Future Sequencing Technologies
Encouraging progress ranges from enhancements of gel-based technologies and the development of novel, gel-less automatable approaches, such as mass spectrometry and DNA chips. New gel-based sequencers in development use tiny (capillary) tubes to run standard electrophoretic separations. These separations are much faster because the capillary tubes dissipate heat well and allow the use of much higher electric fields.

Capillary Array Electrophoresis (CAE). CAE systems promise dramatically faster and higher-resolution fragment separation for DNA sequencing. A multiplexed CAE system designed by Edward Yeung (Iowa State University) has been developed for commercial production by Premier American Technologies Corporation (PATCO). In the PATCO ESY9600 model, DNA samples are introduced into the 96-capillary array; as the separated fragments pass through the capillaries, they are irradiated all at once with laser light. Fluorescence is measured by a charged coupled device that acts as a simultaneous multichannel detector. (Inset circle at upper left: Closeup view of individual capillary lanes with separated samples.) Because every fragment length exists in the sample, bases are identified in order according to the time required for them to reach the laser-detector region.

Novel sequencing methods currently being developed include the use of DNA fragments bound to a solid surface ("DNA chips"), and separation of fragments by mass spectrometry.

How DNA sequencing is accomplished

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 (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.

Current electrophoresis limits are about 500-700 bases sequenced per read. Automated sequencers analyze the resulting electropherograms and the output is a four-color chromatogram showing peaks that represent each of the 4 DNA bases.

After the bases are "read", computers are used to assemble the short sequences (in blocks of about 500 bases each, called the read length) into long continuous stretches that are analyzed for errors, gene-coding regions, and other characteristics.