CGDP - DNA Sequencing
Part 3: Automatic Sequencing

Extraction Amplification Sequencing

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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.