Polymerase Chain Reaction – the PCR technique explained
A frequently used technique in the laboratory of, for example, hospitals, medical universities or factories where food or water is processed and examined, is PCR. This relatively young research method – developed in the early 1980s – has been revolutionary for detecting and discovering organisms such as bacteria and viruses. For tracing and examining genetic material in forensic research, the PCR technique is also crucial for finding a perpetrator.
Since the corona pandemic of 2019, there has been a lot of talk in news and related information programs about the PCR test for SARS-COV-2: the type of coronavirus responsible for the disease COVID. But what exactly is PCR and how does it work? The abbreviation PCR stands for Polymerase Chain Reaction. Polymerase is an enzyme, chain is the English word for ‘chain’ and the word reaction is easily translated to ‘reaction’.
So it is a chain reaction that is controlled by a certain enzyme, namely polymerase. To explain which chain reaction is involved, we have to go back to the basics and that is the carrier of all hereditary material: the DNA. Every cell of a living organism contains DNA (or a derivative thereof) and is made up of four different building blocks: adenine (A), thymine (T), guanine (G) and cytosis (C). Those building blocks can be linked to each other in an infinite number of combinations and those combinations ultimately provide the hereditary properties. On such a strand of DNA – which is made up of those building blocks – a lot of hereditary information will correspond to that of a congener. However, there are always pieces (we call that a gene) on the strand of DNA that are very specific. For example, one bacterium can survive in an acidic environment and another cannot. The acid-loving bacterium will produce substances that make it feel good and that the acid does not affect the cell wall, while another bacterium – which is a family member – cannot and will not survive in an acidic environment.
The PCR technique uses the detection of those pieces of DNA that are very specific. It’s like looking for a needle in a haystack with different types of pins, with which you can pull out that one special pin with a very strong magnet. The problem with DNA is that you cannot see one such piece of strand and that is why the chain reaction was developed. Millions of copies are made of that one piece of specific DNA and with a built-in trick, fluorescent molecules may or may not turn on. And the degree of fluorescence can be measured with certain equipment.
The Principle of the Polymerase Chain Reaction
The aim of the PCR reaction is to multiply the specific piece of genetic material and to make the result measurable using fluorescence.
This requires a number of parts that are put together in a reaction vessel:
- Isolated DNA: DNA released from cells present in, for example, water, blood or milk
- The building blocks: adenine, thymine, guanine and cytosis
- Primers: Fragments of DNA that stick to the beginning and the end of the piece of genetic material to be copied. The fluorescent flags are attached to these primers, which are ‘off’ when nothing is done with the reaction.
- Enzyme: The enzyme polymerase that can link the A, T, C and G building blocks to the primers in the correct order
- Buffer: This is a liquid in which all components are dissolved and which provides an optimal environment for the reaction to take place
The process of copying consists of a cycle of steps that is repeated several times. In general, repeating the cycle 40 to 45 times is sufficient to produce billions of copies. Do the math: if you start from one piece of DNA, you will have two copies after the first cycle, after the third cycle there will be four, then eight, sixteen, etc. The reaction vessel with all components are placed in a device that is very accurate and can heat up and cool down quickly. One cycle consists (generally) of:
- Denaturing DNA: DNA is made up of the building blocks A, T, C and G and these form a double strand of DNA. These connections have to be pulled apart, which can be done by raising the temperature to 90 to 96°C.
- Once the strands are separated, the primers can attach to the beginning and end of the piece of DNA to be detected. To do this, the temperature is reduced to 45 to 65°C.
- Now it is the turn of the enzyme polymerase to couple the A, T, C and G building blocks from the attached primers at a temperature of 72°C. The copy is being made!
After each cycle, the device records how much fluorescence is measured. If the primers can’t find the specific piece of DNA, nothing really happens during all that cycle. The fluorescent molecule attached to the primer remains ‘off’. When a reaction does take place, the enzyme polymerase ensures that that molecule is disconnected during copying. The molecule then starts to emit light, which can be measured at a specific wavelength by the PCR device. The rule of analysis is: the more fluorescence is measured, the more molecules were present at the start of the copying procedure. In the case of a qualitative determination – is the DNA to be examined present yes or no – the analyst reads the Ct (threshold) value. This value represents the cycle where the device starts detecting the fluorescence. A low Ct value therefore means that a lot of specific material was present in the isolated sample, but nothing can be said about exact amounts. There will also be situations where it is important to know not only whether a specific piece of DNA is present in the sample, but also how much. We call this quantitative PCR – also known as qPCR. For this, in addition to the sample to be examined, a series must also be used in which the concentrations are known and a calibration line is determined. On the basis of this calibration curve, it is possible to calculate what the initial concentration was present in the reaction vessel with isolated DNA.
A major advantage of the PCR technique is that in theory it only needs one piece of specific DNA to start the reaction and is therefore very sensitive. So you can detect just that one Legionella bacterium in ten liters of water that can possibly cause a disease. Or you can show that very difficult to culture tuberculosis-causing agent in the patient’s coughed up mucus. In some cases, the disadvantage is that you cannot prove whether it is a still living organism or that you are analyzing a DNA fragment of a virus or bacterium that can no longer cause a disease because it is dead. In general, no distinction is made in the response.This discussion has been frequently instigated in the media when demonstrating the coronavirus and has caused a lot of confusion and criticism regarding the use of this technique for detecting the virus in a nose and/or throat swab. If there is a high Ct value and little initial material was present from the – in this case – coronavirus, is there still a case of contagiousness? And is it also possible that you miss virus particles with the PCR or that the result is positive, even though no virus was present?is it still contagious? And is it also possible that you miss virus particles with the PCR or that the result is positive, even though no virus was present?is it still contagious? And is it also possible that you miss virus particles with the PCR or that the result is positive, even though no virus was present?
The chance that you will get a false positive result with a PCR test is quite small. The chance of a false negative test is higher. This is because there is a much greater chance that errors will be made during sample collection, transport or during laboratory operations. So it is not the PCR test that will necessarily give false negative results, but because of all the actions around it. If you have a PCR test and equipment to detect pathogens, most laboratories will opt for this technique.
Marije, Chief Laboratory Officer.