Part 3: Automatic Sequencing
| Extraction | Amplification | Sequencing |
| Part 1 | Part 2 | Part 3 |
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The Database
*UPDATED*
FLMNH © 2005
Automatic sequencing makes everything easier than
manual sequencing. Instead of using radioactive isotopes to label the DNA fragments, special dyes
are used that fluoresce when a laser hits them. Each of the four nucleotides (G, A, T and C) have their
own unique color. Thus, only one lane per sample is required instead of the four lanes in manual sequencing.
The machine to the right is an automatic sequencer. It is basically an electrophoretic
gel rig with a laser at the bottom. The cycle sequenced samples are loaded at the top and the laser
scans back and forth while the DNA pieces migrate past to the bottom. The information
is passed on to the computer to the left. |
The gel file image below is the result of one automatic sequencing run. This run took
seven hours to complete. Each lane
is a sequence in one of the complementary directions. This gel has 20 individuals sequenced for the COI
gene; thus there are 40 lanes, one in one direction and the other the reverse. The lanes are staggered
because the odd lanes were loaded 10 minutes prior to the even lanes. This helps the detector distinguish
between the signals and track each lane more efficiently. |
The image to the right shows how these gels are read. A computer automatically calls
the bases based upon the signal peaks measured as the fluorescent dyes were excited by the laser while migrating through
the gel. Occasionally, there are problem areas like the "N" in
the bottom read for C. eludens. Fortunately, the complementary strand above is clean and the
proper base is a "T". Without the read in both directions, this portion would be unreliable if
we only had the bottom direction. |