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“Making it Real” Time
Kent E. Vrana
Department of Pharmacology, Penn State College of Medicine
BioTechniques, Vol. 54, No. 6, June 2013, pp. 312–313
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It has been 30 years since the first description of the polymerase chain reaction (PCR)—corresponding, coincidentally, with the year of the first issue of BioTechniques. Ensuing decades have seen remarkable advances in this revolutionary technique. Central to these approaches was the creation of paradigms for monitoring the progress of PCR amplification in real time. One of these seminal reports appeared in BioTechniques in 1997, establishing the double-strand-specific dye SYBR Green as a workhorse tool for continuous monitoring of DNA amplification.

Five years ago, BioTechniques celebrated 25 years of the polymerase chain reaction (PCR) and its role in the genetic analysis revolution (1, 2). Examples of PCR applications are legion—ranging from gene expression analysis to genotyping, SNP analysis, gene cloning, and the creation of mutants.

One of the remarkable advances in PCR was the development of approaches permitting real-time monitoring of the progression of the amplification reaction (3). Up until this point, the PCR process was limited to end-point amplifications in which a reaction was permitted to proceed for a pre-determined number of cycles, the reaction products were resolved on an agarose gel, and the amplicons were then visualized and quantified with an intercalating fluorescent dye (Figure 1A). In their seminal BioTechniques article from 1997, Carl Wittwer and his colleagues built upon their development of the LightCycler (4)—a microvolume fluorimeter permitting rapid temperature control—to enable real-time monitoring of DNA amplification using intercalating dyes or fluorescently labeled hybridization probes. An example of this approach using the dye SYBR Green is shown in Figure 1B. SYBR Green is a cyanine dye that intercalates with high affinity into double-stranded DNA. The resulting complex absorbs light in the blue wavelength (497 nm) and emits an intense fluorescent signal in the green spectrum (520 nm)—hence the name.

Figure 1.  The evolution of quantitative PCR. (Click to enlarge)

Wittwer and colleagues recognized (through the use of their capillary based technology) that they could quantify the amount of product at discrete points during the cycle and so develop an approach for examining amplification progress one cycle at a time. The natural progression of this idea eventually led to the development of technologies that permitted complete monitoring of the amplification process throughout the lifetime of the amplification (Figure 1C). The Wittwer et al. article is, therefore, one of the earliest reports of a real time amplification paradigm, and it hasbeen 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 importance of real-time PCR in 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.

1.) Saiki, R.K., S. Scharf, F. Faloona, K.B. Mullis, G.T. Horn, H.A. Erlich, and N. Arnheim. 1985. Enzymatic amplification 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. Moss, and R.P. Rasmussen. 1997. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22:130-138.

4.) Wittwer, C.T., K.M. Ririe, R.V. Andrew, D.A. David, R.A. Gundry, and U.J. Balis. 1997. The LightCycler™: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques 22:176-181.