CGDP - DNA Sequencing
Part 2: Manual Sequencing

Extraction Amplification Sequencing

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FLMNH 2005


 
    In the old days when we were using radioactivity, all the variable fragment lengths were labelled with the same radioactive isotope. Therefore, we had to run separate lanes for reactions that were terminated with one of the four: G, A, T or C. These thinner gels were run against current and the shorter fragments ran faster than the longer ones. I like to think of it as taking a slinky and stretching out its length. These gels were then exposed to radioactive sensitive film, developed and read on a gel reader like the one seen here. Remember that the same gene was cycle sequenced in two complementary directions so that the reads could be reversed, complemented and compared. Because they are from the same animal, they should be identical.
    Here's a close up of a section from one of those gels. These 6 sets of lanes are from pieces of the 16S gene for three species C. fimbriata, C. cylindrica and C. errones. The code AR at the top is for the sequence in one direction, and BR for the other direction. Differences are marked with arrows. Remember to compare the AR's together and the BR's together, not the AR's with the BR's.
    Here's what the parts of the 16S sequence would look like from this region for these three species. There are five changes seen in this small section. In all of the five positions, C. cylindrica and C. errones have the same base, while C. fimbriata is different.
    This type of sequencing is called manual sequencing because it requires more effort to run gels and read in the sequences from these "autorads". Luckily, technological improvements have advanced these methodologies to Automatic Sequencing.