What‘s Behind the PCR Method?
Methodology and principles of the polymerase chain reaction PCR in simple terms
Three letters, and they’re on everyone’s lips: PCR. Until recently, they were known only to experts in medicine and molecular biology. Since the corona pandemic started, however, almost every child in kindergarten has heard of PCR, short for the polymerase chain reaction. It has become a key component of coping with SARS-CoV-2, the novel coronavirus, along with spacing and hygiene rules, rapid antigen testing and vaccination. Although it is common knowledge that a PCR test can detect an infection with the virus reliably, its principle is still far from clear for most people: an ingeniously simple method of duplicating DNA, the carrier of genetic information in all living organisms, over and over again. The smallest traces of DNA are enough for the test to succeed. Ranging from medicine and diagnostics to food analytics and forensics, the application areas of the PCR method are extremely diverse and go far beyond detecting the coronavirus.
How exactly does the PCR method work? On which principles of molecular biology does it base and which method variants are there? We’ll explain to you one of the most important techniques in molecular biology.
Contents
- The basics: what’s PCR?
- The PCR method in detail: materials, technical equipment and procedure
- Further developments: variants of the PCR method
1.The basics: what’s PCR?
For developing the polymerase chain reaction (PCR), the American biochemist Kary Bank Mullis, along with the Canadian chemist Michael Smith, was awarded the Nobel Prize in Chemistry in 1993, just seven years after his groundbreaking work. This was because the enormous potential of the PCR method had quickly become clear: with just a few materials and an instrument to heat samples, known as a thermocycler, short fragments of the genetic material DNA, even if available in only minute amounts, can be amplified exponentially.
The term polymerase chain reaction refers to the enzyme DNA polymerase used in the method and the fact that a duplication step is repeated many times over with the previously amplified material.
2. The PCR method in detail: materials, technical equipment and procedure
The starting materials
A few materials and functional equipment – that's all it takes to amplify DNA, the key information molecule of life.
The DNA-containing sample does not have to be purified for the PCR method. Amplification is carried out with the DNA fragment of interest in a buffer solution as this keeps the pH value stable over the entire reaction process. A cocktail of nucleotides, the individual building blocks of DNA, is now added to the PCR reaction vessel to create the copies of the original DNA strand. Enzymes that catalyze the amplification, the DNA polymerases, are also required. Heat-stable DNA polymerases, obtained from thermophilic bacteria, are used because they are particularly active at higher temperatures. To enhance the polymerase’s activity, magnesium ions are added. The PCR sample also contains two different primers, short artificially synthesized nucleotide sequences, one specific for each single strand of DNA. The primers allow the polymerase to start replicating.
The reaction steps of the PCR method
Cleavage, annealing, duplication – it takes three steps, repeated multiple times, to amplify the DNA. The polymerase chain reaction is carried out in a so-called thermocycler. This instrument holds a certain number of reaction vessels and independently sets the temperatures required for each of the three steps, repeating them many times over. DNA amplification can thus proceed practically without manual intervention.
To help you choose the right instrument from among the many available on the market today, you can find out about all the relevant manufacturers and their products in the world's largest market overview of thermocyclers.
Step 1: Denaturation: the DNA strands are split
For only a short time of about 20 to 30 seconds the reaction vessels are heated to about 95 °C. At this temperature, the hydrogen bonds holding the DNA double strand together break down, separating it into two single strands.
Step 2: Annealing: hybridization
The thermocycler now cools the mixture. This must happen quickly to prevent the two single strands from re-annealing. Instead, the primers are made to anneal – depending on the primer, specific temperatures somewhere between 50 and 60 °C are needed for this step.
Step 3: Amplification: DNA synthesis (elongation)
The DNA polymerase now uses the nucleotides in the buffer to elongate the DNA strand by attaching complementary nucleotides to the small double-stranded sections that were created by coupling the primers to the single strand. This last step is performed at a temperature of approximately 70 °C.
The sample subsequently undergoes these three steps for between 20 and 50 cycles. Within a very short time the polymerase chain reaction thus produces billions of copies of the original DNA by exponential amplification.
3. Further developments: variants of the PCR method
Since the introduction of the PCR method in the mid-1980s, the technique has developed rapidly, opening the door for a multitude of new applications. Of the many interesting methods available, only a few are mentioned here.
Quantitative real-time PCR (qPCR):
If the objective is to determine the quantity of the amplified DNA, this can be done by quantitative real-time PCR, also known as quantitative PCR (qPCR). The technique is fast and can be carried out fully automatically, with amplification, detection and quantification taking place in a single step. By mixing in an inactive fluorescent dye that is activated by DNA production, fluorescence can be measured at each cycle in real time, allowing conclusions to be drawn about the amount of contained DNA.
Reverse-transcription PCR (RT-PCR)
Not only DNA can be traced by PCR, but RNA as well. This is done indirectly with the help of the enzyme reverse transcriptase because thermostable DNA polymerases do not amplify RNA. In the first step, the reverse transcriptase converts the RNA into cDNA (complementary DNA). This cDNA can then be used to carry out the PCR method in a second step.
RT-PCR is used in research and diagnostics, for example to detect RNA (expression of specific genes in cells; genome of RNA viruses).
Error prone PCR
From a researcher’s point of view, error prone PCR is particularly exciting. During amplification of DNA by DNA polymerases, nucleotides can be incorrectly inserted, resulting in sequence differences from the original DNA. The misincorporation rate increases with the degree of deviation from the optimal reaction conditions. For in vitro evolution experiments, this allows nucleic acids with specific properties to be generated in the lab by directed selection, amplification and mutation.
Conclusion
The PCR method is widely used in laboratory and food analytics, research and diagnostics to amplify DNA, the carrier of the genetic information of all living organisms, needing only tiny traces of DNA. There’s more information about the many different applications of the PCR method and exciting other insights in the PCR Topical Worlds. If you’re looking for the right equipment for your lab, visit the world's largest market overview of thermocyclers.
Glossary of basic terms
DNA
DNA (deoxyribonucleic acid) is a nucleic acid composed of different deoxyribonucleotides, its basic building blocks. DNA consists of two complementary single strands linked to form a double helix.
Nucleotides
Nucleotides are the building blocks that form the nucleic acids DNA and RNA (ribonucleic acid). Each consists of a phosphate residue, the sugar deoxyribose and one of five nucleic bases. In DNA, these are adenine, thymine, guanine and cytosine (abbreviated A, T, G and C). In ribonucleic acid (RNA), thymine is replaced by uracil (U).
Primer
Primers are short, artificially synthesized nucleotides.
(DNA) Polymerase
Polymerases are enzymes that catalyze the polymerization of nucleotides, the basic building blocks of nucleic acids. They link the nucleotides in a specific sequence to form DNA or RNA copies. In nature, polymerases replicate DNA, the genetic information, during cell division. Polymerases are also important for the production of proteins within cells.
There are different types of polymerases and they are found in all living organisms.
