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The Nobel Prize in Chemistry 1993   Kary B. Mullis, Michael Smith  Nobel Lecture Nobel Lecture, December 8, 1993

One Friday night I was driving, as was my custom, from Berkeley up to Mendocino where I had a cabin far away from everything off in the woods. My girlfriend, Jennifer Barnett, was asleep. I was thinking. Since oligonucleotides were not that hard to make anymore, wouldn't it be simple enough to put two of them into the reaction instead of only one such that one of them would bind to the upper strand and the other to the lower strand with their three prime ends adjacent to the opposing bases of the base pair in question. If one were made longer than the other then their single base extension products could be separated on a gel from each other and one could act as a control for the other. I was going to have to separate them on a gel anyway from the large excess of radioactive nucleosidetriphosphate. What I would hope to see is that one of them would pick up one radioactive nucleotide and the other would pick up its complement. Other combinations would indicate that something had gone wrong. It was not a perfect control, but it would not require a lot of effort. It was about to lead me to PCR.

I liked the idea of a control that was nearly free in terms of cost and effort. And also, it would help use up the oligonucleotides that my lab could now make faster than they could be used.

As I drove through the mountains that night, the stalks of the California buckeyes heavily in blossom leaned over into the road. The air was moist and cool and filled with their heady aroma.

Encouraged by my progress on the thought experiment I continued to think about it and about things that could possibly go wrong. What if there were deoxynucleoside triphosphates in the DNA sample, for instance? What would happen? What would happen, I reasoned, is that one or more of them would be added to the oligonucleotide by the polymerase prior to the termination of chain elongation by addition of the dideoxynucleoside triphosphate, and it could easily be the wrong dideoxynucleoside triphosphate and it surely would result in an extension product that would be the wrong size, and the results would be spurious. It would not do. I needed a way to insure that the sample was free from contamination from deoxynucleoside triphosphates. I could treat the sample before the extension reaction with bacterial alkaline phosphatase. The enzyme would degrade any triphosphates present down to nucleosides which would not interfere with the main reaction, but then I would need to "deactivate the phosphatase before adding the dideoxynucleoside triphosphates and everyone knew at that time that BAP, as we called it, was not irreversibly denaturable by heat. The reason we knew this was that the renaturation of heat denatured BAP had been demonstrated in classic experiments that had shown that a protein's shape was dictated by it's sequence. In the classical experiments the renaturation had been performed in a buffer containing lots of zinc. What had not occurred to me or apparently many others was that BAP could be irreversibly denatured if zinc was omitted from the buffer, and that zinc was not necessary in the buffer if the enzyme was only going to be used for a short time and had its own tightly bound zinc to begin with. There was a product on the market at the time called matBAP wherein the enzyme was attached to an insoluble matrix which could be filtered out of a solution after it had been used. The product sold because people were of the impression that you could not irreversibly denature BAP. We'd all heard about, but not read, the classic papers.

This says something about the arbitrary way that many scientific facts get established, but for this story, it's only importance is that, had I known then that BAP could be heat denatured irreversibly, I may have missed PCR. As it was, I decided against using BAP, and tried to think of another way to get rid of deoxynucleoside triphosphates. How about this, I thought? What if I leave out the radioactive dideoxynucleoside triphosphates, mix the DNA sample with the oligonucleotides, drop in the polymerase and wait? The polymerase should use up all the deoxynucleoside triphosphates by adding them to the hybridized oligonucleotides. After this was complete I could heat the mixture, causing the extended oligonucleotides to be removed from the target, then cool the mixture allowing new, unextended oligonucleotides to hybridize. The extended oligonucleotides would be far outnum- bered by the vast excess of unextended oligonucleotides and therefore would not rehybridize to the target to any great extent. Then I would add the dideoxynucleoside triphosphate mixtures, and another aliquot of polymerase. And now things would work.

But what if the oligonucleotides in the original extension reaction had been extended so far they could now hybridize to unextended oligonucleotides of the opposite polarity in this second round. The sequence which they had been extended into would permit that. What would happen?

EUREKA!!!! The result would be exactly the same only the signal strength would be doubled.

EUREKA again!!!! I could do it intentionally, adding my own deoxynucleoside triphosphates, which were quite soluble in water and legal in California.

And again, EUREKA!!!! I could do it over and over again. Every time I did it I would double the signal. For those of you who got lost, we're back! I stopped the car at mile marker 46,7 on Highway 128. In the glove compartment I found some paper and a pen. I confirmed that two to the tenth power was about a thousand and that two to the twentieth power was about a million, and that two to the thirtieth power was around a billion, close to the number of base pairs in the human genome. Once I had cycled this reaction thirty times I would be able to the sequence of a sample with an immense signal and almost no background.

Jennifer wanted to get moving. I drove on down the road. In about a mile it occurred to me that the oligonucleotides could be placed at some arbitrary distance from each other, not just flanking a base pair and that I could make an arbitrarily large number of copies of any sequence I chose and what's more, most of the copies after a few cycles would be the same size. That size would be up to me. They would look like restriction fragments on a gel. I stopped the car again.

"Dear Thor!," I exclaimed. I had solved the most annoying problems in DNA chemistry in a single lightening bolt.