How does Sanger sequencing work?

Sanger sequencing is used to determine the order of nucleotides in a DNA fragment of interest.

A piece of DNA that is to be sequenced is extracted and heated. The heat causes the DNA double helix to unwind into two single strands.

The temperature is then lowered and a DNA primer, which is a short sequence of a single-stranded DNA, is added. The DNA primer binds to one end of the DNA strand and marks where the sequencing will start.

In the next step, the temperature is raised slightly and the enzyme DNA polymerase is added along with free nucleotides that contain one of the four bases – C, T, A, or G. DNA polymerase begins at the primer sequence and adds one nucleotide at a time to build a complementary DNA strand.

Chemically-altered, chain-terminating versions of each of the nucleotides are labeled with different colored dye and added to the mix. This initiates four different sequencing reactions – one for each of the four nucleotides. The different colored dyes allow researchers to identify them when exposed to UV light.

The DNA polymerase stops the DNA sequence when it reaches a chain-terminating nucleotide. Because the DNA polymerase adds the modified nucleotides randomly, DNA sequences of varying lengths are generated.

These DNA segments of different lengths undergo gel electrophoresis, a technique that uses a base gel to separate the DNA fragments by size. On applying an electric current, the smallest pieces move the fastest and travel the most distance through the gel while the largest pieces hardly move any distance at all. The gel is then placed under an UV light to visualize the dark bands in each column. There is one column for each type of nucleotide - adenine, cytosine, guanine, and thymine. The sequence of nucleotides is determined by reading the sequence of the bands.