The Invention of PCR — by Kary Mullis

The concept was not out of the question because in fact one of the natural functions of DNA molecules is to reproduce themselves. They do it every time a cell divides into two daughter cells. A short piece of synthetic DNA could be treated in such a way that it would stick to a longer strand of DNA in a specific way if the sequences matched up somewhere on the long piece. The matching process would not be perfect.

Link to Original | Editor’s note: It will help those curious to understand this process to be aware of three concepts: polymer, enzyme and polymerase. Polymerase is an enzyme that acts as a catalyst to DNA reproduction, accelerating it and reducing the energy needed to sustain the reaction. This makes possible the polymerase chain reaction.

THE SUN HAD been hot that day in Mendocino County. It was May. A dry wind had come out of the east, and nobody knew how hot it had been until, around sunset, the wind stopped. I drove up from Berkeley through Cloverdale headed to Anderson Valley. The California buckeyes poked heavy blossoms out into Highway 128. The pink and white stalks hanging down into my headlights looked cold, but they were loaded with warmed oils that dominated the dimension of smell. It seemed to be the night of the buckeyes, but something else was stirring.

Kary Mullis (1944-2019), inventor of the polymerase chain reaction test for virus detection.

My little silver Honda’s front tires pulled us through the mountains. My hands felt the road and the turns. My mind drifted back into the lab. DNA chains coiled and floated. Lurid blue and pink images of electric molecules injected themselves somewhere between the mountain road and my eyes.

I see the lights on the trees, but most of me is watching something else unfolding. I’m engaging in my favorite pastime.

Tonight, I am cooking. The enzymes and the chemicals I have at Cetus are my ingredients. I am a big kid with a new car and a full tank of gas. I have shoes that fit. I have a woman sleeping next to me and an exciting problem, a big one that is in the wind.

“What subtle cleverness can I devise tonight to read the sequence of the King of molecules?”

DNA. The big one.

There are pressing reasons to want to read this molecule. Children are born with genetic defects, sometimes with tragic consequences like muscles that wither and die. Such things could be predicted and averted if we could read the DNA blueprints.

Then there are the looming but not so pressing reasons for knowing DNA — the ones that extend out to horizons mankind has not yet reached. Understanding the intricate mechanisms of our own genes will have more than just medical impact. It will be one of the long and tangled strands of our future as a growing civilization of the planet Earth. Having a detailed understanding of why children resemble parents will lead to genetic manipulating by those who may prefer alterations over strict duplication. Genetic engineering will not be a new endeavor. Evolution is and always has been a genetic engineer. It’s just that people with eyes and minds and imagination see things in the distance and they get impatient. They will want control and they will want it soon and they will have it. The DNA molecules in our cells are our history, and they are the stuff of which our future will be crafted. All of the organs of all of the plants and animals of Earth and organs that have never been in the light of the moon or the sun, will be ours to explore — to use and adapt to our needs. Our will be done on Earth as we sail off to the stars in heaven.

Yes, DNA is the big one. Tonight I am playing with a fire that will burn as brightly as Antares, descended behind these fragrant mountains several hours ago.

The key to the problem lies in the oligonucleotides that my laboratory at Cetus now easily makes. Like a “FIND” sequence in a computer search, a short string of nucleotides in a synthetic molecule might be able to define a position along a very much longer natural DNA molecule. Finding a place to start is of utmost importance. Natural DNA is a tractless coil, like an unwound and tangled audio tape on the floor of the car in the dark.

What kind of chemical program would be required to “FIND” a specific sequence on DNA with 3 billion nucleotides and then display that sequence to a human who was trillions of times larger than the DNA? Instead of a list of statements in BASIC or FORTRAN run on a computer and displayed on a screen, I had to arrange a series of chemical reactions, the result of which would represent and display the sequence of a stretch of DNA. The odds were long. Like reading a particular license plate out on Interstate 5 at night from the moon.

I knew computer programming, and from that I understood the power of a reiterative mathematical procedure. That’s where you apply some process to a starting number to obtain a new number, and then you apply the same process to the new number, and so on. If the process is multiplication by two, then the result of many cycles is an exponential increase in the value of the original number: 2 becomes 4 becomes 8 becomes 16 becomes 32 and so on.

If I could arrange for a short synthetic piece of DNA to find a particular sequence and then start a process whereby that sequence would reproduce itself over and over, then I would be close to solving my problem.

The concept was not out of the question because in fact one of the natural functions of DNA molecules is to reproduce themselves. They do it every time a cell divides into two daughter cells. A short piece of synthetic DNA could be treated in such a way that it would stick to a longer strand of DNA in a specific way if the sequences matched up somewhere on the long piece. The matching process would not be perfect. I might locate a thousand different places that were similar to the one I was searching for in addition to the correct one. A thousand out of the 3 billion in the human genome would be no trivial feat, but it wouldn’t be enough. I needed to find just one place.

Suddenly, I knew how to do it. If I could locate a thousand sequences out of billions with one short piece of DNA, I could use another short piece to narrow the search. This one would be designed to bind to a sequence just down the chain from the first sequence I had found. It would scan over the thousand possibilities out of the first search to find just the one I wanted. And using the natural properties of DNA to replicate itself under certain conditions that I could provide, I could make that sequence of DNA between the sites where the two short search strings landed reproduce the hell out of itself. In one replicative cycle I could have two copies, and in two cycles I could have four, and in ten cycles.

… I thought I remembered that two to the tenth was around a thousand.

“Holy shit!” I hissed and let off the accelerator. The car coasted into a downhill turn. I pulled off. A giant buckeye stuck out from the hill. It rubbed against the window where Jennifer, my girlfriend and co-worker, was asleep, and she stirred. I found an envelope and a pencil in the glove compartment. Jennifer wanted to get moving. I told her something incredible had just occurred to me. She yawned and leaned against the window to go back to sleep.

We were at mile marker 46.58 on Highway 128, and we were at the very edge of the dawn of the age of PCR. I could feel it. I wrote hastily and broke the lead. Then I found a pen.

I confirmed that 210 was 1,024. I must have smiled. If I repeated this new reaction ten times, I’d get a thousand copies of some piece of DNA, any piece of DNA, the molecule that knew everything about everything. Twenty cycles would give me a million, thirty would give me a billion. I could still smell the buckeyes, but they were drifting a long way off. I pulled back onto the highway, and Jennifer made a sound of approval that we were under way again. She had no idea where we were headed.

About a mile down the canyon, I pulled off again. The thing had just exploded again. A new and wonderful possibility. Not only could I make a zillion copies, but they would always be the same size. That was important. That was the almighty, the halleluja! clincher. The hell with Jennifer. I had just solved the two major problems in DNA chemistry. Abundance and distinction. And I had done it in one stroke. I stopped the car at a nice comfortable turnout and took my time working my way through the consequences. This simple technique would make as many copies as I wanted of any DNA sequence I chose, and everybody on Earth who cared about DNA would want to use it. It would spread into every biology lab in the world.

I would be famous. I would get the Nobel Prize.

“Ten years from now they will know me in Zambia,” I boldly conjectured, “and Alice Springs.”

“Ten years from now I’ll walk into biochemical laboratories at the University of East Jesus, and they’ll ask me to say something wise to the graduate students.

”Somehow, I thought, it had to be an illusion. It was too easy. Someone else would have done it and I would surely have heard of it. We would be doing it all the time. What was I failing to see? “Jennifer, wake up.”

She wouldn’t wake up. I had thought of incredible things before that somehow lost some of their sheen in the light of day. This one could wait until morning. But I didn’t sleep that night. We got to my cabin and I started drawing little diagrams on every horizontal surface that would take pen, pencil, or crayon, until dawn when, with the aid of a bottle of good Anderson Valley Cabernet, I settled into a perplexed semi-consciousness.

Afternoon came — including new bottles of celebratory red fluids from Jack’s Valley Store — but I was still puzzled, alternating between being absolutely pleased with my good luck and clever brain and being mildly annoyed at myself and Jennifer for not seeing the flaw that must have been there. I had no phone at the cabin and there were no other biochemists besides Jennifer and me in Anderson Valley. The conundrum, which lingered throughout the weekend and created an unprecedented desire in me to return to work early, was compelling. If the cyclic reactions that by now were symbolized in various ways all over the cabin really worked, why had I never heard of them being used? If they had been used, I surely would have heard about it and so would everybody else, including Jennifer, who was presently sunning herself beside the pond, taking no interest in the explosions that were rocking my brain.

Why wouldn’t these reactions work?Monday morning I was in the library. The moment of truth. By afternoon it was clear. For whatever reasons, there was nothing in the abstracted literature about succeeding or failing to amplify DNA by the repeated reciprocal extension of two primers hybridized to the separate strands of a particular DNA sequence. By the end of the week, I had talked to enough molecular biologists to know that I wasn’t missing anything really obvious. No one could recall such a process ever having been tried.

However, shocking to me, not one of my friends or colleagues would get excited over the potential for such a process. True, I was always having wild ideas, and this one maybe looked no different from last week’s. But it was different. There was not a single unknown in the scheme. Every step involved had been done already. Everyone agreed that you could extend a primer on a single-stranded DNA template. Everyone knew that it would make a double-stranded DNA molecule that you could heat up and turn into two more single-stranded DNA templates. Everyone agreed that what you could do once, you could do again. Most people didn’t like to do things over and over, me in particular. If I had to do a calculation twice, I preferred to write a program instead. But no one thought it was impossible. It could be done, and there was always automation. The result on paper was so obviously fantastic that even I had little irrational lapses of faith that it would really work in a tube. Most everyone who could take a moment to talk about it with me felt compelled to come up with some reason why it wouldn’t work. It was not easy in that postcloning, pre-PCR year to accept the fact that you could have all the DNA you wanted. And that it would be easy.

I had a directory full of untested ideas in the computer. I opened a new file and named this one “polymerase chain reaction.” I didn’t immediately try an experiment, but all summer I kept talking to people in and out of the company. I described the concept around August at an in-house seminar. Every Cetus scientist had to give a talk twice a year. But no one had to listen. Most of the talks were dry descriptions of labor performed, and most of the scientists left early without comment.

One or two technicians were interested, and on the days when she still loved me, Jennifer thought it might work. On the increasingly numerous days when she hated me, my ideas and I together suffered her scorn.I continued to talk about it and by late summer had a plan to amplify a 400-nucleotide fragment from Human Nerve Growth Factor, which Genentech had cloned and just published an article on in Nature. It would be dramatic. What had taken Genentech months to obtain, I would reproduce in hours.

My friend Ron Cook, who had founded Biosearch and produced the first successful commercial DNA synthesis machine, was the only person I remember during the summer who shared my enthusiasm for the reaction. He knew it would be good for the oligonucleotide business. Maybe that’s why he believed in it. Or maybe he’s a rational chemist with an intact brain. He’s one of my best friends, so I have to disqualify myself from claiming any really objective judgment regarding him. Perhaps I should have followed his advice, but then things would have worked out differently and I probably wouldn’t be here on the beach in La Jolla writing this, which I enjoy. Maybe I would be rich in Tahiti. He suggested one night at his house that since no one at Cetus had taken it seriously, I should resign my job, wait a little while, make it work, write a patent, and get rich. By rich he wasn’t imagining the $300 million that Cetus finally got from Hoffmann — La Roche for PCR. Maybe one or two. The famous chemist Albert Hofmann was at Ron’s that night. He had invented LSD in 1943. At the time he didn’t realize what he had done. It only slowly dawned on him. And then things worked their way out over the years as no one would have ever predicted, or could have controlled by forethought and reason. Kind of like PCR.

I responded weakly to Ron’s suggestion. I had already described the idea at Cetus, and if it turned out to be commercially successful, they would have lawyers after me forever. Ron was not sure that Cetus even had rights to my ideas unless they were directly related to my duties. I wasn’t sure about the law, but I was pretty happy working at Cetus and assumed, innocently, that if the reaction worked big time, I would be amply rewarded by my employer. I was plenty wrong on that one.

The subject of PCR was not yet party conversation, even among biochemists, and it was quickly dropped. Albert’s being there was much more interesting — even to me.

My problems with Jennifer were not getting any better. That night was no exception to the trend. I drove home alone feeling sad and unsettled — not in the mood for leaving my job or any other big change in what was left of stability in my life. PCR seemed distant and small compared to our very empty house.

In September I did my first experiment. I like to try the easiest possibilities first. So one night I put human DNA and the Human Nerve Growth Factor primers in a little screw-cap tube with an O-ring and a purple top. I boiled for a few minutes, cooled, added DNA polymerase, closed the tube and left it at 37 degrees. It was exactly midnight on the ninth of September. I poured a cold Becks into a 400-milliliter beaker and contemplated my notebook for a few minutes before leaving the lab.Driving home, I figured that the reaction would proceed by itself, and I didn’t really care how long it took as long as nobody had to do anything. For a reaction with the potential of this one — especially in the light of the absence of anything else that could do the same thing — time was a very secondary consideration. Would it work at all? The next most important thing was, Would it be easy to do? Then came, How long would it take?

At noon the next day I went to the lab to take a twelve-hour sample. There was no sign by ethidium bromide of any 400-nucleotide fragment. I could have waited another hundred years, as I had no idea what the absolute rate might be, but I succumbed slowly to the notion that I couldn’t escape much longer the unpleasant prospect of cycling the reaction by hand. This meant adding the thermally unstable polymerase after every cycle and a hell of a lot more work for me.

For three months I did sporadic experiments while my life with Jennifer, at home and in the lab, was crumbling. Progress in the lab was slow. Finally I retreated from the idea of starting with human DNA. I settled on something simpler, called a plasmid. The first successful experiment happened on December 16, 1983. It was dark outside when I took the autoradiogram out of the freezer and developed it. There, just where it should have been, was a little black band. A tiny little black band. It meant that I was going to be famous. I remember the date. It was the birthday of Cynthia, my former wife from Kansas City, who had encouraged me to write fiction and bore us two fine sons. I had strayed from Cynthia eventually to spend two tumultuous years with Jennifer. When I was sad for any other reason, I would also grieve for Cynthia. There is a general place in your brain, I think, reserved for “melancholy of relationships past.” It grows and prospers as life progresses, forcing you finally, against your better judgment, to listen to country music.

And now as December threatened Christmas, Jennifer, that crazy, wonderful woman chemist, had finally and dramatically left our house and the lab and headed for New York and her mother, for reasons that seemed to have everything to do with me but that I couldn’t fathom. I was beginning to learn tragedy. It differs a great deal from pathos, which you can learn from books. Tragedy is personal. It would add strength to my character and depth, someday, to my writing. Just right then, I would have preferred a warm friend to cook with. Hold the tragedy lessons. December is a rotten month to be studying your love life from a distance.

I celebrated my victory with Fred Faloona, a young mathematician and a wizard of many talents whom I had hired as a technician. Fred had helped me that afternoon set up this first successful PCR reaction, and I stopped by his house on the way home. As he had learned all the biochemistry he knew directly from me, he wasn’t certain whether to believe me when I informed him that we had just changed the rules in molecular biology.

“Okay, Doc, if you say so.” He knew I was more concerned with my life than with those cute little purple-topped tubes.In Berkeley it drizzles in the winter. Avocados ripen at odd times, and the tree in Fred’s front yard was wet and sagging from a load of fruit. I was sagging as I walked out to my little Honda Civic, which never failed to start. Neither Fred, empty Becks bottles, or the sweet smell of the dawn of the age of PCR could replace Jenny. I was lonesome.