Polymerase Chain Reaction

So to answer the first question, the “How does this work?, let’s talk about what we need first before we get to the steps of how it works . We need the DNA portion that we want to make copies of . We need some kind of buffer to put in it and then we need things that would be necessary to make more copies of the DNA.

Primers: 

We’ll need primers. Recall that primers help DNA polymerase , a building enzyme, know where to go to start its building. 

DNA Polymerase: 

We need DNA polymerase, the building enzyme. 

Fun fact: The DNA polymerase used is recurrently a heat-resistant variety of DNA polymerase as PCR uses heat. Typically, the polymerase chosen for the job is Taq polymerase, a type of heat resistant DNA polymerase. 

Taq polymerase: 

Taq polymerase is originally from a type of bacteria that can handle and live in really high temperatures in nature, in hot springs. We also need DNA nucleotides for the DNA polymerase to build with. 

So, we can look at the PCR sequence in three major steps:

We’ll illustrate with one double stranded DNA molecule here and let’s assume this is what we want replicated. 

Step #1 Denaturation: 

You’ve heard that word “denature” before,right? .One way we had chatter about denaturing enzymes was the use of heat. 

And heat is what we will use in this step. This step requires the addition of heat demanded to disparate the two strands of the DNA molecule. 

Step #2 Annealing: 

Ok, so this word means something a little different in biology- .basically this is when the two DNA strands that now have been disparted by that heat are going to be cooled and be fasten by the primers The temperature for this step should permit the primers to tie to the specific fragment of DNA that you want to boost, which means, make copies of. 

Step #3: DNA Synthesis: 

Remember, synthesis means to make something. With DNA coalescence, we’re going to construct more duplicates of DNA. And to do so, DNA polymerase will begin to work on both of these strands, and it will use the DNA nucleotides as its building material to amplify the DNA. 

Higher Temperature: 

We should record that the temperature at this step may be a compact warmer than the preceding step; it is necessary to be a temperature that is ideal for the specific DNA polymerase used.  

When one cycle is completed: 

Now after finishing one cycle of this, you have two double-stranded DNA molecules, right? Similar to how it would be in DNA replication within a cell. But you can repeat this now! Omitting this time you now have two double-stranded DNA molecules to began with. 

So you restate with the denaturation, annealing, and DNA synthesis steps. At the moment you have 4 double stranded DNA molecules. You ingeminate the steps again. Now you have 8. 

We mention DNA fingerprinting in gel electrophoresis and we mention that DNA fingerprinting can be a part of a crime scene inquiry. 

Well, in order to have enough copies of DNA samples to run in gel electrophoresis to analyze, PCR can be performed to make copies of the fragments of DNA that are found at a crime scene. 

rRT-PCR testing for SARS-CoV-2 (virus that causes COVID-19) :

For an especially relevant example, I can mention one of the testing types done for the virus that causes COVID-19. 

Introduction for COVID-19: 

COVID-19 is a bubonic plague that we’re encountring right now in 2020, and the virus that bring about COVID-19 is called SARS-CoV-2. 

Real-time reverse transcription: 

You might have perceived of one test type that utilize a sample from a nose or throat swab in a “PCR test.” But to be more definite, this test is for this virus is a real-time reverse transcription PCR (rRT-PCR) test. 

Reason: 

The reason it has this fancier “reverse transcription” in there is because this virus uses RNA as its genetic material instead of DNA and you have to use an enzyme called reverse transcriptase to transform the RNA into a DNA. 

Procedure: 

So, before we can do the regular PCR steps we’ve mentioned, we must convert isolated and purified RNA into DNA. 

Addition of Primer: 

A specific primer is added that will bind to an area of viral RNA and then reverse transcriptase is used to convert viral RNA into cDNA (complementary DNA). Using specific primers and the Taq DNA Polymerase, the cDNA can be copied over and over each cycle in the well known steps of what we’ve been mentioning of PCR. 

Taq Polymerase:

See the goal is you need enough copies of the viral cDNA in order for it to be detectable. The idea being if it’s a positive result, you have these selective primers binding and you have the Taq DNA polymerase assembling more and more duplicates of the viral cDNA in each cycle. 

Specific fluorescent probes for Identification: 

In addition, specific fluorescent probes are also used for identification. A certain level is needed for identification of a positive result. And if the virus’s genetic material is not available in the sample, then primers wouldn’t adhere and there would be no cDNA copies producing. 

Conclusion: 

Overall, PCR is such a useful and fascinating technology that will likely remain indispensable for future uses. And it’s a rare day when I pull out that word “indispensable.”