It was put forward by Sanger et al. in 1977, mainly using dideoxynucleotide ddNTP skillfully.

Normal NTP has a hydroxyl group at the 3' position, which is used to form a phosphodiester bond with the next nucleotide, while ddNTP loses hydroxyl groups at the 2' and 3' positions. When DDNTP was added, the chain reaction was terminated.

So when sequencing, the reaction was divided into four groups, and dNTP, primers, templates and DNA polymerase were added. Four kinds of ddNTP labeled with radioisotopes were added to four groups in turn for chain extension. During the reaction, when dNTP is added, it can be carried out normally, but once ddNTP is added, the chain reaction ends, and finally each group produces DNA fragments with different lengths. These DNA chains are separated by denatured acrylamide gel according to molecular weight, and the sequence of DNA can be read directly on the map by autoradiography.

2. Chemical degradation method

In the same year, Gilbert and others proposed chemical degradation. Firstly, the end of DNA is labeled with radioisotopes, and DNA chains are degraded with specific chemical reagents to produce several groups of DNA chains with different lengths. Similarly, the sequence length of DNA to be detected is also directly read by autoradiography after separation by polyacrylamide gel.

After understanding the principle, we can find that Sanger sequencing is in the process of DNA synthesis, while chemical degradation is to sequence the synthesized DNA, which can detect the modification of DNA. The reagents are relatively simple and the requirements for experimental conditions are not high, so chemical degradation method is widely used. Although Sanger sequencing needs primers, DNA polymerase and so on. High-throughput sequencing is based on Sanger sequencing principles, such as Roche 454 and ABI solid-state equivalence technology.