SYBR Green I chemistry based Real time PCR: Melting curve discrepancies

Real Time PCR is a sensitive technique with multidisciplinary applications such as detection and quantification of genetically modified organisms (GMO's), pathogens (bacteria, viruses), high through put screening of Single Nucleotide Polymorphism (SNP) within a population and expression studies. SYBR Green I an intercalating dye, used to discriminate genotypes/strains by melting curve analysis. Present paper discusses the discrepancy in melting temperature (Tm) for the similar PCR product using SYBR Green dye master mixes procured from different suppliers. Genes used in this study are β-globin, Lectin, SP-D a surfactant protein, Salmonella hinH2, Nos terminator and Virus (35S promoter). The article highlights the variation in melting temperature that may not be overlooked while performing SYBR green analysis. (On the basis of our results, one can conclude that proper recording of different kits used in specific experiments, is important for replicating the experiment in different laboratory setting. Tm parameter seems to be a good tool to discriminate between PCR products of various GMO's/strains provided one make sure to use same kit.