Polymerase Chain Reaction
Insights from history
Incipit
Kary Mullis invented the PCR technique in 1985 while working as a
chemist at the Cetus Corporation, a biotechnology firm in Emeryville,
California. The Polymerase Chain Reaction (PCR) technique allowed
scientists to make millions of copies of a scarce sample of DNA.
The technique has revolutionized many aspects of current research,
including the diagnosis of genetic defects and the detection of the AIDS
virus in human cells. The technique is also used by criminologists to link
specific persons to samples of blood or hair via DNA comparison. PCR
also affected evolutionary studies because large quantities of DNA can
be manufactured from fossils containing but trace amounts.
Why inventing PCR?
When completed manually, Mullis' PCR technique was slow and labor-intensive.
Therefore, Cetus scientists began looking for ways in which to automate the
process. Before the discovery of the thermostable Taq enzyme, scientists needed to
add fresh enzyme to each cycle. The first thermocycling machine, "Mr. Cycle" was
developed by Cetus engineers to address that need to add fresh enzyme to each
test tube after the heating and cooling process. Purification of the Taq polymerase
then resulted in the need for a machine to cycle more rapidly among different
temperatures. In 1985, Cetus formed a joint venture with the Perkin-Elmer
Corporation in Norwalk, Connecticut, and introduced the DNA Thermal Cycler.
By 1988, Cetus was receiving numerous inquiries about licensing to perform PCR
for commercial diagnostic purposes. On January 15, 1989, Cetus announced an
agreement to collaborate with Hoffman-LaRoche on the development and
commercialization of in vitro human diagnostic products and services based on PCR
technology. Roche Molecular Systems eventually bought the PCR patent and
associated technology from Cetus for $300,000,000.
Where did the concept come from?
The original concept for PCR, like many good ideas, was an amalgamation of
several components that were already in existence:
1. the synthesis of short lengths of single-stranded DNA (oligonucleotides),
2. the use of these to direct the target-specific synthesis of new DNA copies
using DNA polymerases
3. the development of the electrophoretic gel on which DNA is made to
migrate by an electrical current (the means used to separate out strands
of different sizes)
4. the techniques used to transfer these strands to a membrane and detect
them
were already standard tools in the repertoire of the molecular biologists of
the time (Bartlett and Stirling, 2003).
Where did the concept come from?
What was original, powerful, and significant was the concept that
combined – and reconfigured – these existing techniques (Rabinow,
1996).
Novelty
The novelty in Mullis’s concept was using the juxtaposition of two
oligonucleotides, complementary to opposite strands of the DNA, to
specifically 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 (Bartlett and Stirling, 2003).
Novelty
Mullis said:
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.
(Rabinow, 1996)
Novelty
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.
Traces of prior art
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.
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.
Traces of prior art
Although the PCT patents make no mention of such work, DNA
amplification and cycling reactions were conducted many years before
the filing of the PCR patents in the laboratory of Dr. Khorana.
Dr. Khorana did not patent this work. Instead he dedicated it to the
public.
Traces of prior art
Unfortunately, at the time that Dr. Khorana discovered his amplification
process, it was not practical to use the method for nucleic acid
amplification, and the technique did not take off as a commercial
method.
At the time this work was disclosed, chemically synthesized DNA for
use as primers was extremely expensive and cost-prohibitive for even
limited use. Additionally, recombinantly produced enzymes were not
available. Thus, not until the 1980s, when enzyme and oligonucleotide
production became more routine, could one economically replicate Dr.
Khorana’s method.
Traces of prior art
Techniques tot manipulate DNA were still hierarchically dominated by
concepts and systems in molecular biology and biochemistry. Khorana and
his colleagues were constructing a gene; they wanted multiple copies of it.
Cloning, which emerged in the early 1970s, provided the measn to achieve
that end – by harnessing known biological processes – yielding, if not in vitro
exponential amplification, a sufficient number of in vivo amplified copies for
the purposes at hand. Technology was serving biology.
Although in hindsight it may appear that the scientists in Khorana’s lab were
close to PCR, the historical fact remain that cloning and other techniques
solved their problem for them. Once techniques adequate to the task at
hand became available to Khorana and his co-workers, they stopped
exploring other possible means of amplifying DNA.
Traces of prior art
In an important sense, Mullis had no biological problem to solve.
Khorana was trying to harness a biological process (polymerization) as
part of a larger project to make an artificial version of a biological unit,
the gene.
Mullis’s decontextualization and exponential amplification was the
opposite of Khorana’s efforts at the mimicry of nature. Mullis conceived
of a way to turn a biological process (polymerization) into a machine;
nature served (bio)mechanics.
Traces of prior art and legal history
Dupont also found additional references disclosing the earlier invention
by Khorana, but did not provide them to the court in time and they
were not considered.
Thus, it seems that validity of the PCR patents was never truly tested in
view of the work conducted by Dr. Khorana and his colleagues.
Traces of prior art and
legal history
In USA there are more than 600 patents claiming aspects
of PCR. Such patents cover the basic methods itself
(originally owned by Cetus Corporation and now owned
by Hoffmann-Laroche), thermostable polymerases useful
in PCT, as well as many non-PCR applications (e.g. Taq
polymerase, Tth polymerase, Pfu polymerase, KOD
polymer., Tne polymer., Tma polymer., modified polymer,
etc.), reagents (e.g. analyte-specific amplification primers,
buffers, internal standards, etc), and applications involving
the PCR process (e.g. reverse-transcription PCR, nested
PCR, multiplex PCR, nucleic aicde sequencing, and
detection of specific analytes).
However, the most significant patents (see table),
covering the basic PCR method, the most widely used
polymerase (Taq polymerase), the thermocyclers, are
assigned to Hoffmann-LaRoche and are controlled by
Hoffman-LaRoche or Applera Corporation (previously
known as PE/Applied Biosystems).
References
Rabinow, P. (1996) Making PCR: A Story of Biotechnology. University of Chicago Press, Chicago.
Saiki, R., Scharf, S., Faloona, F., Mullis, K., Horn, G., and Erlich, H. (1985) Enzymatic amplification of beta-globin
genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354.
Mullis, K., Faloona, F., Scharf, S., Saiki, R., Horn, G., and Erlich, H. (1986) Specific enzymatic amplification of DNA
in vitro: The polymerase chain reaction. Cold Spring Harbor Symp. Quant. Biol. 51, 263–273.
Mullis, K. and Faloona, F. (1987) Specific synthesis of DNA in vitro via a polymerasecatalyzed chain reaction.
Methods Enzymol. 155, 335–350.
Saiki, R., Gelfand, D., Stoffel, S., Scharf, S., Higuchi, R., Horn, et al. (1988) Primerdirected enzymatic
amplification of DNA with a thermostable DNA polymerase. Science 239, 487–491.
Lawyer, F., Stoffer, S, Saiki, R., Chang, S., Landre, P., Abramson, R., et al. (1993) Highlevel expression,
purification, and enzymatic characterization of full-length Thermus aquaticus DNA polymerase and a truncated
form deficient in 5′ to 3′ exonuclease activity. PCR Methods Appl. 2, 275–287.
Smithsonian Institution Archives (1992/93). http://siarchives.si.edu/collections/siris_arc_217745
Bartlett, J.M., Stirling, D. (2003). PCR Protocols, vol. 226. Humana Press.
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