The Complete Guide to Sanger Sequencing: Understanding the Chain Termination Method for Accurate DNA Sequencing

Most common used technique for sequencing DNA

Sanger sequencing

The most commonly used technique for sequencing DNA is the Sanger sequencing method, also known as the chain termination method. This method is based on the use of dideoxynucleotides (ddNTPs), which are modified versions of normal nucleotides that lack the 3’ hydroxyl group. When a ddNTP is incorporated into a growing DNA chain, it terminates further chain elongation because it lacks the 3’-OH group that is required for formation of a phosphodiester bond with the next incoming nucleotide.

In Sanger sequencing, a DNA template is first denatured into single strands, which are primed with a DNA oligonucleotide primer complementary to a specific region of the template. Four separate reactions are then set up, each containing a mixture of the four normal deoxynucleotides (dNTPs) and one particular ddNTP (ddATP, ddCTP, ddGTP, or ddTTP). DNA polymerase then extends the primer along the template strand by adding nucleotides onto the 3′-OH group of the primer. However, when a ddNTP is incorporated into the extending chain, it terminates further extension. As a result, Sanger sequencing generates a series of terminated fragments, each of different lengths, each labelled with a different ddNTP and starting from the same primer.

The labelled fragments are then separated by electrophoresis in a polyacrylamide gel that is denaturing, such that ssDNA can be separated based on molecular weight, even if containing similar sequences. A laser excites the fluorophore attached to each ddNTP, which causes it to emit light of a particular wavelength, allowing the sequence of each fragment to be recorded relative to the original primer. The final outcome is a “ladder” of fragments that represent the DNA sequence of the original template.

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