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


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Yi
Shao-dong
SD
Department of Cardiology, Nanfang Hospital, First Military Medical University, Guangzhou 510515, China. ysd@fimmu.com
Meng
Su-rong
SR
Cui
Ying-kai
YK
Chen
Zhe-ming
ZM
Peng
Jian
J
D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D062554:NAV1.5 Voltage-Gated Sodium Channel; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods*; D011995:Recombination, Genetic; D015222:Sodium Channels / Q000235:genetics*; D013577:Syndrome; D017180:Tachycardia, Ventricular / Q000235:genetics*; D014693:Ventricular Fibrillation / Q000235:genetics*
D062554:NAV1.5 Voltage-Gated Sodium Channel; C568320:SCN5A protein, human; D015222:Sodium Channels