How does dideoxy nucleotide (ddNTP) work in Sanger DNA sequencing?

The classical chain-termination method of Sanger sequencing involves using a DNA primer, a single-stranded DNA template, DNA polymerase, normal deoxynucleotide triphosphates (dNTPs), and modified nucleotides (ddNTPs). The chain-terminating nucleotides lack the 3′-OH group needed for the formation of a phosphodiester bond, leading to the termination of DNA strand elongation when a ddNTP is added. The DNA is then split into four separate reactions with all 4 of dNTPs and the DNA polymerase. In each reaction one of the four ddNTPs are incorporated. After template DNA extension, the subsequent DNA segments are heat denatured and separated by size using gel electrophoresis. The process often involves using a denaturing polyacrylamide-urea gel, with each of the four reactions separated into distinct lanes labeled A, T, C, and G. The DNA sequence is determined based on the visualized bands. It can be directly observed from the gel or X-ray image.