The polymerase chain reaction (PCR) won its discoverer a Nobel Prize in 1993. Kary Mullis has recorded the thought process he went through as he conceived of the idea and he states that he knew immediately that he was going to become very "FAMOUS" because of this discovery. Everyone else assumed that he would win the Nobel Prize as soon as they heard of the PCR reaction. The ironic thing about this discovery, is that all the scientific information had been around for approximately 20 years and had been overlooked by the greatest scientific minds in the world. This discovery is now seen as such an obvious concept that some scientists question if Mullis deserved the Nobel Prize for such a "simple" and "obvious" discovery. After you hear the PCR story you can make up your own mind?

The PCR is another one of these discoveries, like that of the structure of DNA, the #transforming factor determining the chemical nature of DNA, and restriction enzymes, that generated a quantum leap in science. Almost the instant after PCR is explained to a biological scientist, ideas as to its uses begin to pour from all but the most dull scientific mind. Even today, years after its discovery, we are still developing new uses for PCR.

The irony of the PCR is that living organisms have been doing it since they #evolved in the primeval organic soup of this planet 3.5 billion years ago and scientists have been aware of the general DETAILS of this process for ~50 years. To put it succinctly, the PCR does in the test tube what every bacterium does in its tube of media or on an agar-plate and each of us do every day; we all produce billions of exact copies of our own DNA; AMPLIFYING our DNA millions of time. The enzyme DNA polymerase was discovered in the 1950s and our knowledge of the process has been increasing ever since. This means that thousands of scientists have studied DNA replication for 40 years without tumbling to PCR.

The basic principle of PCR is shown in Fig. 15. It has been know for a long time that DNA polymerase requires a short strand of DNA or RNA called a PRIMER to "prime" the START OF DNA REPLICATION. Mullis's genius was that he reasoned "That if you added the following components to a test tube containing a single DNA molecule, you could replicate & amplify that DNA molecule many million fold in a short time.

The components are:

A sample of the TARGET DNA to be copied. In theory only a single molecule is needed.

A set of short (15 to 40 bases) single stranded PRIMERS of DNA, in EXCESS, that will bind to complementary regions of the opposing stands of the TARGET DNA molecule . These primers BRACKET the region of DNA to be amplified.

An EXCESS of the 4 nucleotide triphosphates, ATP, GTP, CTP, TTP.

The enzyme, DNA polymerase.

Various buffers and cofactors like magnesium ions required by DNA polymerase.

The final trick was to get the two target DNA strands APART (separated) so the primers could bind and the DNA polymerase could do its thing. It had been known for ~50 years that heat separates DNA strands and that complementary strands then rejoin through base pairing when the temperature is subsequently lowered. So Mullis heated his mixture of target DNA, primers and triphosphate nucleotides to about 90oC for a few minutes to separate the target DNA. He then lowered the temperature enough to allow the primers, which were small and in VAST EXCESS, to bind (ANNEAL) to their respective complementary target DNA base pair-sequences. At this point he added DNA polymerase and allowed the polymerization reaction with the triphosphate nucleotides to occur. That is, the DNA polymerase FILLED IN the missing portion of each strand making TWO NEW DOUBLE STRANDED regions of DNA.


Figure 15. The polymerase chain reaction. For another figure illustrating the PCR click here and view the "PCR" link. Click here and here for brief discussions of PCR.

101PCR animation.gif (151337 bytes)

Summary animation of PCR. For a professional animation that requires Shockwave/32MB memory/Pentium processor click here and load the appropriate file.

Each entire PCR cycle takes only 2 to 10 minutes. However, there was one problem with the system devised by K. Mullis, which was that the DNA polymerase he used was DESTROYED BY THE HEATING process. Mullis had to add new DNA polymerase for EACH ROUND, which was time-consuming and expensive. This problem was solved by using HEAT-RESISTANT DNA polymerase that only needed to be added once. #THERMOPHILIC bacteria that live in boiling hot springs have been described previously. This is another case of SERENDIPITOUS BASIC SCIENCE turning out to be important. The study of thermophiles might seem to be a waste of money and time to many people as these bacteria, while certainly interesting, don't cause disease in man or any other life form. The argument could be made that a scientist could better spend his/her time working on cancer or some other terrible disease that afflicts humankind. However, it turns out that since thermophilic DNA polymerases tolerate high temperatures (e.g. 90oC) for long periods without being destroyed, they are the perfect solution to the PCR DNA polymerase problem. Thus today PCR has become a revolutionary tool because of scientists who studied these odd thermophiles.


Figure 16. This figure represents another perspective of the PCR reaction. Of particular note is the use of the PCR to AMPLIFY SPECIFIC DNA SEGMENTS dependent on the PRIMERS EMPLOYED. By choosing primers with unique sequences one BRACKETS a known length (gene[s])of a DNA molecule. The bracketed segment is indicated by the dashed lines. The red and blue primers, by their COMPLEMENTARY BINDING to base pair-sequences on the two respective parental DNA strands insure that ONLY the portion of DNA between them will be amplified. Five cycles of replication are shown, except that the last cycle is incomplete. Note that the number of DNA molecules increases EXPONENTIALLY with each cycle of replication.

The standard PCR reaction is run through about 30 cycles in a couple of hours which results in the amplification of the original DNA by over a 109 fold. Thus a single specific DNA region can be amplified to yield sufficient quantities to do anything that can be done with bulk-isolated DNA. In the case of crime evidence this means that the DNA in a single hair follicle, a single drop of semen or blood is sufficient to prove that an individual was present at the scene of a crime. Indeed any piece of evidence that contains one or more moderately long DNA fragments (e.g. >200 base pairs) can be amplified with PCR and its RFLP determined for identification purposes---or maybe to build a dinosaur eventually. However, at present the maximum limit for amplification is approximately 42 kilobases (42,000).
You can see more at http://www.slic2.wsu.edu:82/hurlbert/micro101/pages/Chap10.html#History_of_GE