Question

Suppose you want to sequence the following DNA segment:

5’-GATCTCTTGAAATG-primer site-3’

You clone the fragment into a plasmid, transform it into bacterial cells and isolate DNA from one of the bacterial colonies. You use the Sanger sequencing method to determine the sequence, separating the products from the sequencing reactions by gel electrophoresis. Draw the bands that should appear on the gel from the four sequencing reactions.

Answer 1

Given that the primer site is at the 3' end of the DNA to be sequenced, the primer used for sequencing would be a reverse primer that would anneal to the 3' end of the 5' > 3' template strand and the sequence would be that of the 3' > 5' (complementary) strand. Hence the sequence would be:

5'- primer- CATTTCAAGAGATC -3'

In sanger sequencing, the concentration of dideoxy nucleotides is more in the initial stages of the reaction and so the sequence would terminate forming smaller fragments at the beginning followed by longer fragments as the reaction progresses. Now, when the samples are electrophoresced, we get the smaller fragments at the bottom and the longer ones at the top so the sequence is read from the bottom of the gel.