Figure 2. Validation of two cryptic splicing mutation alleles. Validation of two cryptic splicing mutation alleles. Sanger sequencing
verified (A) the coding mutation (c.3793_3794insGAAA) and (B) the deep-intronic mutation (c.2710+233G>A) in MEP_305, (C) the coding mutation (c.934dupC), and (D) the deep-intronic mutation (c.1468–263G>C) in MEP_318. E: Schematic drawing of the minigene splicing assay. F: Using the minigene splicing assay, cryptic splicing induced by the two deep-intronic mutations is observed. Larger reverse
transcriptase (RT)–PCR products are observed in the construct carrying the mutation compared to the wild-type control construct.
G, H: Sanger sequencing of the RT–PCR band further confirms the splicing junction.