The Story of PCR
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We start our story further back, with the discovery of DNA polymerase, the naturally-occurring enzyme that makes PCR possible, and the scientist who has been called the father of DNA biochemistry, Arthur Kornberg.
Arthur Kornberg - Discovery of DNA polymerase
Born in 1918, Arthur Kornberg was a precocious child from an immigrant, Jewish working-class family living in New York, and graduated high school at just 15. When he started studying medicine at the University of Rochester in 1937 he had no intention of becoming a biochemist, and even found the biochemistry parts of his studies rather dull. But that would eventually change.
Kornberg dreamed of working in internal medicine, preferably in an academic research setting. But when the chance came up for the students in his year to obtain fellowships for a year of medical research, Arthur failed to win one. Instead, he chose to plough his own research furrow, following where his curiosity led him.
During his studies, Kornberg noticed that the whites of his eyes appeared to be slightly yellow, which he soon found out was caused by elevated bilirubin levels, a natural substance formed during the breakdown of red blood cells. Wondering how common the condition was, he systematically examined the bilirubin levels of his peers, conducting his measurements on borrowed bench space, late at night and at weekends.”
He discovered that a reduced ability to remove bilirubin from the blood was relatively common and published a paper describing his findings in 1942. We now know this as a benign genetic condition called Gilbert’s syndrome.
After graduating from medical school in the middle of World War II, Arthur took up a post as a doctor on a navy ship. But the job didn’t last long.
At the time, American troops were routinely vaccinated against yellow fever, but the vaccination caused an outbreak of jaundice. Health authorities were concerned and began researching ways to prevent the jaundice side effects. Kornberg’s paper on liver function and bilirubin levels caught the attention of the director of the National Institute of Health and he was summoned to work there despite having no formal training in research.
Immersed in biochemistry at the NIH, Arthur quickly became captivated by the subject. For a while, he worked on nutrition, synthetic diets and vitamins, but he soon found his true calling. He was fascinated by the inner workings of cells, seeking to decipher their molecular machinery, and at the centre of his obsession were enzymes - the molecular machines at the heart of all the chemical reactions of life.
“I responded to the lure of enzymes and have remained faithful to them ever since,” he recalled in his autobiography.
Arthur became a director of the newly formed Enzyme Section in the Nutritional Division at the NIH in 1947, where he became a master of identifying and purifying enzymes involved in respiration - the process of making energy in cells.
Shortly after he moved to Washington University in St Louis, becoming head of the department of microbiology, where he moved his focus to identifying enzymes involved in DNA replication, working together with his wife and fellow biochemist, Sylvy Ruth Levy.
It was 1953 and Watson and Crick had just published the double helix DNA structure. In their paper, they pointed out that the paired strands of the ladder suggested a possible replication method, with each strand of the double helix acting as the template to recreate the matching opposite strand.
Although many at the time thought it would be impossible to recreate DNA synthesis outside the cell, Kornberg had an unrelenting faith in enzymes and the power of biochemistry, saying:
“It always seemed to me, that a biochemist devoted to enzymes could, if persistent, reconstitute any metabolic event as well as the cell does it. In fact, better!”
First, the Kornbergs started looking at the building blocks of DNA, identifying the precursors and enzymes involved in assembling nucleotide bases. By 1955, they could synthesize all four DNA nucleotides in the lab, and began searching for enzymes that could assemble them into DNA strands.
In the search, they broke E.coli bacterial cells apart, separating the proteins inside into smaller subsets, or fractions, and adding DNA and nucleotides tagged with radioactivity, searching for any signs that any of the molecules in each mixture could incorporate the radioactive nucleotides into new DNA chains, indicating replication activity.
It took months to find tiny traces of DNA replication, but in 1956 they did it. Then it was just a case of isolating the enzyme responsible - not a simple task - eventually pinning down the elusive DNA polymerase. By 1957, Arthur and Sylvy could synthesise DNA in a test tube using their isolated polymerase enzyme, but the yields were tiny and the reaction was far from efficient.
At first, they didn’t know if the product DNA was an accurate copy of the original, so they also had to invent a sequencing method using radioactive labelling, enzyme digestion and clever deductions to figure it out.
The experiments confirmed that this DNA polymerase was copying the template DNA accurately, and also coincidentally proved Watson and Crick’s proposal that the two strands of DNA run in opposite directions to each other.
Arthur was awarded the Nobel Prize for his work in DNA replication in 1959
And he continued unravelling the chemistry of enzymes and DNA replication in cells for the rest of his career.
Although in his autobiography “For the love of enzymes”, Arthur said that his wife contributed greatly to the science surrounding the discovery of DNA polymerase, she didn’t get a share in the Nobel Prize.
Reportedly, when a reporter asked Sylvy why not, she joked “I was robbed!”
Har Gobind Khorana – Single primer replication
The next character in our story is Har Gobind Khorana. The youngest of five children, born in 1922 in a poor village in what’s now Pakistan but was then British-controlled India, Khorana’s father strove to provide his offspring with an education and they were among the few people In the village who could read.
His efforts paid off. Khorana studied chemistry at a college in India, before receiving a fellowship to pursue a PhD at Liverpool University. After that, he worked at a number of universities including the University of Cambridge under chemist Alexander Todd, where he developed an interest in proteins and nucleic acids.
In 1960, Khorana moved to the Institute of Enzyme Research at the University of Wisconsin, where he began trying to crack the genetic code. His work earned him the Nobel Prize in 1968, and you can hear that story in episode 16 of series 1, Genetics By Numbers.
As if cracking the code of life wasn’t enough, he then set out on an ambitious project to build the first synthetic gene by stringing together a specific sequence of DNA. Recognising the need to copy their synthetic DNA so they could analyse it, Khorana and his team turned to enzymes.
They used strands of DNA, combined with synthetic primers, DNA polymerase, and nucleotide bases to copy single strands of DNA in a technique they called repair replication.
Taking this idea a step further, a 1971 paper from Khorana’s lab authored by Norwegian post-doc Kjell Kleppe, proposed a two primer system that would double the amount of DNA in a sample, but they didn’t go as far as actually conducting the experiments to find out whether it worked.
Their paper states: “The DNA duplex would be denatured to form single strands. This denaturation step would be carried out in the presence of a sufficiently large excess of the two appropriate primers. Upon cooling, one would hope to obtain two structures, each containing the full length of the template strand appropriately complexed with the primer. DNA polymerase will be added to complete the process of repair replication. Two molecules of the original duplex should result.”
Perhaps nobody in Khorana’s lab got round to testing this idea because it would have been a bit of a faff. AS they note in their paper, DNA polymerase is destroyed at the high temperature of 95 degrees needed to denature DNA and separate the strands, so a fresh dose of enzyme would have to be added every cycle.
Whatever the reason, it didn’t happen. So close, but yet so far.
Kary Mullis – Untamed genius or jerk that got lucky?
By 1980, using DNA polymerase to copy DNA by extending primers through Khorana’s repair replication process was commonplace in many laboratories. But it took one unique mind to provide the spark that brought PCR to fruition, and that unique mind belonged to Kary Mullis.
If you watch a few of Mullis’ many talks that are available on YouTube, you will notice that they usually start the same way, with bizarre stories about launching frogs into space as a child, often including claims that he alarmed the local air force. If that sounds like the far-fetched flight of fancy of an imaginative young boy, I’d like to say that his story gets less bizarre as he reaches adulthood, but unfortunately that is not the case.
Mullis studied chemistry at the Georgia Institute of Technology, before earning his PhD in biochemistry at the University of California, Berkley. He did some postdoctoral work at the University of Kansas before taking up a post at a biotech company called Cetus Corporation, where his job involved making short stretches of DNA called oligonucleotides, which were commonly used as primers for copying DNA by replication repair.
I can’t imagine that Mullis was popular among his colleagues at the company. Reportedly he put locks on pieces of lab machinery to stop other scientists using them, and even threatened to take a gun into work when his co-worker made moves on a woman he was interested in.
His erratic behaviour is perhaps less surprising when you consider that Mullis apparently enjoyed taking LSD and spent his spare time talking to imaginary extraterrestrial raccoons.
As the process of making oligonucleotides became more automated, Mullis found himself with much less to do. So in his boredom, he started thinking about other things. Here’s how his version of the story goes, as recounted by Mullis in an article he wrote for Scientific American in 1990, which starts with a late-night drive along California’s highway 101 to Mendocino County with his girlfriend Jennifer Barnett in the late spring of 1983.
“She was asleep. U.S. 101 was undemanding. I liked night driving; every weekend I went north to my cabin and sat still for three hours in the car, my hands occupied, my mind free. That night the air was saturated with moisture and the scent of flowering buckeye. The reckless white stalks poked from the roadside into the glare of my headlights.”
As he drove, Mullis mulled over an idea that he’d been grappling with in the lab about a new way of sequencing DNA using templates, primers, nucleotides and the Kornbergs’ infamous DNA polymerase. As these molecular components danced around in his head suddenly he saw it.
He realised that by adding two primers matching opposite strands of the DNA double helix flanking a specific sequence instead of one, he could make unlimited copies of DNA using temperature cycling and DNA polymerase. Repeating this routine - separating the strands, attaching primers, extending the strands with DNA polymerase - would exponentially multiply the number of new DNA strands each time.
At mile marker 46.7, on Highway 128, he drew the car to a sudden stop, grabbing a pen and paper from the glove box. He writes in Scientific American:
“I… started drawing lines of DNA molecules hybridizing and extending, the products of one cycle becoming the templates for the next in a chain reaction .... Jennifer protested again from the edge of sleep. "You're not going to believe this," I crowed. "It's incredible." She refused to wake up. I proceeded to the cabin without further stops.”
That night, Mullis could barely sleep, despite the assistance of a good bottle of local cabernet. He describes “deoxyribonuclear bombs exploding in my brain”, scribbling sketches of his new idea on every surface in the cabin he could find to make sure it stayed fresh in his mind through the perplexing night.
Making it work
Back at work on Monday, in the cold light of day, doubt began to set in. The idea seemed so pure and simple that he couldn’t believe no-one had come up with it before, and he asked one of the Cetus librarians to search the scientific literature for anything similar involving DNA polymerase.
Nothing turned up.
Mullis ran the idea past anyone who would listen, and drew a blank - nobody had ever heard of anything like it, although at the same time, nobody seemed particularly excited about it either. But he wouldn’t let it go.
That’s the way Mullis tells the story anyway. It is, of course, plausible that he just read the paper by Kleppe and his colleagues in Khorana’s lab published more than a decade earlier. Either way, he was determined to prove the concept worked.
By 1984, he had some preliminary results that he presented on a poster at the company’s annual conference. Unfortunately, his results were overshadowed by his dubious behaviour at the conference and over the following weeks, senior staff debated whether or not to fire him.
At the same time, Cetus was working on new diagnostic tests for genetic conditions like sickle cell anaemia. Some of the senior scientists at the company thought PCR had potential as a way to increase the amount of DNA, and therefore the sensitivity of detection, of particular sequences.
Mullis must have had some people who believed in him as an untamed genius, so although he ended up relieved of his regular duties at the company, he was given a probationary period to work on PCR as part of a small research group.
Twenty months after Mullis’ original flash of inspiration on the moonlit highway, a technician called Stephen Scharf conducted experiments that actually proved it worked, demonstrating for the first time that the process copied specific sections of DNA, yielding products of the correct size. Scharf triumphantly wrote in his lab book in capital letters: “IT WORKS!”
The team’s experiments continued apace, systematically optimising the conditions and collecting data until they had enough to prove they could amplify specific sections of DNA hundreds of thousands of times. The data formed the basis for the first patent covering PCR, which was filed in 1985.
Mullis snagged a $10,000 bonus from Cetus for the discovery, although he wasn’t as lucky in his love life as his scientific one, as he and Jennifer broke up.
But although the invention of PCR was a breakthrough, the technique still struggled with the same fundamental problem raised by Kleppe’s 1971 paper. Mullis’s PCR method used DNA polymerase purified from E. coli bacteria, which needed to be replaced after each temperature cycle when it was destroyed by the high temperature denaturing step.
The solution to this problem turned out to have already been discovered back in 1969, around 900 miles away from Cetus’s Californian Headquarters.
Taq and Taq polymerase
The hot springs and geysers of Yellowstone National Park in north west Wyoming reach temperatures of up to 95 degrees Celsius, thanks to the volcanic magma below. Such scalding heat was thought to be incompatible with life by cooking the enzymes inside cells, according to the molecular biologists of the 1960s. But one man - Thomas Brock - was determined to prove them wrong.
Setting up a research station in the park, Brock set about sampling bacteria in the steaming springs. Surprisingly, he discovered strange pink bacteria growing in the scalding water at more than 80 degrees Celsius.
He named the species Thermus aquaticus after the hot water it thrived in, and found that these hardy bugs could stand being heated right up to boiling point. Alas, Brock’s heatproof bugs were of little interest at the time. He closed his research station in 1975, after sending a sample of his hardy bacteria to a biobank for safe keeping.
Their time soon came. In 1976, Alice Chien and John Trela from the University of Cincinnati purified the DNA polymerase from Thermus aquaticus - known more simply as Taq polymerase - and showed that it could survive at temperatures over 90°C.
This was exactly the enzyme that the team at Cetus needed to make PCR a game changing laboratory technique. The experiments that proved the incorporation of Taq polymerase into PCR worked, and defined PCR as we now know it, were finally published in 1988, two years after Mullis had left the company.
Bringing PCR to the world
Removing the need to add new enzyme each time meant that a scientist could simply put all the required reagents in a sealed tube, and move it through different temperature cycles.
In the early days this meant painstakingly moving test tubes between different temperature water baths by hand for hours - a task that not even the lowliest graduate student would find appealing.
The invention of thermal cyclers - machines that could automatically run through programmed cycles of heating and cooling - meant that any researcher could pop their tubes in the PCR machine and come back a few hours later to billions of copies of the target DNA. It’s a lot like having an office photocopier, but for DNA.
Kary Mullis won the Nobel prize for the invention of PCR in 1993.
But not everybody was entirely pleased. His colleague Henry Erlich, who led the Cetus PCR group, said:
“On the one hand I was delighted to see that PCR as a technology was recognized by the Nobel Committee, but I was frustrated that awarding the prize to Mullis alone certified an account which Kary had created which was not true.
Mullis had a great idea, which he followed up with years of misrepresentation and self-promotion. Rewriting history was more productive than writing papers. He never really got it to work. It would have been appropriate to include others on the award.”
Mullis quickly adapted to life as a scientific celebrity, and found that the prize money and accompanying fame meant that he could do as he pleased for the rest of his life. Which by all accounts was surf, take drugs, and spout controversial theories about everything from aliens to climate change to HIV.
That is, of course, when he wasn’t busy propositioning female journalists or “shaking up grim-faced women scientists” by adding pictures of naked ladies to his slide shows at lectures.
It’s fair to say that Mullis was a ‘controversial’ character at best. The truth is that the original idea for PCR was published long before Mullis was working in the area. He didn’t invent any of the components, he didn’t conduct the key experiments that proved it worked, and he didn’t develop the method using Taq polymerase that we all know today.
But he did have a key insight, and he could also spin a great story. So perhaps he should have been considered for a Nobel Prize in literature instead?
The exponential growth of PCR
Within a few short years of its inception, PCR had spread to laboratories all over the world. It’s considered one of the most important technological advances of the 20th century, and Taq polymerase was named ‘Molecule of the Year’ by the journal Science in 1989.
Since the 1980s, PCR has steadily and systematically improved, using genetically engineered polymerases and other adaptations that increase the speed, accuracy and control of the process.
PCR is an extraordinarily versatile technique with a huge array of applications from identifying tiny fragments of DNA at crime scenes and disaster zones, to diagnosing diseases, and identifying endangered species.
There’s RT-PCR, which is used to amplify RNA rather than DNA. And there’s also quantitative or real-time PCR, which combines amplification and detection in the same reaction, giving an accurate readout of how much DNA was in there in the first place.
PCR-based technology is at the heart of modern molecular biology and genetic research, and is commonplace in diagnostic tests for conditions such as genetic abnormalities, cancer, and infectious diseases including COVID-19 and Ebola.
The latest iteration of the technique, Loop-mediated Isothermal Amplification or LAMP-PCR uses specific primers that form loops of DNA and enables DNA amplification at a constant temperature, removing the need for thermal cycling and significantly speeding up the process. LAMP-PCR COVID-19 tests are now in use at Heathrow airport, giving results in under an hour.
And as the COVID-19 pandemic has highlighted, the need for rapid, accurate, cheap, portable PCR testing isn’t going away any time soon. Combining clever chemistry with microfluidic devices could bring the time down to a matter of minutes, which could make all the difference to controlling an outbreak.
Three decades on from Mullis’ vision in the night and nearly 50 years after Kleppe and Khorana’s paper, the remarkable story of PCR keeps on amplifying.
Image licensed from Envato Elements
