Multiplex polymerase chain reaction (PCR) and sequence analysis
The SCN5A gene is located on human chromosome 3p21, and the gene consists of 28 exons (Fig. 1) and encodes a protein of 2,016 amino acids with a molecular mass of 227 kDa [7]. Entire coding regions, including exon-intron boundaries of the SCN5A gene, were amplified by multiplex PCR-based direct sequencing. The primers for PCR amplification were designed based on the GenBank reference sequence; primers are shown in Table 1. PCR conditions were as follows: an initial denaturation at 95℃ for 15 min; and denaturation at 94℃ for 30 s, annealing at 68-70℃ for 30-60 s, and extension at 72℃ for 60-90 s, repeated for 30 cycles (Table 1). After multiplex PCR, the reaction mixture was electrophoresed in a 2% agarose gel and stained with ethidium bromide (Fig. 2). Then, amplified PCR products were purified using the QIAquick PCR purification kit (Qiagen) and directly sequenced using the BigDye Terminator ver 3.1 cycle sequencing kit on an ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA).
Sequencing results were compared with the reference sequences (SCN5A/NM_198056.2/ENSG00000183873/ENST00000333535) using the alignment program BLAST 2.0 of NCBI, and we determined the portion of variation that occurred. When a variation was discovered in genomic DNA from patients, we confirmed whether the same variation existed in the control genomic DNA.