PMC:3480677 / 3562-6303 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/3480677","sourcedb":"PMC","sourceid":"3480677","source_url":"https://www.ncbi.nlm.nih.gov/pmc/3480677","text":"Methods\n\nPatient enrollment\nWe enrolled 113 Korean AVB patients who were diagnosed with electrocardiograms and who had inserted permanent pacemakers and 80 normal controls with no cardiac symptoms. All patients and control subjects were recruited from four medical centers - Keimyung University, Yeungnam University, Catholic University, and Fatima Hospital (Daegu, Korea) - from August 2009 to September 2011.\nThis study was approved by the ethical committee at each hospital; consent was obtained from all individuals before enrollment into the study.\n\nDNA extraction\nIn this study, peripheral blood was collected into ethylenediamine tetraacetic acid-containing tubes, and genomic deoxyribonucleic acid (DNA) was extracted from whole blood samples using the QIAamp DNA blood mini kit (Qiagen, GmbH, Hilden, Germany). DNA concentration was determined using a NanoDrop ND1000 spectrophotometer (Thermo Fisher Scientific Inc., Wilmington, DE, USA), and purity of the DNA was assessed based on the 260/280 nm absorbance ratio.\n\nMultiplex polymerase chain reaction (PCR) and sequence analysis\nThe 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).\nSequencing 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.\n\nStatistical analysis\np-values and odds ratios with 95% confidence intervals for each genotype and allele were evaluated by χ2 analysis. A p-value of \u003c0.05 was considered to indicate a statistically significant difference between AVB patients and normal controls of the study.","divisions":[{"label":"Title","span":{"begin":0,"end":7}},{"label":"Section","span":{"begin":9,"end":553}},{"label":"Title","span":{"begin":9,"end":27}},{"label":"Section","span":{"begin":555,"end":1025}},{"label":"Title","span":{"begin":555,"end":569}},{"label":"Section","span":{"begin":1027,"end":2464}},{"label":"Title","span":{"begin":1027,"end":1090}},{"label":"Title","span":{"begin":2466,"end":2486}}],"tracks":[{"project":"2_test","denotations":[{"id":"23105938-1309946-44845438","span":{"begin":1263,"end":1264},"obj":"1309946"}],"attributes":[{"subj":"23105938-1309946-44845438","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#bfec93","default":true}]}]}}