A Chess Story and Testing for Coronavirus

Chessboard (Courtesy: pixabay.com)

Once upon a time, there lived a brilliant sage in a faraway country. One day, inspired by a vision, the sage developed the game of chess. Brimming with euphoria, he went to the king to explain the game. After listening to the sage, the king was intrigued. “I will give you anything you want,” the King declared excitedly, “Chess is marvelous!” The sage, however, shook his head and told the king that he didn’t need anything. The King was not happy; he insisted that the sage asks for a reward. The sage gave up and told the king that he would be happy with some grains of rice distributed in a specific way: one grain of rice on the first square, two on the second, four on the third, eight on the forth, and so on for all the 64 squares on the chessboard. The number of grains, in other words, would double on the adjacent square. The king, baffled by this strange request, summoned his treasury to calculate the number of grains the sage would get. After some calculations, the treasury told the king that the sage was asking for something impossible. Even the entire country’s rice production for a whole year won’t be enough! 

Welcome to exponential growth, something that we are now seeing as the number of COVID-19 patients soars (go here for some exact numbers from the above story, though the story is slightly different there). But here, I am going to focus on another exponential growth that is helping us to test coronavirus patients: Polymerase Chain Reaction or PCR. The “chain” in PCR refers to the exponential growth, and the “polymerase” here refers to an enzyme that can synthesize/replicate DNA strands. Conceptually, the PCR is similar to our chess story: because the production rate doubles in each cycle, it’s possible to make millions of copies of a DNA segment from just one PCR run. Let’s see how the PCR works first before we get into actual coronavirus testing.

The PCR involves repeated heating and cooling of DNA samples and necessary reagents. The heating process “denatures” or separates the two DNA strands so that the replication can proceed. If we think of double-stranded DNA as a zipper, then this heating process is akin to the force we need to open up the zipper. But will our reagents, especially the enzyme we want to use for replication, survive the heat? 

The DNA polymerase that synthesizes our DNA, for example, would start to breakdown in that heat. The solution to this problem came from the fortuitous discovery of a special bacteria named Thermus aquaticus. Discovered from the hot springs in Yellowstone National Park, these bacteria thrive at high temperatures. Since the DNA polymerase used by these bacteria doesn’t degrade in heat, this enzyme is now widely used to perform PCR. This special DNA polymerase is called Taq polymerase. 

Hot Springs in Yellowstone National Park (Courtesy: pixabay.com)

But even with Taq polymerase, we still have to solve two problems. One, DNA polymerases, including the Taq polymerase, can’t synthesize the four building blocks/nucleotides that make up the DNA—these enzymes can only add these nucleotides to extend a DNA strand. A mixture of these four nucleotides, thus, needs to be added to the PCR solution, along with Taq polymerase. Two, DNA polymerase can only add the nucleotides to an existing DNA strand. Hence, we also need a short DNA strand, referred to as a primer. If we want to make copies of a specific DNA segment, we can design a unique set of primers that would only bind to that segment of the DNA. These primers, as well as Taq polymerase, nucleotides, and appropriate buffer needed to run the PCR, can be ordered from several commercial vendors at a relatively low cost. 

The PCR can be run in automated machines for over 25 cycles to generate millions of copies of a DNA sample. These large quantities of DNA can then be easily detected by several methods (more on this shortly). Each PCR cycle goes through three steps:

1. Denaturation (also called melting): As discussed before, this step separates the two DNA strands at a high temperature.
2. Annealing: The reaction mixture is cooled just enough so that the primers can bind to the now-accessible DNA strands.
3. Extension/Elongation: Taq polymerases add nucleotides to the primers to make complimentary copies of both stands. If we had only one double-stranded DNA or two DNA strands in our sample, we’d have four DNA strands after the first cycle.

Steps in PCR (Courtesy: genome.gov)

When it comes to testing for coronavirus, however, we simply can’t run the PCR to detect the presence of viral DNA because coronaviruses don’t have DNA. Instead, the genetic material of the virus is contained within an RNA strand, which why the coronavirus is an RNA virus. This is not a problem though. Initially discovered in some viruses and now commercially available, an enzyme called reverse transcriptase can be used to make complementary DNA or cDNA from an RNA strand. Once we have the cDNA, we can then run the PCR to amplify segments of the DNA that are exclusive to coronaviruses. This whole process, hence, is called Reverse Transcription Polymerase Chain Reaction or RT-PCR.

Steps in RT-PCR (Courtesy: sigmaaldrich.com)

For coronavirus testing that relies on RT-PCR, the reaction progress is monitored by detecting fluorescent signals. In the reaction mixture, special probes are added, and these probes bind to specific cDNA regions. As the Taq polymerase moves along the cDNA template to add the nucleotides, the enzyme degrades the probe. This degradation generates fluorescence that can be detected by a spectrophotometer housed in the PCR machine. After each PCR cycle, more and more probes get degraded, and the fluorescent signal becomes stronger. If there is no coronavirus in a sample, the RT-PCR will not make any DNA copy, and there won’t be any fluorescent signal to detect.

Now, suppose we generate cDNA collected from a person, run the PCR, and detect no fluorescent signal. Does that mean the person is not infected? Of course not! From mishandling of the sample to mistakes in pipetting, there could be many different reasons for a negative result. Conversely, a positive signal could be due to reagent contamination and other factors. To make the results conclusive, hence, we need to use controls, both positive and negative. A negative control could be a tube that contains all the reagents but the sample, which should give no fluorescent signal. A positive control, on the other hand, could be a tube containing the actual coronavirus instead of the actual sample. If we detect a signal on that tube and nothing on the tube with the actual sample, it’s possible that the person is not infected. Once more, the result is not conclusive because the lack of signal could be due to the mishandling of the sample. Indeed, the FDA recommends other controls to ensure that a positive or negative result in detection is conclusive.

There are, of course, other ways to test for coronavirus infection, but I will leave that for another day. In the meantime, if you want to know more about the PCR-based testing, you can read the FDA’s 53-page guidelines.