What are “Primers”? What is “PCR”? How does Gene Sequencing Work?
We have all heard during the pandemic about the PCR Covid-19 tests… What are they? What is the genetics behind it? We will go over these concepts and try to elucidate them.
By Izabela Ninu
Polymerase Chain Reaction (abbreviated PCR)
— is a laboratory technique used in a lab in order to amplify a specific gene sequence many times in order to be able to manipulate it.
We can see in this diagram provided by the genome.gov website the basic usage of the PCR technique: creating millions of copies that we can later analyse or use easily.
Wo wo wo let’s go back for a bit…
Where did all of this start?
PCR dates back to the mid-1980s, which is more or less the time when the Human Genome Project was being considered and then started. (It’s a project from 1990 to 2003, conducted to determine the sequence of the human DNA, and to identify and map all of the genes of the human. It completed to sequence about 92% of the whole genome, and the rest 8% was almost achieved in 2020).
As such, the main technique used was PCR — it is crucial to “amplify” the copy of DNA as we cannot study isolated pieces of DNA.
How does PCR work?
PCR works by firstly heating up a DNA sample so it denatures the DNA, separating the two strands of DNA. Then, an enzyme called “Taq polymerase” will synthetize the two strands of DNA making two distinct copies of it the original. Repeating many times this process (we can go up to 30 or 40 times) will lead to millions of exact copies, that we need in order to analyse the sequence.
As such, you can see above that we need this DNA polymerase, but also the “Primers”
What are Primers?
I’m usually not a fan of Wikipedia… but this time, they show this really clear picture of the influence of primers in the DNA synthetization. As such, there primers are “starting points”, the first origin of polymerization. As such, looking at the blue dotted arrows, they will always start from a primer. But here’s the catch: some arrows point to the left (these are starting from what we call “reverse primers”), and the arrows that point to the right (these start from the “forward primers”).
Let’s go over these primers:
The “forward” and “reverse” names come from a really simple explanation. I need to note here, however, the DNA replication direction, from the 5' strand to the 3' strand.
As such, let’s take this example that I annotated:
The forward primer will run from the left to the right, “the forward” direction in usual reading, and the reverse primer will be reversed, on the other side, from the right to the left. The forward primer attaches to the start codon of the template DNA (the anti-sense strand), while the reverse primer attaches to the stop codon of the complementary strand of DNA (the sense strand). The 5' ends of both primers bind to the 3' end of each DNA strand.
PCR COVID tests?
When I first heard of this method, I was so confused. Why is it used? How is it good?
Amplifying the viral RNA helps to make even small traces of the COVID-19 virus visible in the test sample. Even if you have a small trace of the virus in your system, the PCR test will detect it, without fail. However, this process takes quite some time…
The different temperatures
Small precision, the 3 steps in order to replicate the DNA are:
- denaturation, to break the DNA
- Annealing , adding the primers
- Extension, to polymerize the DNA with the Taq polymerase
All of these steps need to be done at specific temperatures in the lab, in order for the reaction to take place properly.
How did the Human Genome Project relate PCR to Gene Sequencing?
PCR:
- Is a technique used to duplicate specific DNA regions in vitro
- Is often used in the process of DNA sequencing
- Millions or even billions of copies of the target DNA region are produced during this process
- The objective is to produce sufficient quantity of DNA for the next process, which is sequencing
- The process uses dNTPs (Deoxynucleotide Triphosphates)
(wait… what are dNTPs?? - also called Deoxynucleotide Triphosphates or “building blocks” of nucleic acids
Structurally, they contain nitrogenous bases bound to a deoxyribose sugar and three phosphates. They contain a nitrogen base, bound to deoxyribose sugar, and three phosphate groups attached to its 5’ carbon.
During DNA replication, a 5’ phosphate group of one nucleotide forms a bond with the 3′ carbon on another nucleotide via dehydration synthesis. Moreover, the addition of the new nucleotide always takes place at the 3’ end, implying the occurrence of DNA synthesis from the 5’ to 3’ end.
There are four types of dNTPs based on the nitrogenous base it contains in its structure: dTTP, dCTP, dGTP, dATP)
DNA Sequencing:
- Is a technique used to determine the sequence of the bases in DNA
- The process enables researchers to visualize the correct order of the bases in a particular DNA fragment
- The objective is to determine the precise order of the nucleotides in a given DNA fragment
- The process uses ddNTPs (Dideoxynucleotide triphosphates) to terminate strand formation
—wait… and ddNTPs???
I will be going more in depth in another article about Genetic sequencing, stay tuned ;)
