John M. S. Bartlett.pdf - Bio-Nica.info

4 Bartlett and Stirling
The thing that was the “Aha!” the “Eureka!” thing about PCR wasn’t just putting those
[things] together…the remarkable part is that you will pull out a little piece of DNA from
its context, and that’s what you will get amplified. That was the thing that said, “you could
use this to isolate a fragment of DNA from a complex piece of DNA, from its context.”
That was what I think of as the genius thing.…In a sense, I put together elements that
were already there.…You can’t make up new elements, usually. The new element, if any,
it was the combination, the way they were used.…The fact that I would do it over and over
again, and the fact that I would do it in just the way I did, that made it an invention…the
legal wording is “presents an unanticipated solution to a long-standing problem,” that’s
an invention and that was clearly PCR.
In fact, although Mullis is widely credited with the original invention of PCR,
the successful application of PCR as we know it today required considerable further
development by his colleagues at Cetus Corp, including colleagues in Henry Erlich’s
lab (2–4), and the timely isolation of a thermostable polymerase enzyme from a
thermophilic bacterium isolated from thermal springs. Furthermore, challenges to the
PCR patents held by Hoffman La Roche have claimed at least one incidence of “prior
art,” that is, that the original invention of PCR was known before Mullis’s work in the
mid-1980s. This challenge is based on early studies by Khorana et al. in the late 1960s
and early 1970s (see chapter 2). Khorana’s work used a method that he termed repair
replication, and its similarity to PCR can be seen in the following steps: (1) annealing
of primers to templates and template extension; (2) separation of the newly synthesized
strand from the template; and (3) re-annealing of the primer and repetition of the cycle.
Readers are referred to an extensive web-based literature on the patent challenges
arising from this “prior art” and to chapter 2 herein for further details. Whatever the
final outcome, it is clear that much of the work that has made PCR such a widely
used methodology arose from the laboratories of Mullis and Erlich at Cetus in the
mid-1980s.
The DNA polymerase originally used for the PCR was extracted from the bacterium
Escherichia coli. Although this enzyme had been a valuable tool for a wide range of
applications and had allowed the explosion in DNA sequencing technologies in the
preceding decade, it had distinct disadvantages in PCR. For PCR, the reaction must
be heated to denature the double-stranded DNA product after each round of synthesis.
Unfortunately, heating also irreversibly inactivated the E. coli DNA polymerase,
and therefore fresh aliquots of enzyme had to be added by hand at the start of each
cycle. What was required was a DNA polymerase that remained stable during the
DNA denaturation step performed at around 95°C. The solution was found when the
bacterium Thermophilus aquaticus was isolated from hot springs, where it survived
and proliferated at extremely high temperatures, and yielded a DNA polymerase that
was not rapidly inactivated at high temperatures. Gelfand and his associates at Cetus
purified and subsequently cloned this polymerase (5,6), allowing a complete PCR
amplification to be created without opening the reaction tube. Furthermore, because the
enzyme was isolated from a thermophilic organism, it functioned optimally at temperature
of around 72°C, allowing the DNA synthesis step to be performed at higher
temperatures than was possible with the E. coli enzyme, which ensured that the
template DNA strand could be copied with higher fidelity as the result of a greater
stringency of primer binding, eliminating the nonspecific products that had plagued
earlier attempts at PCR amplification.