A thermocycler is a laboratory equipment that allows to carry out the polymerase chain reaction (PCR) efficiently and quickly, by automatic and cyclical realization of the changes in temperature required for the amplification of a chain of deoxyribonucleic acid (DNA), from a thermostable enzyme. PCR enables a small amount of DNA molecules to be amplified many times, exponentially.
The protocol of this technique is based on the synthesis of a fragment of DNA using a polymerase enzyme that can work at very high temperatures, since it comes from the bacterium Thermus aquaticus that lives at high temperatures (79°C to 85°C), known as taq polymerase.
When we do a PCR reaction we simulate what happens inside the cell when the DNA is synthesized and in the tube all the ingredients necessary to do it are mixed: the polymerase, the DNA of the organism we want to study, the oligonucleotides (primers, primers, primers) necessary for transcription to start, dinucleotides (dNTPs), and the conditions for the enzyme to work properly (certain pH, certain amounts of magnesium in the form of MgCl2, KCl, and may need other salts or reagents, depending on each polymerase). This technique has many different applications and has become a very important tool in molecular biology; its applications range from population genetics, molecular evolution and genomics to forensic medicine.
How does the PCR technique work?
If we already have the tubes with everything we need to synthesize the fragment we are interested in (taq polymerase, dinucleotides, DNA, water, buffer with magnesium and other salts, and oligonucleotides). The next step is to place the tubes in the thermo-cycler, which basically serves to heat or cool them to very precise temperatures.
How does that amplify the DNA fragment we want?
The thermocycler heats or cools the tubes to three different temperatures, which are repeated over and over (known as reaction cycles), the first is at 95°C (denaturation) during which the DNA double chains are opened or denatured, remaining in the form of simple chains; then the thermocycler adjusts the temperature in a range between 40 °C and 60 °C (alignment), at this temperature the hydrogen bridges between the oligonucleotides and the DNA are constantly formed and broken, and those more stable bonds (which are complementary) will last longer, leaving the oligonucleotides “aligned” forming a small region of double chain. The polymerase binds to this little piece of double-stranded DNA.)
The polymerase binds to this small piece of double-stranded DNA and begins to copy in a 5’ to 3’ direction; by adding a few more bases, the hydrogen bridges that form between the bases further stabilize the binding and the oligonucleotide remains at this site for the next step. The temperature then rises to 72 °C (a step known as extension), as 72 °C is the temperature at which the polymerase reaches its maximum activity, and DNA fragments are synthesized from the oligonucleotides that have already aligned.
In the first cycle, at these three temperatures, the first fragments will form from the genomic DNA. Then the three temperatures are repeated once again, but in this second cycle, the oligonucleotides, in addition to binding to the DNA we put in at the beginning, will also bind to the newly synthesized fragments of the first cycle, so in this second step the polymerase will synthesize two long fragments copied directly from the DNA and two fragments of the expected size, which is the size that is between the two oligonucleotides we have used. This way, with each cycle, the number of fragments of the size we want will increase.
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