How is DNA sequencing done?
Chromosomes, which range in size from 50 million to 250 million bases, must first be broken into much shorter pieces (subcloning step) Each short piece is used as a template to generate a set of fragments that differ in length from each other by a single base that will be identified in a later step (template preparation and sequencing reaction steps) The fragments in a set are separated by gel electrophoresis (separation step) New fluorescent dyes allow separation of all four fragments in a single lane on the gel. The final base at the end of each fragment is identified (base-calling step). This process recreates the original sequence of As, Ts, Cs, and Gs for each short piece generated in the first step. Automated sequencers analyze the resulting electropherograms, and the output is a four-color chromatogram showing peaks that represent each of the four DNA bases. After the bases are “read,” computers are used to assemble the short sequences (in blocks of about 500 bases each, called
