Sequencing is a method for determining the order of nucleotides in DNA or RNA.Sequencing is the determination of the nucleotide sequence of DNA or RNA. Modern methods include Sanger sequencing (with dideoxynucleotides and capillary electrophoresis) and high-throughput technologies (NGS), such as Illumina (synthesis with reversible terminators), pyrosequencing, and nanopore sequencing. The principle is based on base complementarity and detection of a signal from the incorporation of each nucleotide into the growing chain.
In 1977, Sanger proposed an enzymatic method for DNA sequencing using dideoxy analogs; simultaneously, Maxam and Gilbert developed a chemical method. In the 1990s, automation with capillary electrophoresis accelerated the process. The breakthrough was the advent of NGS in the 2000s (454, Solexa/Illumina), which allowed parallel sequencing of millions of fragments. The latest technologies (PacBio, Nanopore) read single molecules in real time.
General scheme: DNA is fragmented, adapters are ligated to the ends. In Illumina technology, fragments hybridize on a flow cell and are amplified by bridge PCR, forming clusters. Then sequencing-by-synthesis occurs: reversible terminators with fluorophores are added, after each cycle an image is taken, the terminator is removed, and the cycle repeats. Image analysis converts signal intensity into a nucleotide sequence. In nanopore sequencing (Oxford Nanopore), a DNA strand is passed through a protein pore and the change in ionic current, specific for each nucleotide, is measured.
Medical diagnostics of hereditary diseases, creation of personalized medicine, tracking virus evolution, forensics.
The first complete sequencing of the human genome took 13 years and cost nearly $3 billion; now it is done in a day and for less than $1000.
















