SOME ASPECTS OF PRIMER DESIGN FOR REAL TIME PCR WITH SYBR Green AS A DYE

Abstract

This work considers the sequence of steps for designing primers for real time qPCR analysis with SYBR® Green as fluorescent dye. For primer design, we used Primer- Blast software. As an example, we design primers to fum6 gene, a member of fumonisin biosynthetic gene cluster. The importance of a balance between specificity and efficiency of primers obtained with the help of Primer-Blast software is discussed. Challenges of obtaining specific primers are considered, possible solutions are suggested. BLAST analysis of the primers is discussed, potential approaches to choosing an optimal primer pair are considered. The testing results of the designed primers to Fusarium verticillioides fum6 gene in real-time PCR reaction are shown. We used the designed primer pair in a qPCR reaction and analyzed the amplification curve and melting curve of the amplicon. The shape of the amplification curve reflects the kinetics of PCR reaction and gives some approximation about primer efficiency. Melt curve analysis gives the information on primer specificity, with a single melt peak corresponding to a single PCR amplicon.

Keywords

• primer-blast
• primer3
• qpcr
• blast
• fusarium