The dark side of the maverick genius behind a medical revolution
“Kary Mullis was a very smart guy, but he was also very difficult to work with — he was cantankerous, belligerent and hostile.”
Dr Tom White (PhD) is describing his late colleague and friend who won the 1993 Nobel Prize in Chemistry for developing the polymerase chain reaction, or PCR.
The biochemical technique enables scientists to rapidly generate millions to billions of copies of very small DNA samples and allow for more detailed study.
It has been used to track the evolution of Neanderthals from traces of DNA in 50,000-year-old bones and prove that the deformed skeleton found in 2012 by archeologists under a council car park in England was that of King Richard III.
But it’s had a profound effect on medicine allowing for prenatal testing through amniocentesis and the early diagnosis of malignant disease.
And, as everyone knows, PCR testing was fundamental in identifying and treating COVID patients in a pandemic which killed some seven million people.
“Mullis’ story is that he developed this revolution all by himself but was never truly appreciated for what he did,” Dr White tells AusDoc.
“He never credited anybody else at all for what they had contributed. In that he was very wrong.”
Dr Mullis (PhD) said he came up with the idea underpinning PCR in 1983 while driving along the California coast in his silver Honda with his girlfriend.
At the time, scientists could synthesise small pieces of DNA, first by heating up a larger piece of DNA to separate it into two strands, and attaching a shorter piece called a primer at the point where they wanted synthesis to start.
An enzyme called DNA polymerase would keep attaching free-floating nucleotides — the building blocks of DNA — to the strand until it found and then attached a modified nucleotide that would stop the reaction.
But it was difficult to control the length of the synthesised DNA or reliably replicate complex sequences, such as human DNA, since they contained multiple spots where the primer could attach.
Dr Mullis’ solution involved attaching a second primer to the other strand of DNA, at the end of the sequence he wanted to replicate.
Then the polymerase would synthesise the rest of the complementary strand, creating two copies of double-helix DNA with the chosen sequence.
After repeating the steps two more times, fragments that only contained the target DNA sequence would start to accumulate.
The key point was simply the mathematics.
By the 25th cycle, you could create 33 million copies, ‘amplifying’ the target sequence to detectable levels.
“I had just solved two major problems in DNA chemistry,” he later wrote in his 1998 memoir, Dancing Naked in the Mind Field. “Abundance and distinction. And I had done it in one stroke.”
He added: “I would be famous. I would get the Nobel Prize.”
Despite Dr Mullis’ reminiscences of his Eureka moment, Dr White says the reality was that no-one was sure it would work when Dr Mullis first explained his idea.
In fact, many scientists walked out of Dr Mullis’ first presentation. Apparently, they regarded it as another of his many blue-sky ideas.
“He was a chemist, not a microbiologist, and knew nothing about any of the steps that he would have to do to get it up and running,” Dr White said.
At the time they were working at a start-up biotech company called the Cetus Corporation.
But their relationship went back to the University of California, Berkeley in 1967, where Dr White started his PhD in biochemistry and Dr Mullis — who was one year ahead of him — was a teaching assistant.
After Dr White returned from a stint in the Peace Corps during the Vietnam War, he says they became “pretty good friends”.
“We did a repair job on the engine of my old Volkswagen; we spent a lot of time together,” he says.
Then in 1977, Dr Mullis returned to Berkeley after divorcing his second wife, and Dr White helped him get a job at the UCSF Medical Center as a postdoctoral scientist.
“Mullis was working at a bakery at the time, and I said, ‘It’s kind of a waste of your ability’,” recalls Dr White.
He eventually secured Dr Mullis the job at Cetus.
But long before that lifechanging road trip, Dr Mullis was regarded as difficult to work with and soon caused friction among his colleagues.
“Mullis created havoc — affairs with people in his lab, threatening people who were going out with his friend, threatening to kill them, fistfights, threatening the guards in the evening because he didn’t have his badge when he came in the building, problem after problem,” Dr White said in an interview for the 1994 book Making PCR.
At one point, he was even at risk of losing his job after getting into a fight with another scientist at a company retreat.
But Dr White, who was the vice-president of research, decided there was enough potential in PCR not to fire him. Instead, he removed Dr Mullis’ responsibilities for a year so that he could focus on PCR.
Working under another scientist, Dr Henry Erlich from the human genomics team, Dr Mullis failed in his attempts to amplify the human nerve growth factor gene.
He then switched his focus to bacterial DNA with some success, then on to a fragment of beta-globin, the gene that causes sickle cell anaemia.
Part of the problem was that Dr Mullis did not use controls in his experiments — meaning his results, the fragments of DNA he was amplifying, could also be explained as a result of contamination with other DNA.
He used a technique called gel electrophoresis, which separates pieces of DNA based on their size, to show that he had isolated a fragment of beta-globin.
But instead of a distinct band showing the fragment, there was a large smear which could also contain other material.
“I said do the appropriate experimental design with the appropriate controls,” Dr White recalls. “But he just didn’t want to do it properly.”
So Dr White decided to assign another team of scientists to obtain the results to show PCR worked.
The scientist who finally delivered those results was Randall Saiki, a technician in Dr Erlich’s team who had joined Cetus as a research assistant and quickly moved through the ranks.
But with the potential applications of the technique becoming clearer day by day, there was a risk that Mullis’ method would be leaked and published by someone else. The solution was to pressure for Dr Mullis to publish.
“I had asked, pleaded, insisted, demanded that Mullis write up the foundational paper for publication, but he was a flaky guy, so he didn’t do it for almost a year,” says Dr White.
“It was frustrating for me. So finally, I allowed Randy Saiki to give a talk on using the first application of the PCR technique to diagnose prenatal sickle cell anaemia.”
Within a month of Mr Saiki’s talk, he and other scientists in the sickle cell PCR group had written a paper that was subsequently published in the top-tier journal Science in 1985.
“Saiki had done all the work, so he got the first author, and Mullis was in the middle somewhere because, despite the demands we made on him, he had still not written the foundational paper.”
It got worse when Dr Mullis’ paper was eventually written but then rejected by both Science and Nature. Given Mr Saiki’s paper was already out circulating in the scientific community, both journals said it lacked novelty despite fully describing the experimental conditions and other potential applications.
“That was an absolute catastrophe,” Dr White says.
“It made Mullis mad, and he was right to be mad, but not to disparage his colleagues.”
It was two years later when it finally surfaced in a journal called Methods in Enzymology, but Dr White says the disputes and animosity that had preceded its publication led to “hard feelings”.
By then, Dr Mullis had left Cetus, going on to start a company selling jewellery containing pieces of amplified DNA from dead celebrities.
One of the dead celebrities was Elvis, with Dr Mullis having bought the rights to extract DNA from a lock of The King’s hair.
In the meantime, it was left to other scientists to fix two issues making PCR difficult for real world use.
The first was that PCR relies on thermal cycling, where the temperature is raised to about 94°C to denature the DNA, then cooled to 38°C to attach the primer, and finally increased to around 72°C to lengthen the strands.
But the enzyme used initially, a DNA polymerase from Escherichia coli, denatured at 90°C and therefore had to be added after every replication cycle.
When Dr Mullis refused to do the work himself, Dr David Gelfand and Susanne Stoffel from Cetus, took it on, eventually purifying another DNA polymerase, known as Taq polymerase.
Sourced from Thermus aquaticus, a thermo-stable bacterium found in the geysers at Yellowstone National Park, their fix worked.
The second problem was that PCR also required manually moving samples between water baths sitting at different temperatures multiple times per cycle.
A Cetus technician called Robert Watson managed to automate the process with a repurposed pipetting robot.
It was at this point that PCR became a big deal and in the following years it was responsible for a series of major medical breakthroughs.
Scientists at Cetus used it to clone genes, detect HIV viral loads, and began analysing DNA samples from cold forensic cases.
To get an idea of its importance, you can look at the financial numbers. In 1991, Cetus sold the rights to PCR to Roche Diagnostics for $300 million.
But the more informative insight comes from Dr White.
“It had an amazing impact and created entirely new fields of science, including DNA analysis in forensics and molecular ecology,” he says.
“It also had profound effects on the field of evolutionary biology, and it became useful for the detection of ancient DNA — that didn’t exist before PCR, and neither did the ability to study individual variations in the rate of human mutations.
“Without PCR, the Human Genome Project would still not have its first sequences of the human genome.”
PCR was also used to place OJ Simpson at the scene of the murders of Nicole Brown Simpson and Ron Goldman.
Interestingly, Dr Mullis agreed to testify that the DNA evidence should be thrown out because he believed that those charged with conducting the test botched the procedure and contaminated the test result.
Though Dr Mullis was never called to the witness stand due to the risk that he would be cross-examined about his past LSD use, he did pass Mr Simpson a note asking for the phone number of a female witness.
Dr White’s team of scientists would use PCR to develop new tests for sexually transmitted diseases, cancer and hepatitis, as well as a method to study the genetic evolution of species, spurred on by his interest in fungi.
“It was used at the Human Rights Center at the University of California to try to reunite children separated from their parents in the civil wars in El Salvador, Honduras and Guatemala,” he says.
Though other comparable methods were developed at Cetus and other companies, Dr White says PCR dominated because it was made available to researchers everywhere and was easy to use.
“I had children in high school in a remote part of California who were taught how to use it to study their cattle and sheep, and then those high school kids taught elementary school kids how to do it.”
In 1992, Dr White nominated Dr Mullis and two other Cetus scientists for the Nobel and Japan Prizes.
“In the case of the Nobel Prize, Mullis’ PhD adviser, Professor Joseph Nielands at UC Berkeley, had requested my letter of nomination,” he says.
“I nominated Kary Mullis for the concept of PCR, Henry Erlich for proposing the three key experimental test criteria that would prove that the experimental conditions gave an indisputably confirmatory result, and Randall Saiki for discovering the narrow set of experimental conditions that produced an exponentially increasing amplification product,” he says.
However, only three people can share the Nobel Prize each year, and the committee had already decided to recognise another chemist, Professor Michael Smith, for his role in the discovery of a technique to introduce site-specific mutations into genes.
In a second letter to both committees, Dr White nominated Dr Mullis alone for the concept of PCR.
“I suspect, but don’t know, if Nielands actually provided both my letters to the Nobel Prize Selection Committee, or only the one; I never asked him.”
In the end, Dr Mullis was awarded both the Nobel Prize and Japan Prize. It made him famous.
Dr White says it was a “travesty” that Dr Erlich and Mr Saiki were never recognised.
“[They] actually made the method work.”
To celebrate winning the Nobel Prize, Dr Mullis went surfing and partied for two days with friends.
Dr White says his friend was always a “little eccentric”.
In his memoir, Dr Mullis detailed how he was saved from overdosing on nitrous oxide by a woman travelling on the astral plane, and how he learned to control the electrical conductivity of his skin and turn on a lightbulb by looking at a Playboy magazine.
Then there was his encounter with a glowing raccoon in the woods outside his home.
“I remembered the little bastard and his courteous greeting. I remembered his little shifty black eyes. I remembered the way my flashlight had looked on his already glowing face,” he wrote.
Perhaps that alone gives a sense of him. But there was a much darker side to this eccentricity.
Dr Mullis claimed zidovudine — the first antiretroviral medication available to treat HIV — was a “cellular poison” with no benefit, and that the US Food and Drug Administration (FDA) had approved the use of protease inhibitor saquinavir without data showing it cured AIDS.
He was referring to the FDA’s decision to fast-track its review of zidovudine (Retrovir), after drug sponsor Burroughs Wellcome showed that it blocked 90% of detectable HIV replication in the animal cells.
The regulator approved the drug in 1987, following another Burroughs Wellcome trial involving 280 patients with AIDS which showed that only one person in the zidovudine arm died in 16 weeks, compared to 19 in the placebo arm.
Dr Mullis wrote in his memoir that he first came to doubt that HIV caused AIDS while working on a PCR-based test for HIV.
“I did computer searches. Neither Montagnier, Gallo, nor anyone else had published papers describing experiments which led to the conclusion that HIV probably caused AIDS.”
He was referring to Dr Bob Gallo and Dr Luc Montagnier, the researchers who first discovered HIV.
They had only shown that patients with AIDS and HIV antibodies had survived a past HIV infection, not a relationship between the two diseases.
“HIV didn’t suddenly pop out of the rainforest or Haiti. It just popped into Bob Gallo’s hands at a time when he needed a new career,” he wrote.
Then in 1994, Mullis was invited to talk about PCR at the European Society for Clinical Investigation’s annual meeting, but instead spoke about how there was no proof that HIV causes AIDS.
Society president Dr John Martin — who stopped Dr Mullis’ talk about 30 minutes in — later wrote in a letter to Nature that his explanation of AIDS was “incoherent and insubstantial”.
“His talk was in style rambling and in content inappropriate for a public appearance of a leader of science,” Dr Martin wrote.
“His only slides … were photographs of naked women with coloured lights projected upon their bodies.
“[I] asked him to apply the scientific method to the problem, asking him to answer three specific questions about the transmission of AIDS … [but] his reply was again inappropriate both intellectually and in style.”
Dr Mullis proposed that AIDS was the result of a dysfunctional immune system overloaded by multiple latent viral infections, and a result of lifestyle choices made by homosexual men.
“Go up to Los Angeles … where there’s a whole bunch of guys that have had AIDS-type diseases or have seen friends die, and say, ‘I think you guys are suffering from the results of a lot of things that you have done yourselves’,” Dr Mullis said in an interview with anti-vaxxer and AIDS denialist Gary Null.
“A lot of them just get angry if you suggest that it’s got anything to do with their behaviour.”
He also claimed that doctors who prescribed zidovudine were “dumb” and had a financial interest in prescribing the antiretroviral and ordering HIV tests.
The twist came in 2000, when Dr Mullis was among several AIDs denialists invited to a panel that then South African president Thabo Mbeki convened to provide advice on the government’s response to the AIDS epidemic.
It was after convening that panel that President Mbeki declared that HIV did not cause AIDS and denied South Africans treatment, with monstrous consequences.
Over the following five years, an estimated 35,000 babies were born HIV positive, and 300,000 people died because of his decision.
“Tens of thousands of babies were born infected with AIDS because of Mullis and [his view] that HIV did not cause AIDS — that was a criminal aspect of his,” says Dr White.
Dr Mullis spent the remainder of his life — he died in 2019 of complications from pneumonia — boasting about his role in the development of PCR.
“In a sense, I put together elements that were already there, but that’s what inventors always do,” he said in an account given to the Smithsonian Institute’s Archive of Biotechnology.
In his Nobel lecture, memoir and a 1990 piece for Scientific American, Dr Mullis only mentioned technician Fred Faloona, who was the first to help him with his PCR experiments, but not Dr White, Mr Saiki or any of the other scientists who made PCR possible.
The draft manuscript of his memoir did thank Dr White for “not interfering with the development of PCR”, but even that expression of backhanded gratitude did not make the final cut.
“Many of the scientists at Cetus, who were molecular biologists, doubted that he could get it to work, and he was resentful of that,” Dr White says.
Does Dr White think Dr Mullis, who he acknowledges as a “brilliant chemist”, could have developed PCR without the help of his colleagues?
“It’s hard to say,” he says.
“The idea really was unique; even though the three components of it were known quantities, they had never been put together in that order of steps.
“But once it was known, that’s why everyone said, ‘Oh well, it’s so simple, of course somebody must have thought of it, and it’s obvious it would work’.
“I can tell you it was not at all obvious that it would work until it actually worked.
“If the other people hadn’t been assigned to it and gotten that result, Mullis would never have obtained the results and therefore never have obtained the prizes.”
