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

Order of Publishing in Issue: 
10
Volume :2
Issue :4
October, 2008
Page No: 
555 -561
Authors: 
Sudhir K. Goel1*, Shikha Tewari1, Hind lal1, Pramod K. Mishra1, Sudhir K. Agarwal2, U.N. Dwedi2 and Munish Kumar1,3
Address: 
1Petroleum Toxicology Division, Indian Institute of Toxicology Research, Lucknow-226001, India
Address: 
Department of Biochemistry, University of Lucknow, Lucknow, India.
Email-ID: 
skgmun@hotmail.com

Real Time PCR is a sensitive technique with  multidisciplinary applications such as detectionand quantification of genetically modified organisms(GMO’s), pathogens (bacteria, viruses), highthrough put screening of Single Nucleotide Polymorphism (SNP) within a population and expressionstudies. SYBR Green I an intercalating dye, used to discriminate genotypes/strains by meltingcurve analysis. Present paper discusses the discrepancy in melting temperature (Tm) for thesimilar PCR product using SYBR Green dye master mixes procured from different suppliers.Genes used in this study are ?-globin, Lectin, SPD a surfactant protein, Salmonella hinH2, Nosterminator and Virus (35S promoter). The article highlights the variation in melting temperaturethat 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, isimportant for replicating the experiment in different laboratory setting. Tm parameter seemsto be a good tool to discriminate between PCR products of various GMO’s/strains provided onemake sure to use same kit.

Keywords: 
SYBR Green I, Real time PCR, NSP
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