Working principle of protein sequencer

1977 Two rapid DNA sequencing methods were developed, one was the enzymatic method proposed by Sanger et al. (dideoxy chain termination method), and the other was the chemical degradation method proposed by maxam and Gilbert.

The basic principle of chemical degradation method includes three main chemical steps: the coupling of phenyl isothiocyanate with protein and the N- terminal residue of polypeptide, the cyclization and cleavage of phenylthiocarbamate (PTC- peptide), and the conversion of thiazolidinone phenylethylamine (ATZ) into phenylisothiourea amino acid (PTH- amino acid). In each cycle, one amino acid residue is cleaved from protein and polypeptide, and a new free amino acid is exposed for the next ed.

Sanger dideoxy chain termination method is characterized by introducing dideoxynucleoside triphosphate (2ˊ, 3ˊ-ddNTP) as a chain terminator. The difference between 2ˊ, 3ˊ-ddNTP and ordinary dNTP is that the 3ˊ position of deoxyribose is missing a hydroxyl group. Under the action of DNA polymerase, it can form phosphodiester bond with 3-hydroxyl group of polynucleotide chain, but it can't condense with the next nucleotide, which leads to the termination of polynucleotide chain extension. If a small amount of DdNTP is added in addition to the four normal DNTP in the DNA synthesis reaction, the extension of the polynucleotide chain will compete with the accidental but very specific chain, so the reaction products are a series of nucleotide chains with different lengths. In four groups of independent DNA synthesis reactions, four different ddNTP are added respectively, resulting in four groups of nucleotide chains, which terminate at various positions of A, G, C and T respectively. The sequence can be read by polyacrylamide gel electrophoresis of these four groups of nucleotide chains.