DNA Sequence Analysis by Autoradiograph:

After the completion of each reaction, Polyacrylamide gel electrophoresis (PAGE) is performed. Each reaction mix is loaded in a separate lane. The reaction condition should be carefully controlled to separate the strands that differ just by a single nucleotide. PAGE is done in denaturating condition in presence of urea or less frequent formamide. Urea and formamide lowers the melting point of DNA molecule, denatures DNA by disrupting the H bond and the newly synthesized strand separates from the template strand.

Electrophoresis is carried out at high voltage to prevent the renaturation of DNA due to high heat generation in gel.

After complete run, the gel is transferred on nitrocellulose filter and autoradiography is performed so the only bands having the 5' radiolabelled molecule will be visible as bands. In PAGE the shortest fragment moves faster so the bottom most molecule is the first dideoxynucleotide which stopped the chain elongation by its incorporation and thus that should be the first sequenced nucleotide. So by this bottom up approach in all the lanes we can get a combined DNA sequence of the query (Fig .4).

Figure 4: A schematic diagram of Sanger DNA sequencing method with an autoradiograph. The sequence is in the 5' to 3' direction and is the complementary of query.