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Dr. Manuel Varela: Who was Kary Banks Mullis and what did he have to do with PCR?

Aug 22, 2017 by

An Interview with Dr. Manuel Varela: Who was Kary Banks Mullis and what did he have to do with PCR?

Michael F. Shaughnessy –

1) Dr. Varela, a scientist named Kary Banks Mullis won a Nobel Prize back in 1993 for supposedly “inventing” what we now call PCR—First of all, what exactly IS PCR (polymerase chain reaction) and while I know basically what a chain reaction is—why is this important?

The polymerase chain reaction (PCR) invention was developed by an extremely fascinating character and noted biochemist Dr. Kary Mullis. The PCR invention is a molecular biological technique that’s used to enhance the detection of vanishingly minute traces of DNA; that is, PCR greatly amplifies the amount of DNA, however scarce, that may be present within a given sample, in order to further study the amplified DNA using any one of a variety of molecular technologies. The PCR invention is so powerful that it can detect even one molecule of DNA!

Thus, the PCR procedure provides enough DNA in a sample and allows closer investigation. Often the amount of DNA present in a particular environment is simply too low to conduct any useful analysis by conventional molecular approaches. Therefore, the PCR technique is able to find these minute quantities of DNA and enhance the concentrations by orders of magnitude which permit close and careful examination.

The amplification of DNA is only one benefit of the PCR invention.  The procedure may be used to clone novel genes for molecular analysis.

It has been used to determine the sequences of nucleotides along DNA molecules, an early named being given as ‘cycle sequencing.’  Variations of the PCR technique have been used to sequence entire genomes of organisms.

Another application of PCR has been used to construct various mutations within the sequences of bases encoded in DNA in order to study the effects of the mutations in living cells or tissues or even in entire living organisms.

Furthermore, PCR has been used to identify unknown species or individuals or to determine the parental linage in a custody battle. The invention been used frequently as forensic evidence in many crime cases.

2) When exactly when this individual born and where did he go to school?

Kary Mullis was born in the small town of Lenoir, North Carolina, USA on the 28th day of the month of December in the year 1944. The Mullis family lived in this area, located adjacent to the so-called Blue Ridge Mountains, until Mullis turned 5 years of age, at which time the family moved to a larger city. Mullis’ parents were Cecil Banks Mullis and Bernice Alberta Barker Mullis. Mullis went to Dreher high school, which is situated in the city of Columbia, South Carolina. While in high school, one of his main early interests included the subject of chemistry.

After high school graduation in 1962, Mullis entered the Georgia Institute of Technology (Georgia Tech), located in Atlanta, GA, and studied primarily chemistry as his major area of study. While an undergraduate at Georgia Tech, Mullis worked in a chemistry laboratory devoted to the study of light metal hydrides, such as lithium aluminum hydride. Mullis took his Bachelors of Science undergraduate degree in chemistry from Georgia Tech in 1966.

Another area of study for Mullis was his interest of the biological side of chemistry, a field called biochemistry. Toward this, Mullis entered graduate school in 1966 at the prestigious University of California at Berkeley, CA.  Mullis entered the research laboratory of Dr. John “Joe” B. Neilands, professor of biochemistry, and graduate thesis advisor to Mullis. In Neilands’ laboratory Mullis initially studied iron metabolism and transport in bacteria with specialized proteins, called siderophores, that would bind iron more tightly than, let’s say, human iron-binding proteins, would.

However, Mullis and other graduate students in the research group were given carte blanche by Neilands to study whatever interested them. At first, Mullis took many courses outside of the biochemistry department, except molecular biology—which then became a bit of a problem during his thesis defense. He had enrolled in courses pertaining to music, anthropology, math, physics, and sociology.  The story is told that Mullis’ journey into these other non-biochemistry and non-molecular biology fields took him into the study of astrophysics, where his first paper was published, in the prestigious journal Nature. This accomplishment was later to prove most helpful in ultimately helping Mullis to earn his Ph.D.

Interestingly, while still in graduate school and enjoying his intellectual pursuits in other disciplines, Mullis had acquired an intense curiosity with the biochemistry department’s brand new machine, a specialized spectrophotometer called an NMR, for nuclear magnetic resonance, the equipment of which he used to study the structure of an organic molecule with an interesting name, “schizokinen,” related to iron transport systems in a microbe called Bacillus megaterium, a rather large Gram-positive bacterium. With a suitable thesis project complete but the problem of having neglected to enroll in molecular biology re-emerging, it became an obstacle to the final thesis defense approval. This is where the Nature paper had come into play—the idea being that if he knew enough of the scientific method that he actually got a paper accepted in Nature, it ought to be considered that his thesis was conducted in much the same sort of way; thus, the wayward publication greatly helped facilitate the final approval of the thesis by his graduate committee members. Mullis took his Ph.D. degree in the field of biochemistry from UC Berkeley in 1972.

3) What does PCR have to do with DNA?

In short, the PCR procedure makes DNA. The amount of DNA produced, however, is enhanced by orders of magnitude, perhaps a million-fold more DNA can be synthesized, or even a billion-fold, if so desired.  Furthermore, the DNA production is carried out relatively quickly—within just a few hours, in most cases. The PCR invention is also extremely sensitive, having the capacity to detect extremely tiny quantities of DNA.

Remarkably, in his autobiography tilted “Dancing Naked in the Mind Field” Dr. Mullis tells his readers that the idea of the PCR invention came to him almost in a flash while driving in Mendocino County along the California Highway 128 from Berkeley on his way to Anderson Valley, nearby mile maker 46.58. He knew almost immediately that history was about to change as a result of what he had just figured out.

This history-changing invention is described briefly here. The PCR technique itself entails several players in the process.

The first and foremost player is the DNA that is to be detected. This DNA is often called the template or target DNA. The next important player is an enzyme called DNA polymerase, which is a protein that sees the DNA template and uses it to make new progeny DNA.  This new progeny DNA is often called the product—it is the end-result of the PCR process and is the entity that’s produced in an extremely enhanced manner. Important to this process are the four nucleotide bases, denoted as A, G, C and T, which are free floating in a buffer solution prior to the start of the reaction. Another critical player is a set of two so-called oligonucleotide primers, which are short stretches of DNA, varying between 10 and 40 nucleotide bases long and which are identical to the two flanking ends of the DNA template and which anneal to these ends of the single-stranded DNA template. The two annealed DNA primers on the template permit the DNA polymerase to start DNA synthesis along the template using the bases in the template as a guide with which to incorporate into the newly made DNA strands along the template.

At its very core, the PCR procedure has three fundamental steps. The first is called template denaturation, but has also been referred to as melting or DNA strand separation. In this step, the separated DNA strands expose the bases in the template. The second step is called primer annealing, but has also been referred to priming. In this step the two primers bind to the flanking ends of the melted separated DNA strands—one primer binds to the end of one strand of the template, and the other primer binds to the end of the other strand—hopefully not the same strand. The third step is called elongation, or DNA synthesis.  In this stage, the DNA polymerase synthesizes a new strand of DNA along each of the separated primer-annealed template strands, making a double stranded DNA product at the end of the elongation step. Each of the three steps may last only about 15 to 30 seconds or, at most, only a few minutes, depending on the actual length of the overall DNA template molecule.

Then, the three-step process of denaturation, annealing, and elongation, is repeated all over again, making another round of intact double-stranded DNA molecules, but this time, in this second cycle, the end product is doubled over that made in the previously conducted first cycle.

If the three steps (i.e., denaturation, priming and elongation) in a cycle are repeated for about 25 rounds, the original template will have been duplicated about million times over, and if 30 rounds or cycles are performed, then the template will be amplified approximately a billion times the original amount that the investigator had started out with at the beginning of the PCR procedure. In either of these cases, the entire amplification procedure of 25 to 30 cycles may actually take only 2 or 3 hours, and the amount of DNA greatly enhanced, allowing more detailed study via a variety of different ways and for various important purposes.

When Mullis first conducted the PCR process in his laboratory, he merely transferred the test tubes from water bath to water bath, each with their various temperatures set-up beforehand.  Nowadays, these PCR cycles with their various biochemical reactions are performed in automated machines that rapidly switch temperatures and do so at their necessary time-periods, making the process more efficient and less cumbersome.

4) Apparently this has something to do with paleobiology? Can you clarify?

The study of paleobiology has to do with the biology of fossil artifacts formed by life forms, such as plants and animals. Before the advent of molecular biological approaches to the field of paleobiology, experts in paleontology relied mainly on the shapes of fossils, such as bone structures, etc., to identify the origins, species, and the evolution of ancient organisms and, for example, the identities of remains of the dead. It was a slow and cumbersome process, sometimes fraught with inaccuracies, due to lack of complete remains or artificial manipulation of fossils and other artifacts.

With the introduction of PCR, however, and the availability of ancient DNA samples contained within ancient fossils, the study of biologically-based artifacts became a simpler endeavor and could provide valuable information regarding identification, function, and evolutionary relatedness between long dead organisms.

Such implementation of the PCR techniques to the study of, for example, ancient dinosaur fossils, sparked the publication of science fiction novels about bringing back the dinosaurs from the dead to the living, as seen in the famous 1990 book The Jurassic Park by Michael Crichton and feature film of the same name, with their accompanying sequels.

In his science fiction book, Crichton employed PCR in his scheme to clone the dinosaurs and to bring them back to the modern world as living breathing beings.  At a first glance, the process seemed somewhat reasonable. First, the fictitious investigators extracted perfectly intact ancient DNA from dinosaur bone marrow blood preserved the blood meals of ancient mosquitos encased in amber. Next, the fictional process involved using the PCR to amplify the dinosaur DNA. The so-called genomic dinosaur DNA was then used for gene mapping with restriction enzymes and for nucleotide sequencing. The sequenced DNA was compared with those sequences stored in super large DNA databases.  Any dinosaur genes presumably missing from their genomes were then replaced with modern reptilian DNA—most closely related to dinosaurs, according to Crichton—although actual studies showed that some modern birds have been observed to be most closely related to some ancient dinosaurs. The largely intact dinosaur DNA was cloned, restriction enzyme mapped, injected into host eggs, and allowed to hatch the baby dinosaurs.

In reality, the retrieval of intact genomic DNA for any given dinosaur had encountered several problematic issues.  First, ancient DNA was chemically different compared to modern DNA, having been altered with time. Secondly, the extents of the entire genomic dinosaur DNA retrieved was grossly inadequate for cloning at the organismal level.  Sure.  Dinosaur genes may have been able to be cloned using PCR and other modern cloning methods, but cloning of individual genes were a far cry from obtaining entire genomes for dinosaurs, and certainly not for cloning entire dinosaur organisms. Perhaps molecular techniques may someday improve in other ways, such that recovery of long extinct beings, including dinosaurs, may become possible.

Science fiction aside, PCR is used in modern times for forensics, such as for the identification of an unknown species, or of an individual, such as a crime victim or perhaps a criminal perpetrator. PCR became a household word to the laymen during the mid-1990s as a result of the “crime of the century” case with O.J. Simpson murder trial. Recently, PCR was used to identify the microbial species of corpses as a function of time, in order to determine the time of death, as the microbial composition on a corpse will be distinctive as the body decomposes. PCR may be used for identifying genes that are thought to be defective in genetic diseases. The invention may also be used to clear up confusion with paternity claims.

Occasionally, PCR is used after exhumation of a person from their grave to solve a mystery.  For example, PCR was used to study the exhumed bodies of ancient victims of the plague from the medieval times to definitively determine that the ancient bacterium Yersinia pestis was the causative agent of the Great Mortality, also known as the Black Death.

A number of years ago, an idea was proposed in which the remains of President Abraham Lincoln be exhumed from his grave, and PCR would be used to determine whether he had had a genetic disease known as Marfan syndrome, a genetic condition characterized by certain lanky features on the face and limbs.  Apparently, permission to carry out the experiment was refused, and the mystery remains.

5) He also has done some things to “mobilize the immune system”. How can this be done, why is it important, and how does he go about this?

I imagine you are referring to some of the more recent work, published in 2015, conducted by Dr. Mullis and his research collaborators. In short, his laboratory, as you say “mobilized the immune system” by engineering specialized immune system proteins, called antibodies, to redirect them to seek out pathogenic bacteria in order to target them for destruction.

The immune system mobilization works on the following principles. Mullis and his group noticed that humans do not have a certain antigen, a special sugar that the group calls “a-Gal” which is a triple sugar consisting of two types of galactose sugars plus a modified glucose sugar called N-acetyl-glucosamine, the latter of which is often found in the cell walls of most types of bacteria. The a-Gal triple sugar antigen is lacking in humans and will induce an immune response if found in humans.  Exposure to a-Gal in humans will cause them to mount an immune response, making specific anti-a-Gal antibodies.

The Mullis group connected the a-Gal antibody molecule to another molecule called a “DNA aptamer” to form a new conjugate molecule they called an “alphamer.”  The DNA aptamer is an adapter molecule that has a specific DNA or RNA sequence which is capable of binding to certain bacterial antigens. The alphamer in turn now has the ability to bind the undesirable bacterial antigen and to recruit human antibodies to the site where the pathogenic bacteria are so that the recruited specific antibodies mediate the destruction of these infectious disease causing bacteria.

In the published work, Mullis and his collaborators were able to mobilize antibodies using their novel alphamer directed against a virulence factor, called M protein, that permits evasion of macrophage-mediated phagocytosis and is made in sufficient quantities by Group A Streptococcus (GAS) microbes, which normally cause a plethora of quite severe diseases. The 2015 work was conducted in Petri dishes (in vitro), and future work is aimed at applying the invention to an in vivo model, such as in laboratory animals and eventually to application in humans.

The work holds tremendous promise in aiding the immune system to fight infections, like GAS and others, in the future.

6) What is he currently working on?

The immune system mobilization described above, represents, as of this writing, some of his most recent work.

Other relatively earlier works include a critical refinement of his PCR technique that changed the course of its usefulness and in a rather captivating way, involving bacteria that live in extreme environments.

The particular refinement process began with a problem, which was that during the PCR steps, the DNA polymerase enzyme itself would melt.  It’s properly folded amino acid string would unravel, losing its correct shape and ceasing to function in its DNA synthesis function. This prevented the proper DNA amplification process.  The solution was found in an extremophile microbe.

That is to say, the usefulness of the invention process for the PCR technique by Dr. Mullis was greatly enhanced due to the ability of certain bacteria to grow and thrive in very high temperatures. The heat-loving (i.e., thermophilic) bacterium called Thermus aquaticus (denoted colloquially as Taq) harbors a specialized DNA-making enzyme, called Taq DNA polymerase. This enzyme can function at its best to make DNA at a rather high temperature of 72 °C (which is about 160 °F) and can actually resist being destroyed itself by the very high temperatures which are needed to denature the template DNA molecules.  Such denaturation temperatures may run as high as 95 °C, or over 200 °F.  This Taq DNA polymerase heat resistance property allows investigators to easily denature DNA templates and to make new DNA quite efficiently at 72 °C.

If using conventional, heat-sensitive DNA polymerase enzymes from moderate temperature-loving organisms to do PCR, then these molecular helpers themselves would be destroyed while trying to separate the double-stranded target DNA templates, making it impossible to successfully amplify the DNA. Using Taq DNA polymerase, however, made this issue go away during the critical heat denaturation step.

7) He was also awarded the Japan Prize- what do we know about this and to whom is it awarded?

The Japan Prize, also known as the Japan International Prize is awarded to individuals who make an outstanding contribution to technology and to science and who have made an extremely significant advancement along the frontiers of new scientific developments.

The very prestigious and lucrative award (over half-a-million US dollars or 50 million yen) was established in the mid-1980s and is funded primarily by the Japan Prize Foundation and administered by the Japanese Ministry of Technology. The Prize recognizes individual inventors and scientists from all over the world.

In his autobiography, Mullis recounts the award ceremony in 1992 and his enjoyable conversation with the Empress of Japan, whom he colloquially called “sweetie” and inquired whether she had any available Empress girlfriends, all without, apparently, causing any awkward international incidents.

After receiving the Japan Prize, Mullis was approached by a German TV program host, who claimed to accurately predict future Nobel Laureates, and informed Mullis that he was next up for the award in 1992. Mullis began preparing himself for the occasion while trying to be humble about it, while at the same time allowing himself to be the focus of a documentary about his story and his invention. To his dismay, however, Mullis did not actually receive the Nobel in 1992 and actually gave up thinking about it.

All hope was not lost as he was called by Stockholm in 1993, informing him that he had been bestowed the Nobel in Chemistry.  He replied “I’ll take it!”

Mullis later reported jokingly that receiving the Nobel gave him a ticket into just about any office and a new license as an expert on just about any topic, as long as one does their homework on these new topics beforehand, of course.  As a result, Mullis has had several public disagreements regarding, for instance, HIV as a causative agent of AIDS, even though very few of the investigators in each of their fields hold similar views.

8) What have I neglected to ask about this awesome scientist?

Like other awesome investigators before him, and likely after him, too, I surmise, two of the most prestigious journals in the world, Nature and Science both summarily rejected his Nobel Prize worthy manuscripts about his history-making PCR invention.

Likewise, as had occurred with other famous and dedicated scientists who have had their hard work and efforts rejected by journals, Dr. Mullis was quite upset about these rejections for quite some time. Further, like all investigators who are in the same boat, he published elsewhere, this time in Methods in Enzymology.

Another controversy involved Mullis’ admitted use of the drug called LSD during the 1960s. He apparently became enamored with its use, studying its biochemistry and its physiology, even synthesizing ‘legal’ variants. He also dabbled with electric shocks upon himself, attempting to control his body’s physiology with his mind, even claiming to have accomplished telepathy using his electrotherapy methods. Also, he recounts in his autobiography that while alone in the woods in Mendocino Valley, CA, a raccoon that glowed a green color had spoken to him and that he awoke the next morning not having remembered any events in the interim. He attributed the encounter to possibly a non-earthling alien. No one can argue that Dr. Mullis is not an outrageous character.

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