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.

How does it work? The procedure requires placing a small amount of the DNA containing the desired gene into a test tube. A large batch of loose nucleotides, which link into exact copies of the original gene, is also added to the tube. A pair of synthesized short DNA segments, that match segments on each side of the desired gene, is added. These "primers" find the right portion of the DNA, and serve as starting points for DNA copying. When the enzyme Taq DNA Polymeras from the bacterium, Thermus aquaticus is added, the loose nucleotides lock into a DNA sequence dictated by the sequence of that target gene located between the two primers.

How does it work? The test tube is heated, and the DNA's double helix separates into two strands. The DNA sequence of each strand of the helix is thus exposed and as the temperature is lowered the primers automatically bind to their complementary portions of the DNA sample. At the same time, the enzyme links the loose nucleotides to the primer and to each of the separated DNA strands in the appropriate sequence. The complete reaction, which takes approximately five minutes, results in two double helices containing the desired portion of the original. The heating and cooling is repeated, doubling the number of DNA copies. After thirty to forty cycles are completed a single copy of a piece of DNA can be multiplied to hundreds of millions.

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). Bartlett, J.M., Stirling, D. (2003). PCR Protocols, vol Humana Press. …