Total genomic DNA was isolated from peripheral blood using DNeasy blood DNA extraction Kit (Qiagen) according to the manufacturer’s instructions. DNA purity and concentration were measured using a NanoDropTM spectrophotometer.
Polymerase chain reaction reactions were performed on genomic DNA, following standard protocols. Sanger sequencing was performed using an automated sequencer (ABI 3500; Applied Biosystems, Foster City, CA, United States) and a cycle sequencing reaction kit (Bigdye Terminator v3.1 kit, Applied Biosystems). Data was analyzed using BioEdit software version 7.2.5.
Sanger sequencing technique was first used to screen the c.211dupA mutation on exon 5 of BRCA1 (NM_007294.3) among all 122 participants. It was also used to validate the identified variants resulting from the NGS assays. Non-carriers of c.211dupA mutation were tested for other recurrent BRCA1/2 mutations in Tunisia namely BRCA1-c.798_799delTT, BRCA1-c.5266dupC, and BRCA2-c.1310_1313delAAGA (Mahfoudh et al., 2012 (link); Fourati et al., 2014 (link); Msolly and Kassab, 2015 ; Riahi et al., 2015 (link)).
Polymerase chain reaction reactions were performed on genomic DNA, following standard protocols. Sanger sequencing was performed using an automated sequencer (ABI 3500; Applied Biosystems, Foster City, CA, United States) and a cycle sequencing reaction kit (Bigdye Terminator v3.1 kit, Applied Biosystems). Data was analyzed using BioEdit software version 7.2.5.
Sanger sequencing technique was first used to screen the c.211dupA mutation on exon 5 of BRCA1 (NM_007294.3) among all 122 participants. It was also used to validate the identified variants resulting from the NGS assays. Non-carriers of c.211dupA mutation were tested for other recurrent BRCA1/2 mutations in Tunisia namely BRCA1-c.798_799delTT, BRCA1-c.5266dupC, and BRCA2-c.1310_1313delAAGA (Mahfoudh et al., 2012 (link); Fourati et al., 2014 (link); Msolly and Kassab, 2015 ; Riahi et al., 2015 (link)).
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