Bigdye terminator kit

Long-range PCR was performed to detect and sequence SPAST gene breakpoints. To select the deletion-specific products, as narrowly as possible, sets of primers complementary to the intronic sequence that presumably contains breakpoints were designed and long-range PCR was performed. Candidate PCR products were extracted from the gel, purified, and sequenced in both directions using the BigDye Terminator Kit (Thermo Fisher Scientific).

Genetic study of the patients was conducted using direct sequencing of the entire coding and exon-intron boundaries of the ITGA2B and the ITGB3 genes [3 (link), 4 (link)]. Primer sequences are available upon request. PCR amplification was performed in a total volume of 25 μl containing 2.5 μl of 10X buffer (Kawsar Biotech Co., Tehran, Iran, KBC), 2.5 μl DMSO (Sigma-Aldrich, USA), 0.96 mM dNTPs (KBC), 4 mM MgCl2 (KBC), 6 pmol of forward and reverse primers, 1 U Taq polymerase (KBC), and 30 ng of genomic DNA as a template. The thermocycling consisted of 1 cycle of initial denaturation at 95 °C for 5 min, followed by 30 cycles of 1 min denaturation at 95 °C, 1 min annealing at 64 °C, and 1 min extension at 72 °C, following a final extension at 72 °C for 10 min and storing the PCR product in 4 °C.
Sequencing.
PCR products were directly sequenced using BigDye Terminator kit (Thermo Fisher Scientific, USA, TF) according to the manufacturer’s protocol. Then the samples were run on an ABI3130XL Genetic Analyzer (TF). Sequences were compared with human genomic and cDNA sequences of the genes with the accession numbers of ITGA2B (NM: 000419, NC:000017) and ITGB3 (NM:000212, NC:000017). Mutations nomenclature was conducted according to the recommendations of Human Genome Variation Society [11 (link)].