Single-generation sequencing

First-generation sequencing, also known as Sanger sequencing, was invented by Frederick Sanger in 1977. It is the earliest gene-sequencing technology and is regarded as the gold standard for genetic testing. The principle behind Sanger sequencing lies in incorporating dideoxynucleotides (ddNTPs) into the newly synthesized DNA strand. Since these ddNTPs lack a 3'-hydroxyl group, they cannot form phosphodiester bonds with the next nucleotide, thereby terminating the DNA synthesis reaction. Sanger sequencing consists of a set of four separate reactions; each reaction system contains the four deoxynucleoside triphosphates (dNTPs), which can normally support DNA synthesis. To each reaction system, one of four different dideoxynucleoside triphosphates (ddNTPs) is added. These ddNTPs randomly substitute for their corresponding dNTPs. Because ddNTPs lack the essential 3'-OH group required for chain extension, further elongation of the growing oligonucleotide is prevented, causing the synthesis to terminate specifically at G, A, T, or C residues. The identity of the terminating nucleotide is determined by the type of ddNTP incorporated. To facilitate identification, these ddNTPs are typically labeled with fluorescent dyes or isotopes. By means of gel electrophoresis, fragments of varying lengths can be separated, and the terminal bases of these fragments can then be analyzed to determine the exact base sequence of the target DNA segment.

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