big dye terminator v1.1 cycle sequencing kit  (Thermo Fisher)

Journal: Frontiers in Medicine

Article Title: Case Report: Beyond type 1 diabetes: a case of delayed MODY1 diagnosis and successful transition to sulfonylurea therapy

doi: 10.3389/fmed.2025.1590935

Figure Lengend Snippet: Pedigree of proband and segregation analysis of the novel HNF4A variant. Sanger sequencing was used to validate the presence of the novel HNF4A variant identified by NGS and to verify the segregation in the proband's family. As shown by the chromatograms, the substitution was inherited from the affected father.

Article Snippet: Briefly, PCR products, corresponding to the genomic region of interest, were purified by enzymatic digestion with Exo/SAP-IT (Thermo Scientific ® , Massachusetts, USA) and sequenced with the Big Dye Terminator Cycle Sequencing Kit (Thermo Scientific ® , Massachusetts, USA) according to the provided protocol; sequencing reactions were run on a 3,130 × l Genetic Analyzer (Thermo Scientific ® , Massachusetts, USA) and analyzed with the Sequencer 4.7 software (Genecodes ® , USA).

Techniques: Variant Assay, Sequencing

Journal: Frontiers in Medicine

Article Title: Case Report: Beyond type 1 diabetes: a case of delayed MODY1 diagnosis and successful transition to sulfonylurea therapy

doi: 10.3389/fmed.2025.1590935

Figure Lengend Snippet: The novel HNF4A variant affects the splicing of the gene. RT-PCR products obtained from Hek-293 cells transiently transfected with the different pSPL3 constructs. A band of 263 bp was detectable both in cells transfected with the empty vector (EV) and with the construct carrying the third HNF4A exon 3 and flanking intronic sequences with the c.391G>A variant (V). This is due to the skipping of the HNF4A exon induced by the presence of the substitution with combination of the two artificial exons as observed in cells expressing the empty vector. A band of 358 bp is detectable in cells expressing the wild-type minigene construct (WT) and corresponding to the correct splicing combining the exon 3 of the HNF4A gene with those provided by the splicing vector, as schematically represented in the middle panel. The splicing events were also checked by Sanger sequencing of the RT-PCR products as shown by the chromatograms reported in the right panel. 1kb and 100bp, molecular weight markers; -, PCR reaction negative control; SA and SD6, artificial exons provided by the pSPL3 vector.

Article Snippet: Briefly, PCR products, corresponding to the genomic region of interest, were purified by enzymatic digestion with Exo/SAP-IT (Thermo Scientific ® , Massachusetts, USA) and sequenced with the Big Dye Terminator Cycle Sequencing Kit (Thermo Scientific ® , Massachusetts, USA) according to the provided protocol; sequencing reactions were run on a 3,130 × l Genetic Analyzer (Thermo Scientific ® , Massachusetts, USA) and analyzed with the Sequencer 4.7 software (Genecodes ® , USA).

Techniques: Variant Assay, Reverse Transcription Polymerase Chain Reaction, Transfection, Construct, Plasmid Preparation, Expressing, Sequencing, Molecular Weight, Negative Control