- [Voiceover] Have you ever wondered how we sequence DNA?
Well, let's just take a quick look at DNA sequencing.
We're going to break down DNA
sequencing into three different steps.
The first step is you take the sample
of DNA that you are interested in sequencing
and you basically use PCR to amplify the sample.
By using PCR in order to amplify the sample,
you're able to generate lots and lots of DNA fragments.
The next thing that you do is normally in PCR
you have to add nucleotides, you have to give
the growing strand the substrate from which it can grow.
Normally you add in regular deoxynucleotides
and those look something like this.
You've got an OH group here.
You've got an H group here.
You have a base...
And then you've got a carbon group...
And oxygen-hydrogen.
So, this is what a normal nucleotide looks like...
But interspersed in the PCR, what you also want to add is
you want to add in something known as a dideoxynucleotide.
A dideoxynucleotide looks something like this.
It's basically exactly the same thing
but it only has a hydrogen here,
so this oxygen is removed.
And what that basically does is if this
dideoxynucleotide, we can abbreviate ddNTP,
if this incorporates into the growing strand,
since there's no oxygen group here,
the strand can no longer elongate.
You basically have termination of strand elongation,
as soon as this ddNTP incorporates.
What you can do is you can actually fluorescently
label the different dideoxynucleotides.
For example, we have four different options.
We can label all the G's blue,
we can label all the A's red,
all the T's green,
and all the C's orange.
And so basically what you have is you have
these dideoxynucleotides with different fluorescent labels
getting incorporated into the growing strand
and since PCR is able to amplify creating millions
and million of DNA fragments, you can basically,
what you can do is you'll have strands of different lengths.
Let's just kind of look at an example.
Let's imagine that we've got a nucleotide being
incorporated here, a regular nucleotide,
and then another one incorporated here
and then another one and then just randomly,
all of a sudden, we have a dideoxynucleotide
being incorporated here and this would stop
the elongation of the strand.
So, you would have a DNA strand
which that's just four nucleotides long.
And after another round of PCR,
what we might have is we might have,
one, two, three, four, five, six,
it's just growing, it's growing, it's growing,
and all the sudden, whoa, what happened?
You got a dideoxynucleotide being incorporated.
And so basically, you just do this
and after you've got millions of samples,
you will eventually be able to have
something that looks like this.
You'll have maybe just one regular nucleotide
and you've got a dideoxynucleotide incorporated,
or you might have maybe, let's say,
two of them, so you'll have two
and then you've got a...
Let's use this color suite we've got here.
What you can basically do is you can see you have strands
and they're elongating and different strands are terminated
at different points by a dideoxynucleotide.
And so, basically, the next step,
is you use gelelectrophoresis...
Electrophoresis...
In order to separate the strands by size.
So, when you run all the different fragments on a gel,
it will separate them by size
and then you can just have a computer go in
and analyze all the fluorescent labels.
So if it sees here, that you've got this blue fluorescent
light, then it knows that the second nucleotide
in the sequence is a G, so it'll say G.
And then, it'll look here, it'll say, okay well this is a C.
It'll look here, it'll say we have another G
and so on and so forth.
And basically computer is able to, by reading
these fluorescent labels, these fluorescent tags,
it's able to give you a DNA sequence.
And so this is basically an overview
of how DNA sequencing works.