Long range planning

Eisenstein, Michael

doi:10.1038/nmeth1105-884

Classic Protocol
Published: November 2005

Long range planning

Michael Eisenstein

Nature Methods volume 2, page 884 (2005)Cite this article

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A late-night drive to Mendocino, California, USA, some 20 years ago gave a young researcher at Cetus the opportunity to dream up a solution to a problem that had been much on his mind lately—a process for generating large quantities of scarce DNA sequences. Kary Mullis had conceived the polymerase chain reaction (PCR)—that now-ubiquitous process in which a mixture of DNA, nucleotides, primers and polymerase is cycled repeatedly through temperatures appropriate for strand denaturation, primer annealing and polymerase-driven extension, leading to exponential amplification of the target sequence. “I had solved the most annoying problems in DNA chemistry in a single lightning bolt,” he would later write of his revelation, with typical modesty. “I could make as much of a DNA sequence as I wanted and I could make it on a fragment of a specific size that I could distinguish easily.”¹

Of course, in those heady early days before PCR achieved full commercial exploitation, many elements of this useful technique presented quite a chore. In its earliest incarnation², PCR was performed by transferring samples to different heating blocks, and was driven by DNA polymerase I from Escherichia coli—necessitating the addition of fresh enzyme after each denaturation step, owing to the enzyme's temperature sensitivity. Within a few years, however, both of these obstacles would be overcome; programmable thermocyclers saved scientists the trouble of continuous tube trafficking and, more importantly, Taq polymerase—derived from the thermophilic bacterium Thermus aquaticus—had appeared on the scene. This polymerase had been isolated years before by researchers who had then contemplated its potential as an engine for reverse transcription, where high-temperature synthesis could eliminate the obstacles posed by RNA secondary structure³. But Taq's ability to thrive at temperatures well over 90 °C made it a natural choice for the rigors of PCR cycling, and it performed admirably⁴.

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References

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