Recall: Regular nucleotide "building blocks" of DNA have an "-OH" (hydroxyl group) at the 3' end to which the new, incoming DNA nucleotide attaches via the phosphate-group 5' end.

(If you don't remember this, please click here to review DNA replication.)

Here's a picture of a normal (deoxy) DNA nucleotide:

As compared to a dideoxy DNA nucleotide:

Note the difference!

Either of these two nucleotides can be added to a growing, replicating DNA strand. However, once a dideoxy DNA nucleotide has been added, DNA polymerase no longer recognizes the free end. Replication STOPS.

In the next picture, the top diagram shows a normal DNA nucleotide. DNA polymerase can add a new nucleotide to this one, but not to a strand like the one shown directly below it:

The diagram below shows a portion of a hypothetical strand of DNA that you want to sequence. Note that it's single-stranded at the "-OH" point, and ready for replication by DNA polymerase. DNA polymerase will now add complementary bases to the available (single-stranded) sequence "TATGGCATG"

How do we sequence it? Let's follow the steps...

The Recipe for Success:

  • Place goodly quantity of your single-stranded DNA of interest (the one you want to sequence) into four different test tubes, each containing:

  • Allow the substances in the four test tubes to undergo DNA replication for a period of time, and then stop the reaction (chemically).

  • The picture below shows that in each of the test tubes, you'll get a certain quantity of dideoxy nucleotide-ended fragments in each test tube. In tt#1, for example, since there are only two sites with adenine available for pairing, you'll get some dideoxy-arrested fragments of each length.

    Study the four following drawings carefully, noting how many different sizes of fragments you'll get in each of the four test tubes.

    Note that:
    tt#1 has two different sizes of fragments.
    tt#2 has three different sizes of fragments.
    tt#3 has one different sizes of fragments.
    tt#4 has two different sizes of fragments.
    ...each of which corresponds to the number of complementary bases found in the original fragment you were trying to sequence.

    (Do you see where this is going? If not, study the drawings carefully!)

    We use special enzymes to digest away the "old" DNA template, leaving only the newly synthesized fragments, each ENDED with a radioactive dideoxynucleotide of known identity (T,A,G or C, depending on which well it was in).

    You'll now have four samples of the same thing you got above, but single-stranded:

    Now all that's left is to sort the fragments by size. This is done via a process known as ELECTROPHORESIS (explained in class):

    Fragments migrate from the negatively-charged to the positively-charged poles of the electric field, and separate at different levels because fragments of different sizes migrate at different speeds (long fragments are slower; small fragments are faster).

    The result from the DNA fragments we just made in our four test tubes would look something like this:

    The largest fragments are at the bottom (which corresponds to the 5' end of the DNA), and the smallest are at the top (which corresponds to the 3' end of the DNA).

    NOTICE THAT THE FOUR COLUMNS MAKE A SORT OF "MATRIX"--WITH EACH OF THE FOUR NUCLEOTIDES OCCUPYING ONLY ONE OF THE VERTICAL SPACES IN THE GRID.
    The scale on the left side of the diagram labeled "bp" is simply the number of the base pair, from 1 through 9, of our fragment.

    By reading the base at the lowest part of the gel from its bp line (bp #1) across to its actual base (T, A, G or C) you can determine the sequence of the bases in your nine-base fragment, from 5' to 3' direction.
    The sequence of our fragment is shown on the far right hand column of the drawing.