Sanger method

Detecting the DNA

The products are detected using fluorescent dyes. Most commonly, each dideoxy base is given a different 'coloured' tag: we can imagine that A is green, C is purple, T is red and G is orange. Each time a chain-terminating dideoxy base is added, a coloured tag is added to the DNA molecule.

At the end of the reaction, there is a set of DNA copies, each copied molecule with a coloured tag corresponding to the last base added. Now the molecules must be ordered to read the DNA sequence.

The sequence is read by separating the DNA copies by size. In modern DNA sequencing, the DNA sample is applied to one end of a capillary tube filled with a viscous gel. An electric field is then applied across the tube. DNA is negatively charged (it has lots of phosphate groups) and will move towards an anode - the positively charged terminal. Separating molecules in this way is called electrophoresis.

The DNA molecules move through the liquid according to their size: the largest DNA molecules get 'caught up' in the molecules of the gel and move relatively slowly; the smallest molecules are hampered less and move more quickly. The DNA copies emerge from the end of the capillary tube smallest molecules first.

Reading the sequence is done by illuminating the DNA, just before it emerges, with a laser to detect the 'coloured' tag on the dideoxy base at the end of the DNA copy. The colour of the emitted fluorecence is read by the detector and a base is assigned. The result is stored and assessed by software designed to test how reliable the base assignment is.