The sequencing technique

The sequencing method of Sanger, the DNA that is to be sequenced, called the template, is hybridised with a short, complementary strand of 20 to 25 nucleotides, called the primer. DNA polymerase and deoxynucleotides are added to the mixture. The enzyme catalyses the synthesis of the complementary DNA strand, starting at the primer and extending in the 3' direction. Sanger incorporated a chain terminating nucleotide into the reaction mixture. This reagent is a modified deoxynucleotide triphosphate, called a dideoxynucleotide (ddNTP). When the sequencing reaction proceeds in the presence of a small amount of the dideoxynucleotide, the resulting chain-extension reaction products will consist of a set of molecules. All starting at the primer, all extending in the 3' direction, but terminating wherever the dideoxynucleotide happened to be incorporated. This set of reaction products is called a sequencing ladder. Four such ladders were synthesised by use of the four ddNTPs in separate chain extension reactions. Using gel electroforesis to seperate the different molecules on their size, you can read de DNA code.

In 1986, scientists discovered a means of detecting the ddNTPs with fluorescent tags, which required only a single test tube instead of four. This gives the results in peaks, visible in this pictures. Each colour indicates the existance of one of the four nucleotides. There is more about fluorescence sequencers in the techniques page.