IG1_02_20smc48-capillary
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Automated DNA sequencing by capillary electrophoresis

    Separation 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.

    An alternative separation method uses 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]. The end-label dyes are activated by a laser in the detector window [right], 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 side-by-side 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 sequencers 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 directly 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.

HOMEWORK: Popular accounts (like "Jurassic Park") suggest that "DNA sequences are read by a laser'" Explain why this is incorrect.


All text material ©2014 by Steven M. Carr