A sufficient amount of a small DNA fragment is available for dide...

Question

A sufficient amount of a small DNA fragment is available for dideoxy sequencing. The fragment to be sequenced contains 20 nucleotides following the site of primer binding:

5'-ATCGCTCGACAGTGACTAGC-[primer site]-3'

Dideoxy sequencing is carried out, and the products of the four sequencing reactions are separated by gel electrophoresis. Draw the bands you expect will appear on the gel from each of the sequencing reactions.

Accepted Answer

Hi, everyone. Welcome back. Let's look at our next question. It says, how do D D N T P differ from D N T P in their structure? And how does this fati facilitate the Dixy sequencing? Well, let's look at our answer choices. Choice. A says D D N T P lacks a hydroxyl group on the three prime C and therefore hinders the formation of phospho dier bods leads to the formation of fragments. Well, this is correct. They have this hydroxyl group on the three prime carbon and therefore cannot form a fossil dier bond. So when one of the D D N T P s incorporates into the DNA strand that terminates the strand in oxy sequencing, you makes a small amount of this D D N T P in your mixture and synthesize a DNA strand, these D D N T P s will be randomly incorporated in there creating fragments of different sizes ideally at every single base in the chain. If you label these D D N T P S uh fluorescently or otherwise, you can actually then identify the identity of the base where the strand terminates, get enough of these fragments and you can read off the sequence of the DNA strand that you're looking at. Let's continue on to the other answer. Choices to understand why they're not correct. Choice. B says D D and T P have an additional carboxyl group at the five prime C and therefore hinders the formation of phosphor dier bods leading to the formation of fragments. Well, as we discussed, this does hint hinge on preventing the formation of those phosphor dira bods. But that does not happen at the five prime carbon but at the three prime carbon. So choice B is not our answer. Choice. C says D D N T P lacks a sugar phosphate group which vital facilitates the termination of DNA replication. And this is not correct and eliminate that because again, it's the lack of a hydroxyl group on the three prime carbon, not the lack of a sugar phosphate group that leads to the termination of the DNA replication. Finally, choice D D D N P D D N T P has DNA primer attached to its proximal end which facilitates the action of DNA polymerase. Well, this isn't what the D D N T P does. Again, that happens at the very end of the chain terminating replication where it incorporates no choice D is not correct. So again, how do D D N T P differ from D N T P and their structure? And how does this facilitate the Dixy sequencing? Choice? Ad D N T P lacks a hydroxyl group on the three prime C and therefore hinders the formation of phospho dier bonds leading to the formation of fragments. See you in our next video.

Key Concepts

Dideoxy Sequencing

Gel Electrophoresis

Primer Binding Site

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