Interviewer: Lydia Morrison, Technical Writer and Podcast Host, New England Biolabs, Inc.
Interviewee: Becky Kucera, Principal Development Scientist of Applications & Product Development, New England Biolabs, Inc.
Today I am joined by Becky Kucera, Applications and Product Development Scientist at NEB. Thanks so much for speaking with me today Becky.
Thanks for having me.
So, please tell our listeners how long you have worked at New England Biolabs?
I've been here for 30 years now. I started out in production, working with DNA polymerase, but then had a wonderful time in research and now I've been in products and applications development for quite a while. Before that, I was in academic science for 10 years, and you know, I'm very thankful that even after four decades of working at the bench, it's still exciting to come to work every morning.
Today, you're here with me to tell the story of how the polymerase chain reaction, better known as PCR, was invented. Firstly, can you explain what PCR is for our listeners who aren't bio-chemists?
Certainly. PCR is short for Polymerase Chain Reaction and it's a technique using a polymerase, to make exponentially increasing copies of a specific region of DNA you care about, starting from that very small amount, in a type of chain reaction. You can think of it as “molecular DNA xeroxing.” And the reason for having these large numbers of copies, is that having those large numbers allow scientists to have enough for all kinds of crucial analysis that just aren't possible when that important DNA sequence is varied amongst a very large amount of complex DNA, when it's literally a needle in the haystack. Now, PCR requires certain things to make those copies. It requires DNA starting material of course, and the small building blocks of DNA called deoxyribonucleotides, that become the familiar A, C, G, and T bases of DNA.
You also need short pieces of DNA called primers that mark the beginning and end of the DNA sequence that you care about, and the DNA polymerase that uses those individual building blocks to extend those primers into making completely new DNA copies. Now, this isn't all done at once. Instead, PCR uses about 30 cycles of switching to temperatures that favor different stages of PCR. So, there's a very high temperature that's needed for the double stranded DNA to literally be pulled apart, allowing the primers, at a different temperature, to anneal to what is now the single stranded DNA, at the beginning and end, and then, the polymerase finds those primer ends, and reads the template strand, and expands that primer into a full copy of that DNA template. So where once there was one copy of say, a gene, now you have two. And the next cycle, you have four copies, and then eight copies, and then sixteen copies, and on, and on. It's such a powerful technique, because this doubling with every cycle means 10 cycles will take one copy and turn it into 1,000. Thirty cycles turns it into 1,000,000,000. That's an incredible, powerful technique, and absolutely is a chain reaction on a molecular level.
And, how was PCR used over the next 20 years or so?
Well, it was very interesting. The research community responded so quickly to the potential of this technique. It was put to work in so many different ways, and it was really, really exciting time. It allowed medical diagnostics. It allowed research focusing on specific gene mutations that were linked to certain diseases in the research labs. It even increased food safety because for the first time, we could identify easily bacteria, or fungal contaminants that are on the surface of foods that are detrimental to human health.
It even gave us information on the history of life itself because all of a sudden, we had a way to make copies in sequence, the DNA from such ancient animals, and plants, preserved in amber, that had been unavailable to us, as they sat in that amber for hundreds of millions of years. PCR also became a powerful tool in forensics, leading to the apprehensions of a lot of bad guys. Also, to ending false imprisonment of people who were innocent. In fact, I remember one of the earliest crime fighting cases solved through PCR tied a suspect in an Arizona murder case to his victim's body, that was found under a Palo Verde tree in California, through PCR, that was done on seed pods from the tree, that fell into the bed of his truck.
Have there been any further developments to PCR?
Oh, absolutely. Science never stands still. For polymerases, what came next was a discovery of very accurate so-called proofreading DNA polymerases that made fewer mistakes when they were making those DNA copies. And then, mixes of DNA polymerases, each having its strengths and weaknesses, and, functionally, they would complement each other in a positive way during PCR. Then, came specifically engineered DNA polymerases, where changes in a gene would be designed to alter the amino acids making up the polymerase in a very specific way to gain a certain functionality that was sought.
Now, in addition to changes in DNA polymerases, there were also changes in the process itself. PCR developed into qPCR, sometimes called real-time quantitative PCR, that allows exact determinations of how much of a certain DNA sequence is present, and this can be very useful when the level of an infectious agent's DNA, like a bacterial cell's DNA, correlates with the seriousness of the disease. And, also, digital PCR was the last development, and that detects low-abundance DNA variants to be detected amongst a vast excess of DNA having the normal sequence. So, imagine this, a medical researcher who finds a small amount of DNA released from a single dying tumor cell in a person's bloodstream, even when it's pretty much a needle in the haystack again, in terms of all the normal sequence DNA around.
So, this is a really hot area right now in science, as you can imagine, as it would be so great to be able to screen for many different serious health challenges from a single, easy to obtain blood sample, early enough for effective treatments to be started.
So, what are the events that came together to enable the development of PCR?
Well, like most things, it did start with a great idea, in this case, from the mind of Kary Mullis, but it rose from just a useful technique to a useful technique that was found in every molecular biological lab in the world, because of two additional things: a wonderful, heat-loving polymerase called Taq DNA polymerase, from a bacterium that flourished in the hot springs of Yellowstone, and a piece of equipment called the thermal cycler. So, it was mostly a good idea, but also a hot springs enzyme and a new laboratory device.
So, I’ve heard a lot of stories about Kary Mullis. Can you tell us how he came up with the concept of PCR?
Certainly. Kary is a very charismatic individual, according to anyone who has been able to meet him. He related the story of PCR's discovery in a really entertaining article in an issue that published in Scientific American in 1990. It's really kind of become kind of a legend since then.
The story goes, he was working in the San Francisco area back in 1983. On weekends, he would regularly drive his little Honda Civic up to Mendocino County, Red Woods area, to kind of get away from the lab and relax a bit. Recharge his batteries. One late night, as he was driving up Route 101 with a friend named Jennifer, on a very familiar stretch of road with little traffic, his mind wandered into the current lab project challenges he was facing. He needed a way to identify what nucleotide was present at a certain position in a person's genomic DNA. Remember, this was way before genomic DNA sequencing was possible. That would come much later.
For Kary, the early ideas of PCR started coalescing in his mind so suddenly, he pulled over to the side of the road to focus on the mathematics of that DNA copying without getting in an accident. When he realized the process could theoretically lead to millions and billions of copies, he woke Jennifer up and enthusiastically told her about his idea. Jennifer was reported not impressed at being awakened in the middle of the night, and promptly went back to sleep.
But, he was persistent. The following Monday, back at the lab, his colleagues were also skeptical. But he just didn't let it go. Instead, he put all of his energy into designing the early PCR experiments that would prove the method worked, which they eventually did in a very clear cut manner. Even at later scientific meetings, when he would describe his theory of PCR and have early experimental data to back it up, the discussions still didn't go well. Sometimes, allegedly, it came close to actual physical altercations. You should understand, this is not how scientists are supposed to behave, who are supposed to be always rational and show respect for the ideas of others. One can't help but wonder whether part of the problem was people thinking, "If it was that simple, why wouldn't someone else have already discovered it by now?"
After facing such harsh criticism of his new idea, what attributes of Kary's personality, do you think, led him to be able to continue his efforts to win over the scientific community?
Kary absolutely believed his data and was extremely tenacious. Some might say, arrogantly stubborn, in believing in that idea and his data that came from his early experiments. His tenacity was such that when he was met with the skepticism of others and outright rejection, in some cases, it just didn't stop him.
The second event you mentioned was the discovery of taq DNA polymerase. Can you tell us a little bit about that?
Early PCR required a person to stand by and add DNA polymerase called the Klenow fragment with each cycle because the high temperatures that you needed earlier in the cycle to pull that double-stranded DNA apart would heat and activate, or kill, the polymerase. Standing by and manually adding fresh enzyme in every cycle became very, very tedious. It quickly became clear that what was needed was an enzyme that could handle the high temperatures of PCR. Where better to look for such an enzyme than in the bacteria that normally grow in very hot areas of our planet?
As luck would have it, earlier in 1969, a scientist named Tom Brock was working on understanding how living bacterial cells managed to survive and thrive at extreme temperatures like those in the hot springs of Yellowstone. In these hot springs, he discovered a bacterium called Thermus aquaticus, which translates to "living in hot water."
In 1976, another scientist named Alice Chen isolated and characterized, on a small scale, the enzyme that would come to be called taq DNA polymerase. And that polymerase could tolerate temperatures up to 95 degrees C, very close to boiling water temperatures, perfect for PCR, although it hadn't been discovered by Kary yet. It was just very fortunate that when PCR did come along, taq DNA polymerase was waiting in the wings, so to speak. The PCR-driven need for a thermostable enzyme focused attention fast on the potential of taq.
Were all new enzymes discovered like that in the early days?
In a way, yes. Remember this was way before the present age of bioinformatics and data mining from sequenced genomes. In those early days, new enzymes were discovered from the environment, from all kinds of environments, like the Yellowstone Hot Springs. In fact, at NEB, employees returning from vacations took scrapings from the bottoms of their shoes or boots and gave them to the enzyme discovery group at the company.
They would dilute the scrapings, spread it out onto an agarose nutrient plate, and wait to see what new and interesting bacterial colonies might grow on the plate overnight. Sometimes, if they were lucky, these new bacteria would show new and useful enzyme activities.
Our new enzymes came from all over the place. One came from a scientist's brother's backyard in Minnesota while another one came from a waterfall in Colorado where animals tended to gather and poop in the water. But, one could argue one of the most important sources for a new enzyme was that Hot Springs in Yellowstone.
I know you were working at the bench at NEB during the development of PCR. Could you tell us what that was like?
It was crazy and exciting and a little bit exhausting. The funny thing is every new person on the Taq DNA polymerase came to do the same thing, pull out their yellow pads of paper and grab a pencil, and draw out all the steps of PCR to convince themselves that, my goodness, this could actually work. Then it was all about the challenge of purifying it on a large scale.
The phone was ringing quite a bit in those days, mostly academic scientists who wanted to get their hands on Taq DNA polymerase so they could do PCR, and the enzyme wasn't cloned at the time. There were a lot of challenges. Getting large fermentation yields of the bacterial cells themselves were difficult to come by plus there were no easy ways to purify the polymerase on a large scale even if you have the cells.
Scientists worked oftentimes way past midnight and weekends. I remember one Christmas where three of us were at the lab joking that this wasn't exactly where we expected to be on Christmas Day. Nowadays the purification of enzymes from bacterial cells is more straightforward but it wasn't back then. Purifying any one enzyme requires a way to separate that enzyme from the hundreds of other enzymes and cellular material present in any bacterial cell.
So little was known about all the individual enzymes that were attempting to be purified at the time, that often times it was just a matter of trying one thing after another, which would be pouring lysed or broken open bacterial cells over material packed in a cylinder, this was called a chromatography column, and hopefully that chromatography material would bind your enzyme while everything else flows through the column allowing you to then add something like high salt to release your enzyme from the column and you would have achieved a level of purification from that single chromatography step.
For any one enzyme it would take two, three, four columns, all the way up to ten or fifteen, to get an enzyme like Taq polymerase purified enough for PCR application. But, we did it. In March of '87 New England Biolabs was the very first company to have Taq DNA polymerase available to scientists. Believe me, it took a village to accomplish that.
The need was so great that sometimes there was just one day between one batch of polymerase being sold out and the next batch passing quality control. We all worked very hard but looking back on it, I think we really benefited from a sense of real camaraderie that occurs whenever people are working together on a common goal.
It must have been so amazing to be a part of that team.
It certainly was. I enjoyed it.
So, that was how the first naturally occurring thermophilic DNA polymerase became available for PCR. How did that lead to a race to develop and produce a recombinant form of the enzyme?
Well, to answer that question we need to understand why you want to clone an enzyme in the first place instead of just using the one produced from its natural source, and the answer is to gain convenience and reliability. Not all species of bacteria can be easily grown in the lab, either because the right nutrients aren't identified or they're there but at the wrong levels or the oxygen level isn't right or the temperature needs are extreme, like that Thermus aquaticus.
However, there's one bacterium that is well-characterized, well-behaved almost always, and has become the real workhouse of molecular biology. That bacterium is Escherichia coli or E. coli for short. This is a laboratory strain derived from the harmless E. coli found in the intestinal tract of all warm-blooded animals, including humans.
Now, E. Coli for lab use has also been intentionally weakened in such a way that it can't grow outside of the laboratory environment. It needs certain nutritional components that we put into the growth medium for the cells. This gives us an extra level of safety.
While we're talking about safety, it's important to realize this is not the O157:H7 strain that sometimes makes the headlines for making people seriously sick. That strain of E. Coli only became dangerous because it was infected by a virus called a bacteriophage that incorporated its gene for a really bad toxin into the E. Coli bacterium.
Cloning any gene into E. Coli allows us to have this very reliable bacterial strain do all the work of transcribing the cloned gene into messenger RNA and translating that RNA into the protein, in this case, the enzyme Taq DNA polymerase. So, all we have to do is harvest the bacteria and purify the enzyme. Since E. Coli reproduces by one cell dividing into two every 20 minutes or so, its own kind of chain reaction, a single cell can lead to a million cells within seven hours.
Not only does it grow fast, it isn't fussy in terms of the nutrients it needs to grow, they're not expensive, they're not complicated, it doesn't care whether oxygen is present or not, and it grows at a much more reasonable temperature of 98.6 degrees Fahrenheit, body temperatures. This contrasts with Thermus aquaticus bacteria that requires a temperature around 176 degrees Fahrenheit, which is hard to maintain in a laboratory. So, there's some very, very good reasons everyone wanted to clone Taq polymerase and that particular race was won by Francis Lawyer at Cetus in 1988.
The last element you mentioned that brought PCR to fruition was the development of a thermocycler. Can you tell me about the impact that invention had?
That was the final enhancement that led to the explosive growth of PCR. A thermocycler is simply an instrument that allows you to set temperatures and times for the stages of a PCR cycle, a number of cycles you want to use and even how long you want the reaction held in a nice, cold, stable temperature until it's convenient for you to work with those DNA copies.
Now, to fully appreciate this you need to understand that before thermocyclers people and water baths were the thermocyclers. You would stand at your lab bench in front of three water baths set at the three temperatures needed for the three stages of PCR and at intervals of about a minute or so you would reach in and manually move the tube from one water bath to the next one. This would go on for about four hours. It got old really fast. At NEB, everyone knew when I was doing a PCR because I'd be very grouchy by the end of the day. Thermocyclers changed all of that.
According to the salesperson who sold us our first thermocycler, we had one of the very first ones in the United States and no one at NEB was happier about that than me. No more needing to function as a human thermocycler.
I can imagine that must have been super frustrating and super gratifying when the thermocycler was delivered.
It was. There was cheering in the hallway.
So, what lessons can we draw from the advent of PCR?
That's an interesting question. There actually is a major lab and life lesson that emerges from the story of PCR. By far, looking back on it, Kary's tenacity, his belief in his thoughts and his experimental results in the face of really overwhelming initial skepticism and near hostility, teaches us to be champions of our ideas, even in the face of adversity. Of course, sometimes a bit of luck and timing is important. I think about Taq DNA polymerase there waiting in the wings to make PCR easier. Lastly, technology is here to make our lives simpler, as a thermocycler certainly did for us.
I think the main theme here is not giving up on something you believe in. In a beautiful case of real poetic justice, Science magazine, one of the premiere scientific journals in the United States that had soundly rejected Kary's first PCR paper in 1986, would just three years later declare PCR the molecule of the year. From Kary's initial thoughts that night, driving up through the red woods, to his being awarded the Nobel Prize for chemistry in 1993 for developing PCR, was only 10 years. That speaks to not letting negativism stand in your way and believing in yourself and your ideas.
It absolutely does. Thank you so much for joining us today, Becky.
It's been a pleasure.
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