But at the start, the technique wasn't really quantitative. PCR, in its original form, was an endpoint assay, a plus/minus readout where data are interpreted after the reaction has finished using gel electrophoresis. Band size indicates a positive or negative reaction, yet band intensity has little quantitative value because the reaction often isn't linear. To circumvent that problem, researchers like Vrana had to take extreme measures, such as constructing standard curves and tediously titrating cycle numbers.
This all changed in 1993. Russell Higuchi, at Roche Molecular Systems, and colleagues described a method for real-time quantitative PCR (qPCR). This method used an intercalating dye (ethidium bromide) and a CCD camera to record reaction progress as cycling proceeded, producing a reliably quantitative analysis of PCR amplification.
Other methods soon followed. SYBR Green supplanted ethidium, and other strategies were introduced, too, most notably Life Technologies’ popular TaqMan chemistry, which uses a fluorescently labeled oligonucleotide probe to assess target abundance, rather than a non-specific DNA-binding dye.
Vrana became an early adopter. “I was bright enough to see…the power with which it could be used,” he says. Yet PCR was not just about RNA quantitation. Vrana also used it for creating mutants, genetic analysis, validation of microarray data, and more. “It has turned out to be a bedrock in the field,” he says.
Today, qPCR is a hugely popular PCR variant, and RNA quantification is among its most common applications. In 2008 Vrana co-authored a review on PCR's 25th anniversary that tracked the number of qPCR papers over time (3). The figure resembles a typical qPCR amplification curve, he says. “They could be superimposed on each other.”
Of course, researchers knew long before the invention of qPCR that dyes such as ethidium bromide can bind DNA. And they also knew about PCR's quantitative limitations. Why, then, did it take so long for someone to put the two together?
“It truly was an engineering solution,” he says. “I don't think people understood the potential for real-time quantitative PCR. To think back on it now, the draconian things we used to do in terms of quantifying amplicons on ethidium bromide gels—I'm just stunned that we would do that.”Breaking the Diffraction Barrier
About the time that Higuchi was introducing the world to qPCR, Stefan Hell was preparing to upend the world of microscopy.
At the end of the 1980s, Hell was a doctoral student at the University of Heidelberg. Though his interests lay in more fundamental aspects of physics, his thesis advisor directed him towards an applied project, studying the application of confocal microscopy to inspect photolithographic structures. It wasn't where Hell wanted to go.
“After a while, I became kind of frustrated, and I was thinking about what else could you do with light microscopy,” Hell, now Director of the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany, recalls.
As he contemplated the fundamentals of light microscopy, Hell began to think about the so-called diffraction (or Abbe) limit, which says two objects cannot be distinguished under a light microscope if they are closer than half the wavelength of light used to illuminate them.
That limitation is a function of the properties of light and lenses, and since the invention of the microscope it has been practically inviolable. And indeed, it is inviolable—the wave properties of light mean optics can only be so precise and can focus only so sharply. But that doesn't mean it isn't possible to make the limit irrelevant.
Hell started exploring “molecular transitions,” the ability of fluorescent molecules to occupy one of two states, active or silent. Consulting textbooks on everything from quantum optics to nuclear physics, he noticed a phenomenon he had learned about as a first-year student: stimulated emission.
“If you realize that you want to keep things dark, if you're trained as a physicist, then you know the most fundamental way of turning a fluorophore off is to instantly send it down to the ground state by [using] a beam inducing stimulated emission,” he says.