Referenced in: none

Automatically associated channels: SK2

Title: [Construction and identification of the expression plasmid of SK2 (KCNN2) gene from human atrial myocytes with overlapping PCR].

Authors: Xiao-qiu Tan, Gui-Lan Chen, Tao Li, Liang Mao, Isao Inoue, Yan Yang, Xiao-Rong Zeng

Journal, date & volume: Zhongguo Ying Yong Sheng Li Xue Za Zhi, 2012 Jul , 28, 381-4

PubMed link: http://www.ncbi.nlm.nih.gov/pubmed/23156743

Abstract
Small conductance calcium activated potassium channels type 2 (SK2) play a crucial role in atrial repolarization. It is difficult to acquire the full-length of its coded gene KCNN2 by RT-PCR with one step. We aim to get the full-length of KCNN2 gene and construct the plasmid by Overlapping PCR, and further more discuss the application of Overlapping PCR.Total RNA was extracted from human right atrial tissue and cDNA was acquired with reverse transcription. Overlapping PCR was conducted with three pairs of primers which were designed according to the sequence of KCNN2 (AY258141) gene. The expression plasmid of pIRES-hrGFP-SK2 was constructed by directed cloning with restriction enzyme site and identified by enzyme cutting and sequencing.Three parts of PCR amplification were consistent with predicted size. The sequence of the plasmid was consistent with the gene-bank data except two sites, however, which were the same as gene in different tissues.The expression plasmid pIRES-hrGFP-SK2 was constructed successfully. Overlapping PCR is a good choice for amplifying these genes with long size or low expression.