Question

Please explain how to identify which lane corresponds to which ddNTP.

44. For the Sanger sequencing of a template DNA sequence 3'-CATGGGCTTTAGTCCT-5', which lane of the agarose gel below represents the reaction mix containing ddATP? a) Lane A b) Lane B c) Lane C d) Lane D e) None of these lanes

Answer 1

Given the sequence 3'- CATGGGCTTTAGTCCT -5' which is a template DNA sequence (3' > 5' strand) used for the sequencing reaction. So the sequencing result would actually be a sequence complementary to the given sequence, which would be a 5' > 3' strand of DNA:

5'- GTACCCGAAATCAGGA - 3'. A simpler way of looking at this is that the number of adenine bases in the sequence is 6 and the lane that has 6 bands would be that which corresponds to adenine bases. The right answer here is option b) Lane B.

Another way to look at it is that the Sanger reaction proceeds with shorter sequences in the beginning followed by longer ones. This is because the concentration of ddNTPs is more in the initial stages of the reaction (polymerase stops the addition of nucleotides after a ddNTP is added, leading to chain termination. When the reaction is run on a gel, the shorter DNA are found at the bottom of the gel and the longest on the top. So the sequence is read from bottom-up.

In the sequence 5'- GTA ....-3' we can easily make out that the lowest band on the gel corresponds to the base G, followed by T and then A, and A appears to be in the lane B.

Option b is the right answer.