Slamdunk Callahan wasn’t buying it. He had just asked me about the meaning of PCR, and I had flippantly answered, “Polyvinyl chlorinated RedAngus.” I liked my poetic license. Slamdunk was less than pleased.
We had been strolling down the street on a warm summer evening and were now at the entrance of the Confused Chromosome Club. The door was propped open to allow a breeze, and the darkened interior looked cool and inviting. We were about to go inside for a couple of root beers when suddenly, out of the blue, he asked me about PCRs. Although Slamdunk is my friend, he’s not exactly predictable. And my answer did not exactly satisfy him. At first he just shook his head, but then his response was stunning: “Do all academics think in such obfuscating terminologies?”
I was impressed. I had never heard the words “obfuscating terminologies” in the same sentence. “Fair enough," I answered. “Let’s go inside. PCR it is.”
We walked to a back table and ordered a couple of root beers. I scooped up a handful of peanuts.
“So,” I asked, “why do you want to know about PCR?”
“Some guy came by the farm last week and tried to sell me some cows based on their PCR results. At first, I thought it was EPD spelled badly, but he insisted that it was all about genes. He also babbled about a piece of equipment called a ‘thermal cycler.’ I shooed him off right smartly enough, but he got me to wondering. So I checked Wikipedia, Facebook, Craigslist and, of course, Dr. Google. PCR is something about genes and DNA, but … so what?”
“Well,” I said, “PCR is indeed important. It’s one of the main reasons we have so many advances in gene technology, and today it’s used in hundreds or even thousands of research projects. But I’m not sure what that fellow was trying to sell you, unless it was a set of reaction tubes and a thermal cycler. Maybe he was trying to sell you a polyvinyl chlorinated RedAngus.”
“Nice, but you still haven’t answered my question.”
“OK,” I said. “PCR stands for polymerase chain reaction. It’s a very clever laboratory procedure for creating millions of copies of DNA so the molecule can be sequenced and analyzed. The American scientist Kary Mullis developed it in 1983, and he received the Nobel Prize for it 10 years later.”
“You remember something about DNA, don’t you? The double helix and all that – two strands coiling around each other like a twisted ladder? On each strand, each rung of this ladder contains a nucleotide – adenine, thymine, guanine or cytosine – and each is linked to the other strand in specific pairs. Namely, if one strand contains adenine, it is always linked to a thymine in the other strand. If one strand contains a guanine, it is always linked to a cytosine. Scientists shorthand these linkages as A-T and G-C. These pairs are the building blocks of DNA code.”
“So?” Slamdunk wanted to know.
“Well, this pairing is very convenient because if you know the nucleotide at any point on one DNA strand, then you automatically know the corresponding nucleotide on the other strand. But this pairing solves only one issue about DNA – sequencing the code. The other big issue is that you can’t analyze a single DNA molecule. One molecule is obviously not enough genetic material for the analysis. You need millions and millions of exact copies of this molecule so you can apply laboratory techniques. That’s where the PCR process comes in.”
We stopped talking for a moment and looked around. A couple of folks from the bar had walked over and were standing nearby, watching, eavesdropping. They didn’t look like reporters, so I continued.
“The PCR process begins by putting the sample DNA or the piece of DNA you want to analyze into a tube along with lots of individual nucleotides. Then the tube is placed in a thermal cycler and heated to between 200 and 208 degrees Fahrenheit – just below the boiling point of water.”
Slamdunk jumped in. “Thermal cycler? Isn’t that the equipment that the salesman talked about?”
“I don’t know about your salesman, but a thermal cycler is a metal block with holes for the tubes,” I answered. “The block can be heated and cooled in precise increments and programmed to cycle through those temperatures again and again. On your farm, I don’t know how you would use it, unless you feed your cattle with hot test tubes.”
I was on a roll now, so I continued: “In any case, the high temperature denatures the DNA so it unravels into its two separate strands. Then the block temperature is lowered to 120 to 150 degrees, and a special enzyme called DNA polymerase is added to the tube. The enzyme then works on each single strand of DNA. Now things get interesting: The DNA polymerase enzyme goes down the strand from one end to the other – kind of like a zipper – and at each nucleotide, it adds the paired one. Which means that it systematically builds the corresponding second strand from the first strand. Once it completes its route to the end of the strand, we have a new double-stranded DNA molecule that is an exact copy of the original DNA molecule. And since the original DNA was denatured into its two strands, and the DNA polymerase enzyme worked on each one separately, now we have two full, double-stranded DNA molecules in the tube, rather than just one. In effect, one cycle of this PCR process has doubled the number of DNA molecules.”
“Big deal,” said Slamdunk, still unimpressed. “Now you have two molecules of DNA. Whoopee. Two molecules do not an assay make.”
“Well yes,” I said, “but this is only one cycle of the PCR. Now the cycle starts again. The thermal cycler reheats to 200 to 208 degrees, which denatures both molecules into their single strands. Then it’s cooled to 120 to 150 degrees again, and the polymerase enzyme goes back to work. After the second cycle, we now have four DNA molecules. And we do this again and again and again. Which means that every cycle through the PCR process doubles the number of DNA molecules.”
“How many cycles?”
“The PCR process is one of exponential amplification,” I answered. “After four cycles, we have 16 molecules. After eight cycles, 256 molecules, and so on. You know, two to the X power, where X is the number of cycles. The PCR process is often run for 30 cycles or more. For example, 35 cycles results in, uh …” I did a quick calculation, “34,359,738,370 molecules. Give or take a few. I’d say that’s enough material for laboratory analyses.”
“This takes weeks, I presume.”
“No. Actually, modern PCR equipment is highly automated and incredibly fast. Although the specifics depend on the length of DNA that we want to amplify, the entire PCR process can take only a few hours.”
“Whoa! I’m impressed.” Slamdunk took another swig of root beer. “But in the end, isn’t this just a laboratory curiosity? I can’t buy a thermal cycler. What good is this PCR process to us in the field?
"Especially in a cattle field?”
Slamdunk knew how to turn a phrase.
“Well,” I began, “PCR gives us enough copies of a DNA molecule so we can sequence it and add the data to our growing genetic library. We use this information in all sorts of fields. In cattle production, we routinely use it in DNA testing for parentage tracking and disease resistance. In humans, we use it for genetic mapping and police work and prenatal tests and genetic fingerprinting, just to name a few.
“And in livestock nutrition research,” I continued, “PCR has really opened a new world. It’s given researchers tools for identifying and recording things that were far out of reach just 15 years ago.”
“Like … ?”
“Like finding ways of controlling methane production in the rumen, which has major implications for greenhouse gases. Other active investigations currently focus on things like rumen acidosis and its cousin subacute acidosis, on the effects of different weaning methods on rumen function and development, on beginning to understand lower tract fermentation, on the ecology and survival of E. coli in the gut, on how different rumen microbes can change animal feed efficiency, on . . .”
“OK, OK! I get it!” Slamdunk was pretty excited. “But anything really practical?”
I paused for a moment and looked around the room. Then I looked back at Slamdunk.
“Well, your ranch is not on Neptune, so you know about COVID. What do you think the PCR test is?”