Automated DNA sequencing by capillary electrophoresis
of the products of a radioactively-labelled
or fluorescent dideoxy
sequencing reaction has historically been
done in a poly-acrylamide
slab gel, for example the kind used in a 96-lane ABI
377. Drawbacks off this method include the
necessity of preparing a fresh gel for each sequencing run,
manual loading of samples into the gel, relatively long run
times (8 ~ 10 hrs for resolution of a 1Kb DNA fragment), and
difficulties in automated and/or manual tracking of lanes.
separation method use capillary
electrophoresis. A series of standard dideoxy DNA sequencing reactions
are prepared, each in one well of an 8 row x 12 column 96-well plate. [Left, A]
A series of ultra-thin capillary tubes (0.1 mm inside
bore, 50 ~ 80 cm length) are filled with a resin bead mixture
and placed in a multi-capillary array. One end of the
array is mounted in a negatively-charged Cathode plate [right] so
that their ends align with each well of the sample plate.
Application of high voltage causes the labelled DNA in each sample well
to enter the corresponding capillary, and migrate though it
toward the positively-charged Anode
reservoir. Because of the high voltages used,
electrophoretic separation requires only 1 ~ 3 hrs. As in
slab-gel electrophoresis, the smaller fragments move more
quickly than the larger [Left, B]. A laser in
the detection window
[right] activates the end-label dyes, and the fluorescence
wavelength of each fragment is read by a photometer as
it passes a fixed point. Unlike the slab gel system, the laser
and photometer are fixed and immovable, and can activate and
read multiple capillaries simultaneously as separate data
streams: this avoids the problem of tracking separate lanes in a
slab gel. Mobility data are sent to a computer, and a chromatogram
is generated for each sample that is essentially
identical to those of the slab gel system.
Alternative configurations of the capillary array can read 1, 4, 16, or 48 wells simultaneously, moving between the wells of the sample plate automatically in a regular pattern. Capillary machines typically use a robot to load a stack of sample plates automatically. The capillary array is flushed with buffer between plates, so that the machine can be left to run unattended for many runs. In the largest facilities, PCR reactions may also be linked directlty to sequencing plates in robotic workstations that service dozens or hundreds of capillary sequencers. The chromatogram data are placed on a server, and can be downloaded by users hundreds or thousands of miles away. Economices of scale can make it cheaper to outsource DNA sequencing rather than buying, maintaining, and staffing an in-house machine.
Popular accounts (like "Jurassic Park") suggest
that "DNA sequences are read by a laser'"
Explain why this is incorrect.
All text material © 2013 by Steven M. Carr