+44-7360-538437- Biomedical Research (2013) Volume 24, Issue 4
Asymmetric real-time PCR improves the SNP detection of VEGF+ 936C>T by melting curve analysis in LightCycler.
Vascular endothelial growth factor (VEGF) plays a critical role in angiogenesis. Several VEGF polymorphisms were associated with development of different diseases in human. More commonly studied VEGF single nucleotide polymorphisms (SNPs) are -2578C/A, - 1154G/A, +936C/T. Our aim was to develop an asymmetric PCR on Light Cycler melting curve analyses for VEGF +936C/T to remove hook effect which is commonly observed in routine real-Time PCR with melting curve analyses. Real–time PCR with equimolar and asymmetric primer concentration and hybridization probes in LightCycler was performed and melting peaks for hook effects were compared. Melting curve was used for the genotyping. Asymmetric primer concentration (sense: antisense=3:1) neutralized the hook effect and resulted in the easy genotyping for the detection of VEGF SNP +936C>T. An asymmetric real-time PCR with melting curve analysis could be applied when hook effect is observed with common real-time PCR protocol.
Author(s): Binod Kumar Yadav, Sun-Young Oh and Byoung-Soo Shin