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Title: [PCR-based site-directed mutagenesis and recombinant expression plasmid construction of a SCN5A mutation (K317N) identified in a Chinese family with Brugada syndrome]

Authors: Shao-dong Yi, Su-rong Meng, Ying-kai Cui, Zhe-ming Chen, Jian Peng

Journal, date & volume: Di Yi Jun Yi Da Xue Xue Bao, 2003 Nov , 23, 1139-42

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/14625171

Abstract
To perform PCR-based site-directed mutagenesis of a new SCN5A mutation (K317N) identified in a Chinese family with Brugada syndrome and construct the recombinant expression plasmid pRc/CMV-Hh1 containing the human cardiac sodium channel alpha subunit (hH1), mutant cDNA.A pair of primers was designed according to the restricted sites Sse 8387I and Age I of the SCN5A sequence with the mismatches introduced into primers. Mutagenesis was performed in a single-step PCR, and the fragments amplified by PCR containing the mutation site were subcloned into the pRc/CMV-hH1 vector.Sequence analysis confirmed the presence of the desired mutation site, and a mutation from K (Lys) to N (Asn) in codon 317 was identified in the SCN5A gene, indicating the successful induction of the mutation at K317N of the SCN5A gene.PCR site-directed mutagenesis is accurate and highly efficient, and the successfully constructed recombinant expression plasmid pRc/CMV-hH1 (K317N) may provide a molecular basis for further functional and genomic investigation of SCN5A.