## Sequencing determines the order of nucleotides in DNA.

Knowledge of the exact sequence, i.e., the order of the DNA nucleotides, is not only important from a research perspective, but also forms a very important part of the diagnosis of many diseases or disease predispositions, is used in criminalistics, and is an important aspect of evolutionary studies to determine the relationship of organisms. The very first molecule sequenced was the tRNA for alanine (this was back in 1965), and a few years later (1977) the two most famous sequencing methods were published. The first, named after its authors Allan Maxam and Walter Gilbert, was the Maxam-Gilbert method. It is also called "chemical sequencing" because various chemical compounds were added to the reaction that changed the nitrogen bases, and based on this change, the analysed DNA segments were then cleaved. Since it is more complicated and has several shortcomings compared to the second successful sequencing method, it is no longer widely used. The second method, which is still widely used today, is the Sanger method, which in turn is named after its author Frederick Sanger. For the discovery of the sequencing methods, both authors - Gilbert and Sanger - received the Nobel Prize in Chemistry in 1980.

The key component of the Sanger sequencing reaction is special variant of nucleotides – dideoxynucleotide triphosphates (ddNTPs). These differ from ordinary nucleotides in that they do not have a free hydroxyl group (-OH) on the 3'-carbon of deoxyribose, so DNA polymerase cannot attach additional nucleotides to them (see chapter 3 for an explanation). If such a special nucleotide is added to the DNA chain, the DNA polymerase gets stuck at this position and the synthesis is terminated. This is why they are also called dideoxyterminators. Within the sequencing reaction, there are the following main components: DNA template (the DNA segment we want to sequence), dideoxynucleotide triphosphates (each of the four, A,T,C, G, labelled with a different fluorescent dye), primer, DNA polymerase and standard nucleotide triphosphates. After the primer binds to the DNA template, synthesis begins, and if we have a suitable ratio of dideoxynucleotide triphosphates to deoxynucleotide triphosphates, a dideoxyterminator is occasionally incorporated at each site, depending on complementarity with the template. When this happens, synthesis is stopped at that DNA molecule. In the end, a mixture of synthesis products of different lengths is obtained, depending on when termination occurred. Since the last nucleotide is a dideoxynucleotide, each product is also fluorescently labelled. This is followed by electrophoresis in a special device (called a sequencer), in which the individual fragments are divided according to their length. The shorter the fragment, the faster it moves. The device is able to split DNA molecules that differ in length by even a single nucleotide. A special detector then reads a fluorescent signal when these fragments pass a certain point. The result is called electrophoretogram, from which we can determine the sequence of nucleotides in the analysed DNA. The entire analysis is performed with the aid of software (Figure 4.5).

Sanger sequencing can sequence about 1,000 nucleotides per reaction, which is why second-generation sequencing methods - NGS (Next Generation Sequencing) - are already being used to sequence entire genomes, but often also in diagnostics. These technologies came onto the market between 2005 and 2007, the best known of which include Illumina, GS FLX system, Ion Torrent or SOLiD. Their common feature is that they are based on the principle of initial amplification (synthesis) and consequently differ in approaches such as "reading" the DNA sequence. This type of sequencing is much more efficient than the Sanger method because NGS uses micro-nanotechnologies, minimizes the amount of input sample, and allows parallel sequencing of a large number of DNA sequences at once.