frame to both the FLAG (F) and Myc (M) epitope tags of the expression vector. b Identification of positive minigene constructs by agarose electrophoresis of plasmid DNA isolated from 22 ampicillin-resistant bacterial clones. Plasmids with bold numbers, displaying reduced electrophoretic mobility as compared to the empty vector (EV), were identified as positives (containing the insert) and sequenced. Plasmids with the reference T or alternative C nucleotide, corresponding to the variant c.1686−9 T > C, were classified as REF or MUT, respectively, as indicated on the top of each lane. c DNA sequencing chromatogram of REF 1517 and MUT 1521 plasmids. The arrows mark the position of the affected nucleotide. d KCNQ1 variants identified by Sanger sequencing of 19 positive plasmids. For each variant, the presence of the reference (−) or the alternative (+) nucleotide in each plasmid sequence is indicated. Variants are labeled with the same tag (#) ID numbers used in Table 1 and Fig. 2.