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Features Special Commentary “Making it Real” Time

Kent E. Vrana Department of Pharmacology, Penn State College of Medicine

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 devel-

opment 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 ampli- fications 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 devel- opment 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. 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 has

Vol. 54 | No. 6 | 2013 A B


Figure 1. The evolution of quantitative PCR. (A) Prior to the advent of real-time PCR quantifi- cation, end-point PCR applications would measure the amount of amplified DNA by perform- ing a pre-determined number of PCR cycles and then resolving the amplicon products on an agarose gel. The binding of an intercalating dye—in this case ethidium bromide—permits visualization of the DNA and can be quantified as a surrogate measure of the amount of DNA. Thanks to Izel Tekin for the image. (B) This image, from the original Wittwer et al. article, demonstrates the use of the intercalating dye SYBR Green to measure the amount of fluorescent amplicon resulting from a serial dilution of the template DNA. In this case, each cycle was monitored for 100 ms in the middle of the elongation segment. (C) In the ensuing years, engineering solutions have been developed that permit the real-time monitoring of the amplification reactions; here is an example of SYBR Green fluorescence monitoring in a 96 well microtiter plate format. Thanks to Jim Jefferson for this image.


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