How does Sanger Sequencing Work? – Seq It Out #1

Overview of Sanger Sequencing by capillary electrophoresis as the gold standard method, named after Dr. Frederick Sanger who invented it in the mid-1970s.

A short primer binds next to the region of interest and DNA polymerase extends it by adding complementary nucleotides from the template DNA strand in the presence of all four nucleotides.

Dideoxynucleotides with removed oxygen atoms act as chain terminators when incorporated by the polymerase, stopping DNA extension and allowing identification of the terminal base.

Each of the four chain-terminating nucleotides is labeled with a specific fluorescent dye color to identify which base terminated each DNA fragment.

Extension products are injected into a gel-filled glass capillary by electrical current, and an applied electrical field causes negatively charged DNA fragments to migrate toward the positive electrode, separating by size.

A laser excites the fluorescent dyes on DNA fragments as they pass a detection window, and emitted light is detected by a sensor and converted into base calls by software.

The complete sequencing reaction produces a pool of DNA fragments measured base-by-base, generating a colorful electropherogram and text file containing the DNA sequence.