00:01
So let's talk about sequencing.
00:03
So in the original singer sequencing, we use our dna template.
00:11
We have primers, and that's this.
00:15
In the original case, we use radioactive primers.
00:19
Dna polymerase plus deoxy ribonucidid trifosates.
00:25
So d.
00:26
Ntps and dye deoxy trifosates.
00:30
D -8 -ntps.
00:34
So we want to know what the reason to add these d -d -ntps to the sequencing procedure, and what happens if we add 10 times less.
00:44
So first let's talk about what these we mean by di -deoxy -charyphosphate, so dd -ntp.
00:51
So in the normal d -ntp, this will have a, in the hydroxyl group here on the sugar.
01:05
And this hydroxyl group is going to be the site where the next base adds on to the strand.
01:17
So this is on the three prime side.
01:21
And tryphosphate because it has three phosphates.
01:23
So deoxyribon nucleotid trigonotidchifosate means that it has one of these hydroxyl groups.
01:33
However, the dye -deoxy nucleotide tryphosphate means that it doesn't have this hydroxyl group here on this 3 -prime end, and therefore no phosphodiasopine can be formed, therefore nothing can be added to this.
01:51
So this is what we call a terminator.
01:54
So the di -deoxy nucleotriiposphates are terminated, in the reaction.
02:01
And in the cases now, they are also fluorescently labeled, as we see here in this picture.
02:07
So here is our template with our primer and our mix of dntps, and our ddntps.
02:13
And our ddntps have different colors so we can see them.
02:18
So what happens? why do we add them? so in this reaction, they're at a ratio where there's more dntps than ddntps.
02:28
And so as the plymase is going along and incorporating the bases as it's reading the template and making the copies, every once in a while it will add one of these ddntps instead of a dntp.
02:44
And when this happens, the reaction will be terminated because nothing can be added because it doesn't have that hydroxyl group.
02:53
So no phosphideiaspora, diastrobin can form...