How does DNA sequencing work?

A biochemical method that is used to determine the order of the DNA oligonucleotide bases, i.e., adenine, guanine, cytosine, and thymine. This finds the hereditary genetic information in the cell nucleus, plasmids, mitochondria, or chloroplasts, which supplies the basic program for the functioning of all living organisms.

Let’s talk a little bit about the DNA first. DSN was discovered in 1869, but its role in genetic inheritance was not demonstrated until 1943. Later, in 1953, with the help of biophysicists James Wat-son and Francis Crick, Rosalind Franklin and Mau-rice Wilkins found that the structure of DNA is a double helix polymer, a helix made up of two strands of DNA wrapped around each other. This breakthrough has led to advances in scientists' understanding of replication and the hereditary regulation of cellular activity. Each strand of the DNA molecule consists of a long chain of monomeric nucleotides. The nucleotides in DNA consist of a deoxyribose sugar molecule with a phosphate group and one of four nitrogenous bases attached, two purines, which are adenine and guanine, and two pyrimidines, which are cytosine and thymine. They are linked together by co-valent bonds, such that between the phosphate of one nucleotide and the sugar of the next nucleotide, they form a phosphate-sugar backbone from which the nitrogenous bases protrude. One strand is held to the other by hydrogen bonds between the bases; these bonds are specific, so adenine can only bind to thymine, while cytosine can only bind to guanine. The configuration of the DNA molecule is extremely stable, so it allows to it serve as a template for replicating new DNA molecules, such as the production of the associated RNA molecule. A gene is the segment of DNA that codes for the synthesis of a particular protein in the cell.

The first DNA sequencing techniques appeared as early as the 1970s, the so-called first-generation sequencing techniques. These included the Maxam-Gilbert method, discovered, and named after two American molecular biologists, Allan M. Maxam and Walter Gilbert. The second method, discovered by the English biochemist Frederick Sanger, was the Sanger method, also known as the dideoxy method. Of the two methods, the Sanger method became the more widely used because it allowed the rapid and exact sequencing of long DNA sequences. For this he was awarded his second Nobel Prize in Chemistry in 1980, which he shared with Walter Gilbert and Paul Berg. Eventually, the dideoxy method was used to sequence the entire human genome. The significance is that the DNA chains were synthesized on a template strand, but the growth of the strand was stopped when one of the four possible dideocxy nucleotides, lacking the 3’ hydroxyl group, was incorporated, preventing the addition of another nucleotide. This created population of nested, truncated DNA molecules, which represented each site of the nucleotide in the template DNA. The molecules were separated by size, a process known as electrophoresis, and the nucleotide sequence deduced was deduced using a computer. This method was later carried out using automated sequencing machines, in which the truncated DNA molecules were labelled with fluorescent tags, separated by size in thin glass pellets and detected by laser excitation

Next-generation sequencing technologies have already largely superseded first-generation sequencing technologies. These newer approaches now allow the simultaneous sequencing of many DNA fragments, sometimes millions of fragments. They are much more cost-effective and faster. The usefulness of new technologies has been enhanced by advanced in bioinformatics, which have enabled larger data storage made it easier to analyze and manage very large data sets, often in the gigabases.

Knowing the sequence of DNA segments has many uses. First, it can be used to identify the DNA segments that code for genes or phenotypes. When a particular DNA region is sequenced, it can be used to look for the characteristic features of genes. Human genes are located adjacent to so-called CpG islands, clusters of the two nucleotides that make up DNA, cytosine, and guanine. If a gene with a known phenotype, such as a human disease gene, is known to be located in a region of a sequenced chromosome, then genes not associated with that region will be candidates for that function. Second, homologous DNA sequences of different organisms can be compared to draw evolutionary relationships within and between species. Third, a gene sequence can be screened for functional regions. Namely, to determine the function of a gene, different regions can be identified that are common to proteins with similar functions. To give an example, certain amino acid sequences within a gene are always found in proteins spanning the cell membrane. These are called transmembrane domains. When transmembrane domains occur in a gene with this function, it indicates that the encoded protein is in the cell membrane. Other domains characterize DNA-binding proteins. There are many public DNA sequence databases available for analysis.

Information researched by Dezső Sándor