An important technique for increasing one's knowledge of gene structure is rapid, accurate DNA sequence analysis. The first method developed for rapid sequence analysis of cloned DNA fragments involved chemical modification and cleavage of specific nucleotides, followed by electrophoresis on high-resolution denaturing acrylamide gels[117, 118]. This chemical sequencing method was developed in 1977 by MAXAM and GILBERT.
An alternative method for rapid DNA sequencing, the dideoxy sequencing
method, was used in this
study[119, 120].
Once the clones have been generated and selected, the primer extension is
performed to determine the sequence of interest.
A synthetic oligonucleotide primer is annealed to the pUC DNA genome
immediately adjacent to the cloned insert or to the insert itself. Four
separate primer extension reactions employing Taq polymerase
are subsequently initiated. Each
reaction contains all four deoxynucleotide triphosphates (dNTPs), but one dNTP
is labeled with 
S to allow subsequent detection of elongated chains using
autoradiography . In each of the four reactions, one
dideoxynucleotide triphosphate (ddGTP, ddATP, ddTTP, or ddCTP) is added in
low concentration. Thus, in each tube numerous primer extension reactions are
taking place simultaneously; however, at any given point of the extension, a
small percentage will incorporate ddNTP at the end of a newly extended chain.
For this small portion of reactions in the tube, the nascent chain cannot be
further elongated because the 3'-hydroxyl group is absent. However, other
elongation reactions will continue until a ddNTP is incorporated. Thus, in the
four tubes, there will be a mix of all possible lengths of the sequence, all
starting at the primer region and extending up to the dideoxynucleotide in each
specific tube. These fragments will be radioactive due to the labeled
nucleotide inserted at multiple points along its length. When the reaction
products of the four tubes are run in adjacent lanes on a high-resolution sequencing
gel, each fragment, from a few nucleotides in length to the maximal length
completed by the primer extension, will be visualized as separate bands. The
shortest fragments will run farthest down the gel.
Thus, for example, in the G tube with the dNTPs plus ddGTP, only those extended sequences ending with a G will be present, but all possible lengths of extended DNA ending in G will be in the tube. So, if C nucleotides exist in the DNA template inserted in the vector at positions 15, 17, 21, and 28 past the primer (position 1), the G tube will have fragments of dideoxynucleotide terminated at positions 15, 17, 21, and 28. Moreover, if six nucleotides of the template DNA contain the sequence 5'-ATTGCG-3', the newly synthesized primer-extended DNA fragment would carry the inverse complementary sequence that could be read 5'-CGCAAT-3'. The autoradiograph of the sequence gel would show a C signal, a G signal, a C signal, two A signals, and a T signal, as the gel is read from bottom to top.
This method is rapid, accurate, and simple, requiring less labor than chemical sequencing. In addition, less radioactive material and fewer hazardous chemicals are used in the dideoxy sequencing method. Autoradiographs of the gels can be read after one to two days, allowing the rapid collection of sequence data. Although only 200 to 400 bases are typically read from any given set of reactions, by using different starting primers (see table tab:seqprimers) the whole gene can be accessed.