Seqscape 4

Tumor DNA samples were analyzed for mutations in exons 9, 10, 11, 12, 13 and exon 14 of POLE (NG_033840.1) by Sanger sequencing. Exons were amplified using M13-tailed oligonucleotides, as previously published [61 (link),62 (link)]. After the purification of PCR products using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany), bidirectional Sanger sequencing of PCR products was performed on an ABI 3500 Genetic Analyzer (Thermo Fisher Scientific, Bremen, Germany) using the BigDye Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific, Bremen, Germany) according to standard protocols. Finally, sequence analysis was carried out using SeqScape 4 software (Thermo Fisher Scientific, Bremen, Germany).

The blood samples were taken from the patient and their parents, and high-quality genomic DNA was purified from peripheral blood leukocytes using the Wizard® Genomic DNA Purification Kit from Promega. The quality and concentrations of DNA samples were examined by Nanodrop 1000 Spectrophotometer (Thermo Fisher Scientific). We sequenced all exons and flanking intronic sequences of ITGB2 using the primers and PCR conditions of Yassaee et al. [13 (link)]. PCR products were sequenced using BigDye™ Terminator v3.1 Ready Reaction Cycle Sequencing Kit, electrophoresis was performed on SeqStudio Genetic Analyzer, and sequence chromatograms were analyzed by SeqScape 4 software (Thermo Fisher Scientific). Ninety-six DNA samples from healthy individuals all of Moroccan origin were sequenced for ITGB2 exon 7 to verify the frequency of the novel mutation identified.