title: [Construction and identification of the expression plasmid of SK2 (KCNN2) gene from human atrial myocytes with overlapping PCR].
abstract: OBJECTIVE
  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.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Key Laboratory of Medical Electrophysiology of Ministry of Education, Luzhou Medical College, Luzhou 646000, China.
authors.forename: Xiao-Qiu
authors.initials: XQ
authors.lastname: Tan
authors.forename: Gui-Lan
authors.initials: GL
authors.lastname: Chen
authors.forename: Tao
authors.initials: T
authors.lastname: Li
authors.forename: Liang
authors.initials: L
authors.lastname: Mao
authors.forename: Isao
authors.initials: I
authors.lastname: Inoue
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Xiao-Rong
authors.initials: XR
authors.lastname: Zeng
mesh_terms: D001483:Base Sequence; D015870:Gene Expression; D006801:Humans; D032383:Myocytes, Cardiac; D010957:Plasmids / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D051640:Small-Conductance Calcium-Activated Potassium Channels / Q000235:genetics*
chemical_list: C496897:KCNN2 protein, human; D051640:Small-Conductance Calcium-Activated Potassium Channels