Exonuclease 1 en 0582

All mutations (I157T, IVS2+1G > A, and delC1100) detected by TaqMan PCR and allele-specific PCR were confirmed by capillary sequencing. PCR amplification products, purified by incubation with 10 U of exonuclease I (EN 0582) and 1 U of phosphatase Fast-AP (EF 0651) (both from ThermoFisher Scientific, Waltham, MA) for 15 min at 37 °C, followed by 20 min at 80 °C, were used as template for Sanger sequencing reactions. Sequencing reactions were performed using forward and reverse sequence-specific primers (CHEK2_EK3_F & CHEK2_EK3_R; F2 CONTROL & R2 CONTROL; CHEK2_1100delC_3_F & CHEK2_1100delC_3_R) and an ABI PRISM Big Dye Terminator Kit, version 3.1 (catalogue number: 4337450, Applied Biosystems/ThermoFisher Scientific), according to the manufacturer’s instructions. Sequencing results were analyzed using a 3130 Capillary Sequencer (Applied Biosystems/ThermoFisher Scientific). The sequences generated were compared with the reference sequence (NM_007194.4) using the NCBI BLAST Nucleotide tool. We decided to use Sanger sequencing instead of next-generation sequencing tests (NGS) in the analysis because the sensitivity of Sanger sequencing is at the level of 20% of allelic frequency so it is sufficient to detect germline mutation which are at the level of about 50% allelic frequency [37 (link)].