Sanger sequencing works by synthesizing complementary DNA strands using a template strand. The process involves four main steps:
1. DNA Denaturation: The double-stranded DNA is heated to separate it into two single strands. 2. Primer Annealing: A short primer binds to the single-stranded DNA template. 3. Extension and Chain Termination: DNA polymerase extends the primer by adding nucleotides. The reaction mixture contains normal deoxynucleotides (dNTPs) and a small proportion of fluorescently labeled dideoxynucleotides (ddNTPs). When a ddNTP is incorporated, it terminates the chain. 4. Separation and Detection: The resulting DNA fragments of varying lengths are separated by capillary electrophoresis and detected based on their fluorescent labels.
