TANDEM RECOMBINANT PLASMID CONSTRUCTION AS POSITIVE CONTROL FOR PIK3CA H1047R DETECTION BASED ON SYBR GREEN I qPCR

Abstract

PIK3CA H1047R mutation is found in breast cancer in high frequency and its detection could be applied as prognosis and predictive factor for trastuzumab therapy. qPCR is one of the simplest and robust method for PIK3CA H1047R detection. Provision of positive control for PIK3CA H1047R detection based on qPCR will support data analysis efficiently and avoid false negative result. In this research, we constructed a tandem recombinant plasmid (pGEM-tandPIK3CA) as positive control for Tm Shift SYBR Green I qPCR-based of PIK3CA H1047R detection system by ligating wild-type and PIK3CA H1047R fragments tandemly into pGEM-T Easy. The tandem plasmid was confirmed by restriction, DNA sequencing and qPCR. As a result, pGEM-tandPIK3CA has been successfully constructed and confirmed. Statistical analysis shows high repeatability and reproducibility with % CV of <25%. The main advantage of this tandem positive control is its ability to serve as positive control for both wild-type PIK3CA and PIK3CA H1047R simultaneously, therefore improving the efficiency of the detection system.