PCR analysis, what is it, and why is it popular?

PCR analiza blog

PCR analysis, what is it, and why is it popular?

Real time PCR analysis is a term that entered everyday life quite unexpectedly. What is the real time PCR method and why has it been talked about so much in the last couple of years?

Going by the logic behind the name, it should be real-time PCR, but then what is PCR?

PCR stands for polymerase chain reaction. It actually means successive duplication reactions, specifically in this case, duplication of the DNA sequence of interest.

In order to really understand the genius of the PCR method, you need to know a few things, which are already common knowledge.

All the information about the building blocks that make us who we are is written in our genes, more precisely in our DNA.

DNA, although it carries an incredible amount of information, is written with only 4 different nucleotides. Essentially everything that makes up our body and ensures its functioning is written with only 4 letters. Combinations of those nucleotides, or rather their sequence, is the basic element of a gene.

What does this specifically mean for us when explaining the term PCR and PCR analysis, well still nothing.

In order to connect this with replication, one should familiarize oneself with the very structure of the DNA molecule and how it is packaged and how it is naturally replicated.

Nucleotides, the letters with which the code is written, are arranged in a sequence and thus form a chain, which for stability is always packed in pairs, double-stranded. This is made possible by the unique property of nucleotides, they attract each other in precisely determined pairs. One nucleotide, a letter, will always be paired with a precisely determined second letter, and this will ensure not only the stability of the chains themselves, but also the information they carry.

This packaged DNA is stored in our cells. When it is used, more precisely when the information it carries is used, the double-stranded structure must be separated into individual strands. Purely due to easy physical access to the nucleotides of interest.

Now connecting this to our original topic and the concept of PCR analysis, this is exactly what happens during natural DNA replication. Chains must first develop, and then the nucleotides can be transcribed without interruption. In our cells, these processes will be carried out by enzymes, after which the chains will return to their double-stranded structure and everything will return to its original state except for the duplicated segment.

The PCR reaction reproduces exactly those processes, only under controlled conditions, and this is the basis of all PCR analyses.

An elevated temperature is used instead of an enzyme to separate the chains. While for the actual process of incorporating nucleotides into a new chain, rewriting the sequence, a thermostable enzyme, polymerase, is used.

Now let’s go back to the very beginning and the polymerase chain reaction sounds significantly more understandable, but we still cannot say that the concept of real time PCR analysis is any clearer.

So by simulating the natural sequence duplication we can multiply the DNA chain as many times as we want. But the DNA strand is written with over 3 billion nucleotides, we simply have to know what we want to multiply.

This is where the ingenious feature of nucleotides that they mutually attract other precisely determined, complementary nucleotides comes to the fore.

In replication, the first step is always the binding of the initial, complementary sequence to the strand to be transcribed. Perhaps it’s best to look at it as if someone has given you an entire book to rewrite, but they’ve written the headings for you, and it’s up to you to continue

In cells, this important role is performed by enzymes, and during the PCR reaction, you have the opportunity to choose the titles. By choosing the starting sequence of nucleotides, you also choose which part of the DNA chain will be copied.

Now we can already say that we have enough information to assume why this method is so popular. In addition to giving us the ability to amplify segments of DNA, we can also choose exactly which sequences we want to amplify and study later.

Polymerase chain reaction is a very important weapon of every molecular biologist. Any living organism to investigate, even some organisms that can’t really be called alive. By multiplying the desired part of the chain, we obtain numerous copies of the sequence we intend to investigate or identify. By properly selecting primers, copy titles, we can accurately distinguish genes, gene parts, gene variants, etc.

Only the element of real time is missing and we can say that we fully understand the name and function of real time PCR.

This is a great continuation of the story, since what exactly does a large number of copies of the desired sequence mean to us, certainly it will not be visible to the naked eye, or maybe it will.

The real-time component of the method name refers to the ability to observe changes in real-time, live.

This is accomplished by adding fluorescently labeled probes, short sequences of nucleotides, letters that use the same phenomenon of complementarity to bind to the target sequence and incorporate it into the new strand being synthesized.

Probes do not emit a signal unless they bind to the target sequence, which would mean that if they have nothing to bind to there will be no signal, and if they do there will be. Also, the more they bind, the stronger the signal.

Therefore, by multiplying the desired sequence and incorporating fluorescently labeled probes, which bind to even more specific nucleotide sequences within these already multiplied segments, we get information about the very composition of the chains, more precisely, we know which letters are in the positions that interest us.

Let’s look now, for the last time, at the very beginning of the text to see if we can now answer both questions in one sentence.

Real time PCR is a method by which we obtain detailed information about genes and DNA sequences in real time. Its distinctive features and simplicity have made it irreplaceable and undoubtedly one of the methods of the future.


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