Amplification and Sequencing of TEP1-TED Domain

A 456 bp fragment of the TEP1-TED domain was amplified with primers OB2996F (5'-CACGGTCATCAAGAACCTGGAC-3') [19 (link)] and EMTep1R (5’-TCCAGCAATGCCATCAACACAT-3'), the latter specifically designed for the aim of this work in order to avoid co-amplification of other TEP-related paralogs, which were instead pervasively co-amplified with other primer couples available in literature. Amplifications were performed in a 15 μl reaction-mix using 0.5–1.0 μl of template DNA using the High Fidelity AccuPrime Taq DNA Polymerase kit (Life Technologies) following manufacturer's guidelines. Thermocycler conditions were as follows: initial denaturation at 94°C for 2 min followed by 35 cycles of 94°C for 30 sec, 54°C for 30 sec, 68°C for 1 min, with a final elongation at 68°C for 7 min. The resulting products were analysed on 1–2% agarose gels stained with GelRed (Biotium), purified with the SureClean Kit (Bioline) and sequenced at the BMR Genomics s.r.l. (Padua, Italy). Sequences are available in GenBank under Accession Numbers KR091079—KR091309.