Sanger sequencing is a method of DNA sequencing that relies on the selective incorporation of fluorescently labelled chain-terminating dideoxynucleotides in an in vitro DNA replication system.
The DNA sample containing the sequence to be determined is denatured in the presence of a primer, a mixture of normal deoxynucleosidetriphosphates (dNTPs) and fluorescently labelled dideoxynucleosidetriphosphates (ddNTPs), and DNA polymerase. This reaction mix is subjected to several cycles of amplification, during which the primer, annealed to the single stranded DNA, is elongated by the addition of dNTPS by DNA polymerase. The ddNTPs, which lack the 3’-OH group required for the formation of a phosphodiester bond between two nucleotides, cause DNA polymerase to stop extension of DNA every time a ddNTP is incorporated. The end result is a mixture of DNA fragments of different lengths that all contain a fluorescently labelled ddNTP at their 3’-end. Each of the four ddNTPS (A, G, C, T) has a different fluorochrome attached to it.
Upon removal of the unincorporated dNTPS and ddNTPS, excess primer and template, the sequencing reaction products are subjected to capillary electrophoresis, during which the DNA fragments are separated by size and their fluorescent tag identified by a laser detector. A computer software then determines the original DNA sequence based on the length of each DNA fragment and the type of fluorochrome attached to its 3′-end.
For an animation of the Sanger sequencing method visit Sanger Sequencing Animation – Life Technologies (Applied Biosystems)