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been recognized by the citation of the original paper 825 times (Thomson Reuters Web of Knowledge). From a practical standpoint, the


advantage of the SYBR Green approach is that the non-specific intercalating dye allows for the low cost development of a real-time PCR assay that does not require synthesis (or purchase) of much more expensive fluorescently labeled gene-specific hybridization probes. As a result, this tool has enjoyed widespread adoption throughout the molecular biology community and across a variety of different thermocycler technologies. While the lack of sequence specificity can present technical problems, careful experimental design, coupled with melting curve analysis to document a single amplicon species, will permit effective quantification. It would be hard to overstate the real-time PCR in


importance of


molecular biology. The introduction of readily accessible and easy-to-use tools for quantifying the amount of DNA or RNA (following cDNA production) in a sample has provided important insights into a variety of biological processes and pathological conditions. The work of Wittwer et al. played an


important role in the development of this technology and the creation of new vistas in genetic analysis.


References


1. Saiki, R.K., S. Scharf, F. Faloona, K.B. Mul l is, G.T. Horn, H.A. Erl ich, and N. Arnheim. 1985. Enzymatic ampl if i- cation of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230:1350- 1354.


2. VanGuilder, H.D., K.E. Vrana, and W.M. Freeman. 2008. Twenty-five years of quantitative PCR for gene expression analysis. BioTechniques 44:619-626.


3. Wittwer, C.T., M.G. Herrmann, A.A. Mos s, and R.P. Rasmus sen. 1997. Continuous f luorescence monitoring of rapid cycle DNA amplif ication. BioTech- niques 22:130-138.


4. Wittwer, C.T., K.M. Ririe, R.V. Andrew, D.A. David, R.A. Gundr y, and U.J. Balis. 1997. The LightCyclerTM


: a micro-


volume mult isample fluorimeter with rapid temperature control. Biotechniques 22:176-181.


BioTechniques 54:312-313 (June 2013) doi 10.2144/000114042


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