## In the polymerase chain reaction (PCR), a limited section of DNA is amplified

When analysing DNA, we are often not interested in the sequence of the entire molecule, but only in a specific part of it, for example, a gene under investigation (for an explanation of the term gene, see Chapter 2 - Getting to Know DNA). Within the DNA molecule, this section often occurs in only one copy, so we would need a huge amount of input DNA to obtain the desired section in sufficient quantity for various experiments. This problem was solved by a revolutionary method that is now an almost everyday part of DNA work - the polymerase chain reaction, or PCR for short. Behind its invention is the American scientist Kary Mullis, who began working on this method in 1983, but it was not published until 1985, when he and his colleagues succeeded in amplifying the human gene for beta-globin (the protein subunit of haemoglobin A). He was awarded the Nobel Prize in Chemistry in 1993 for the discovery of PCR.

As input components of the reaction, we need the template DNA to be amplified, free deoxynucleotide triphosphates (dNTPs), primers (short, single-stranded DNA segments, usually 20 nucleotides long that are used to start the synthesis), DNA polymerase, suitable buffer solution, and water. In the first step of the reaction, the DNA strands are separated from each other (denatured) by high temperatures (94-98 °C). In the second step, after lowering the temperature (50-60 °C), the primers are bound to the single strands of the template DNA according to the principle of base complementarity. The binding of primers is called annealing. After increasing the temperature to 68-72 °C, the third step is carried out by the action of DNA polymerase, the synthesis of a new DNA strand – polymerization, or extension. All these steps are repeated 20-40 times, resulting in amplification of desired DNA segment (Figure 4.1).

The optimal amount of DNA added to start the PCR is 1-50 ng. However, the PCR method is very sensitive, and it is possible to begin the reaction with an amount of about 15 pg (15 pg of DNA can be obtained from about 2 cells). Since the PCR method is basically an exponential reaction, if the input amount is one double-stranded DNA molecule, we can obtain 2 molecules after the first cycle, 4 after the next, 8 after the third, or 2nDNA molecules after the nth cycle under ideal conditions of reaction.

To perform PCR, we use a device called a thermocycler because it cyclically repeats the selected temperature program. The first prototype of such an automatic device was built in 1986 and became possible only when a thermostable DNA polymerase isolated from the high-temperature organism Thermus aquaticus was used for PCR purposes. The first automatic PCR cycler was launched in 1988, and in 1989 the thermostable Taq DNA polymerase was named Molecule of the Year by the journal Science.