Hitchhiker's Guide to the Genome

Last updated on Aug 19, 2020 6 min read

Dethroning the Coronavirus King: Covid-19 and the Techniques Scientists are Using to Find it

Picture this: you just finished working at the hospital treating patients. You’re tired, but your nephew’s wedding is in a few weeks. So to get ready, you check in at the Metropole hotel in Taiwan. Unfortunately for Dr. Liu Jianlun, attending that wedding never became a reality. One night at the Metropole Hotel changed to two weeks at the hospital. Without much warning, Dr. Jianlun passed. The disease’s name? SARS, or Severe-Acute Respiratory Syndrome.

In early 2003, the virus SARS-CoV began its frightening sojourn to overrun the world, starting a pandemic that infected more than 8,000 and killed over 770. Like a bad sequel, a sibling to the original SARS virus has arrived, known as SARS-CoV-2 (the disease that it causes is known as Covid-19); it has currently trumped its older brother in the number of infections and deaths, numbering at more than 22 million infections and close to 783K deaths.

Because such viruses spread fast via close contact, understanding how they look internally becomes vital to figuring out who is infected and preventing further spread. How do scientists achieve this? Here is a tale behind some of the science used against two terrors not to be trifled with, one of which has been conquered, and the other still on its conquest.

Decoding DNA

Deoxyribonucleic acid – or DNA for short - has never been lacking for fans to study it. Edwin Chargaff showed that DNA is what is inherited from our parents. Francis, Crick and Franklin proposed the double-helix model in the mid-1950s. Their research has shown us that nucleotides - the base unit of DNA - can give us a boon of information to collect and study about life. But in the 1960s and 1970s trying to explore the innards of the genome and thus DNA was like exploring the Wild West. How do you figure out the order of DNA? The first one to accomplish this was a British biochemist by the name of Frederick Sanger.

Sanger Sequencing

Sanger was already well known for his scientific achievements by the time he developed the Sanger sequencing method. Having won a Nobel Prize in Chemistry for determining the sequence of insulin, he then pivoted to studying DNA with Watson and Crick. His prior research helped him sequence the first genome.

Sanger’s method required several ingredients: nucleotides, a template DNA strand for nucleotides to attach to, a protein called DNA polymerase to add nucleotides to the DNA strand, and primers, which were like signposts that attached to the strand to show where DNA polymerase should start. Yet there’s a problem. If unchecked, DNA Polymerase loves to keep adding nucleotides to the sequence. How do you get it to stop?

The trick was to mix in a modified version of nucleotides called dideoxynucleotides or ddNTPs. They lacked a part that prevented DNA polymerase from adding another nucleotide to the end of the strand. Imagine making many sequences of Lego bricks where each sequence ends in a deformed brick where nothing can attach.

Scientists used gel electrophoresis – a tool to separate DNA sequences by weight – to sort the soup of sequences. In gel electrophoresis, samples are poured into separate lanes in a Jello like substance. When the process completes, dark bands show up for each sequence of a different length. Since each sequence differs by one nucleotide, each band represents the nucleotide at the end. To identify the individual bases, scientists must add unique fluorescent dyes to each ddNTP. Thus, determining the nucleotide order just requires reading the order of the colors!

Knowing how sequencing works, you can probably imagine why it was pivotal to characterizing SARS. Scientists collected SARS’s info and then compared it to the vast amount of virus sequences already published; they were able to build a tree of sorts that showed how closely related SARS to viruses like it. In SARS’s case, they found that it belonged to a group of viruses shaped like a crown – otherwise known as coronaviruses. These days, the word corona virus is everywhere. Just the name of it has spread faster than the virus itself; its presence is pervasive. Regardless, the infection rate is still quite high, and researchers are doubling down on getting more testing kits. But then, you might ask: how do scientists quickly diagnose cases of Covid-19? The answer lies in an enzyme called reverse-transcriptase.

Like Identifying a Book by its Quotes

In order to figure out if someone has Covid-19, we need to determine if any of their samples contain traces of the virus’s RNA, or ribonucleic acid, because from there we can test if. Like DNA, RNA is also composed of bases, with two differences: there’s only one strand of nucleotides in RNA, and instead of the letter T for the base Thymine found in DNA, uracil is used instead. RNA is important because the proteins that work to make your body go are translated from the sequence of RNA, which in turn is transcribed from the DNA sequences in your genome. Think of it like abridging a book to get the important parts. You start cutting the chapters you think aren’t necessary and then you get the RNA. Splice further by editing the words to get a precise meaning, and those are your proteins. From DNA, the starting genetic material, to RNA to proteins – that is the central dogma of biology.

However, in the late 1970s, this generally accepted dogma was upended by two researchers, Howard Temin and David Baltimore. They found that some viruses were special in the way they integrated themselves into the host; these ones quite literally inserted their RNA into the host’s genome. Baltimore and Temin, though from different labs, arrived at a similar conclusion that these viruses, dubbed retroviruses, used an enzyme to turn the RNA into DNA and insert that into the host’s genome. That enzyme would be called reverse transcriptase. The idea that the central dogma was no longer a linear sequence of events turned many heads. For virologists and epidemiologists, it meant that they could identify viruses easier.

RT-rtPCR

Because RNA is an edited version of DNA, we cannot assume that the RNA we see is from the virus. RT RT-rtPCR, or reverse-transcriptase real-time polymerase chain reaction, takes advantage of reverse transcriptase to make many fluorescently labeled copies of the virus’s DNA. Once the virus’s genome has been sequenced, we can mix in primers that partially identify the virus into a sample, add in reverse transcriptase that adds nucleotides, and voila, create a DNA strand! If these viral DNA strands are created, they should light up – thus scientists can tell that the viral DNA does exist and that a patient does test positive for Covid-19.

Sources:

Videos (not mine!), for education purposes (I know I don’t have visuals of mine, but oh well):

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Kritin Karkare
Bioinformatics Staff Research Associate

I bring bioinformatics to beginners. San Diego based, Thai-Indian American science journalist. Likes Pokemon, Science communication and asking too many questions, not necessarily in that order.

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