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 genotyp-ing. 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.