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From: Methods in Molecular Biology, Vol. 226: PCR Protocols, Second Edition
Edited by: J. M. S. Bartlett and D. Stirling Humana Press Inc., Totowa, NJ
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A Short History of the PoIymerase Chain Reaction
John M. S. BartIett and David StirIing
The development of the polymerase chain reaction (PCR) has often been likened
to the development of the Internet, and although this does risk overstating the impact
of PCR outside the scientifc community, the comparison works well on a number
of levels. Both inventions have emerged in the last 20 years to the point where it is
diffcult to imagine life without them. Both have grown far beyond the confnes of
their original simple design and have created opportunities unimaginable before their
invention. Both have also spawned a whole new vocabulary and professionals literate
in that vocabulary. It is hard to believe that the technique that formed the cornerstone of
the human genome project and is fundamental to many molecular biology laboratory
protocols was discovered only 20 years ago. For many, the history and some of the
enduring controversies are unknown yet, as with the discovery of the structure of DNA
in the 1950s, the discovery of PCR is the subject of claim and counterclaim that has
yet to be fully resolved. The key stages are reviewed here in brief for those for whom
both the history and application of science holds interest.
The origins of PCR as we know it today sprang from key research performed in
the early 1980s at Cetus Corporation in California. The story is that in the spring of
1983, Kary Mullis had the original idea for PCR while cruising in a Honda Civic on
Highway 128 from San Francisco to Mendocino. This idea claimed to be the origin
of the modern PCR technique used around the world today that forms the foundation
of the key PCR patents. The results for Mullis were no less satisfying; after an initial
$10,000 bonus from Cetus Corporation, he was awarded the 1993 Nobel Prize for
chemistry.
The original concept for PCR, like many good ideas, was an amalgamation of
several components that were already in existence: The synthesis of short lengths of
single-stranded DNA (oligonucleotides) and the use of these to direct the target-specifc
synthesis of new DNA copies using DNA polymerases were already standard tools in
the repertoire of the molecular biologists of the time. The novelty in Mullis`s concept
was using the juxtaposition of two oligonucleotides, complementary to opposite strands
of the DNA, to specifcally amplify the region between them and to achieve this in a
repetitive manner so that the product of one round of polymerase activity was added
to the pool of template for the next round, hence the chain reaction. In his History of
PCR , Paul Rabinow quotes Mullis as saying:
History of PCR 3
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 amplifed. 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
fnal 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 95C. 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
purifed and subsequently cloned this polymerase ,, allowing a complete PCR
amplifcation to be created without opening the reaction tube. Furthermore, because the
enzyme was isolated from a thermophilic organism, it functioned optimally at tem-
perature of around 72C, 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 fdelity as the result of a greater
stringency of primer binding, eliminating the nonspecifc products that had plagued
earlier attempts at PCR amplifcation.
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However, even with this improvement, the PCR technique was laborious and slow,
requiring manual transfer between water baths at different temperatures. The frst
thermocycling machine, 'Mr Cycle, which replicated the temperature changes required
for the PCR reaction without the need for manual transfer, was developed by Cetus
to facilitate the addition of fresh thermolabile polymerases. After the purifcation of
Taq polymerase, Cetus and Perkin-Elmer introduced the closed DNA thermal cyclers
that are widely used today .
That PCR has become one of the most widely used tools in molecular biology is
clear from Fig. 1. What is not clear from this simplistic analysis of the literature is the
huge range of questions that PCR is being used to answer. Another scientist at Cetus,
Stephen Scharf, is quoted as stating that
.the truly astonishing thing about PCR is precisely that it wasn`t designed to solve
a problem; once it existed, problems began to emerge to which it could be applied. One
of PCR`s distinctive characteristics is unquestionably its extraordinary versatility. That
versatility is more than its applicability` to many different situations. PCR is a tool that
has the power to create new situations for its use and those required to use it.
More than 3% of all PubMed citations now refer to PCR (Fig. 2). Techniques have
been developed in areas as diverse as criminal forensic investigations, food science,
ecological feld studies, and diagnostic medicine. Just as diverse are the range of
adaptations and variations on the original theme, some of which are exemplifed in
this volume. The enormous advances made in our understanding of the human genome
(and that of many other species), would not have been possible, where it not for the
remarkable simple and yet exquisitely adaptable technique which is PCR.
Fig. 1. Results of a PubMed search for articles containing the phrase 'Polymerase Chain
Reaction. Graph shows number of articles listed in each year.
History of PCR 5
References
1. Rabinow, P. (1996) Making PCR. A Story of Biotechnology. University of Chicago Press,
Chicago.
2. Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985) Enzymatic
amplifcation of beta-globin genomic sequences and restriction site analysis for diagnosis
of sickle cell anemia. Science 230, 1350-1354.
3. Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specifc
enzymatic amplifcation of DNA in vitro: The polymerase chain reaction. Cold Spring
Harbor Symp. Quant. Biol. 51, 263-273.
4. Mullis, K. and Faloona, F. (1987) Specifc synthesis of DNA in vitro via a polymerase-
catalyzed chain reaction. Methods Enzymol. 155, 335-350.
5. Saiki, R., Gelfand, D., Stoffel, S., Scharf, S., Higuchi, R., Horn, et al. (1988) Primer-
directed enzymatic amplifcation of DNA with a thermostable DNA polymerase. Science
239, 487-491.
6. Lawyer, F., Stoffer, S, Saiki, R., Chang, S., Landre, P., Abramson, R., et al. (1993) High-
level expression, purifcation, and enzymatic characterization of full-length Thermus
aquaticus DNA polymerase and a truncated form defcient in 5a to 3a exonuclease activity.
PCR Methods Appl. 2, 275-287.
7. http://www.si.edu/archives/ihd/videocatalog/9577.htm
Fig. 2. Results of a PubMed search for articles containing the phrase 'Polymerase Chain
Reaction. Graph shows number of articles listed in each year expressed as a percentage of
the total PubMed citations for each year.
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