The process begins with the denaturation of double-stranded DNA to obtain single strands. A primer is then annealed to the template strand, and the DNA polymerase enzyme initiates the synthesis of a new DNA strand by adding nucleotides. The key feature of Sanger sequencing is the inclusion of dideoxynucleotide triphosphates (ddNTPs) in the reaction mixture. These ddNTPs lack a 3' hydroxyl group, causing termination of DNA strand elongation when incorporated. By labeling these ddNTPs with fluorescent dyes, the resulting DNA fragments can be separated by capillary electrophoresis and detected based on their fluorescence.
