DNA (Sanger) sequencing using dideoxy chain termination.
- A primer is annealed to the target template DNA.
- A mixture with DNA polymerase, the four dNTPs, and one
dideoxynucleotide
(such as ddATP), is added.
- During primer extension,
the polymerase occasionally inserts a ddNTP instead of a dNTP,
which lacks the 3'-OH needed to attach the next nucleotide, terminating the chain.
- Gel electrophoresis separates the synthesized fragments
that terminate at the dideoxynucleotide.
