The potential mutation of the proband and the remaining family members were validated using Sanger sequencing. DNA was extracted from peripheral blood samples from the proband and other family members. DNA sequence including the candidate mutation was amplified using the following primers: MYOC forward, 5′-TGTAGTCTCGGCTCACAG-3′ and reverse, 5′-TGATAGGATAGAGGGCTTT-3′. The PCR cycle consisted of an initial denaturation step of 5 min at 98°C followed by 35 cycles of 30 sec at 98°C, 30 sec at 53°C, and 45 sec at 72°C, and a final step at 72°C for 5 min. All PCR products were separated and then directly sequenced using BigDye Terminator v.3.1 Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.) and analyzed by capillary electrophoresis using an ABI Prism 3500 Genetic Analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.).
Bigdye terminator v 3.1 mix
Manufactured by Thermo Fisher Scientific
The BigDye Terminator v.3.1 Mix is a reagent used in DNA sequencing applications. It contains the necessary components for performing cycle sequencing reactions, including fluorescently labeled dideoxynucleotides, DNA polymerase, and other essential reagents.
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Lab products found in correlation
2 protocols using bigdye terminator v 3.1 mix
1
Validation of Proband Mutation
2
Genetic Profiling of One-Carbon Metabolism Genes
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Genomic DNA was extracted from peripheral whole blood or dried blood spots obtained from the proband and his family members, as well as control subjects. The coding region and flanking intron sequences of GLDC (NM_000170.2), AMT (NM_000481.3) and GCSH (NM_004483.4) genes were amplified using standard PCR (polymerase chain reaction) conditions and bi-directional DNA sequencing. All the primers used are listed in Additional file 1 : Table S1. The PCR cycle consisted of an initial denaturation step of 2 min at 95 °C followed by 36 cycles of 30 s at 95 °C, 1 min at 60 °C, and 1 min at 72 °C, and a final step at 72 °C for 2 min. All PCR products were separated and then directly sequenced using BigDye Terminator v.3.1 Mix (Applied Biosystems, Foster City, CA) and analyzed by capillary electrophoresis using an ABI Prism 3500 Genetic Analyzer (Applied Biosystems).
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