Before we move in to how sequencing works.
I need to introduce you to one more player
in the game. That is the dideoxynucleotide.
You will recall that the three prime OH handle
is really key for DNA to continue synthesis.
If there is no three prime OH handle on that
ribose sugar, then DNA polymerase cannot add
new nucleotides to the chain. Dideoxy, DNA deoxyribonucleic
acid is missing the three prime OH handle
to grab onto and add new nucleotides. When
we sequence DNA, we are going to be replicating
DNA in vitro. We are unzipping it, asking for
it to replicate and continually making more
strands in order to see what the sequence
is. Termination of replication occurs every
time a dideoxynucleotide shows up. Now dideoxynucleotides
have been made for all of the nucleotides.
We can have As,Ts, Cs, and Gs.
Let us take a closer look at what happens
when we have each of these nucleotides in
a vial and we are asking replication to happen.
First of all, the ingredients for replication
that are pretty key are going to be our template
strand of DNA. We better have some DNA polymerase.
We probably need to have some primers. We
need normal nucleotides for certain and a
few dideoxynucleotides. In the case of DNA
sequencing, we are going to have four seperate
vials. This is the enzymatic method where
we have one vial that has all of the regular
ingredients and dideoxy-As and another one
that has dideoxy-Gs and then another one that
has dideoxy-Cs and then another one that has
dideoxy-Ts. There are four different reactions
going on in which we were asking for multiple
rounds of synthesis of DNA. In the container
that contains just dideoxy-C for example,
we will see that every so often DNA replication
will terminate, but it will terminate when
one of these dideoxynucleotides is put down.
For example, here you have three different
lengths at which synthesis stopped at the
first G. Synthesis stopped at the second G
so on and so forth. You got to keep in mind that there
are hundreds of different fragments, thousands
probably of different fragments of variant
lengths depending on how far DNA polymerase
went along before picking up a dideoxynucleotide,
which had no three prime OH handle, so termination
of replication happened. In the tube that
has dideoxy-Cs, we see the same thing happen,
replication happens, thousands of copies were
made. Some of them stop early. Some of them
stop later and then we will look at dideoxy-A.
We have the same thing going on in that
vial and in the fourth container, we have
the same thing going, but now they all end
in Ts. Now we can use these fragment lengths
and the endings and guess what, to figure out the
lengths of the fragments, we are going to
do gel electrophoresis. Each of the different
reactions or vials are put into a different
lane in a gel electrophoresis and we will
let the process go, run our current across
the gel. The DNA fragments move towards the
positive pole because they are negatively
charged. Now we can read our gel and it is
important to think about what is going on.
Each lane has fragments that end in G, C,
A, and T respectively and because of the position
in the gel, we can read the fragments. Now
the shortest fragments go the farthest and
the longest fragments go the least far.
The ones that are closest to the wells are going
to be at the three prime end of the DNA strand.
We can read the sequence like this. The first
one we know is in the T lane and so it must
be a T. The second one in the sequence as
we have read down the gel is AG and we so
can see that it's a G because of its position,
there is lots of them in there and so the
next one in the line is a G. Following that,
we can see the next one down the line is the
C so on and so forth. We can put the A in
position and we can read the entire gel till
we get to the end and we have a sequence. Now you
can only so many or so long of a sequence
in one gel. Clearly we cannot have a gel that
goes all the way around the world. We have
to chop it into pieces, which is why we had
that chopping into pieces part.
This whole technique, the enzymatic method
is pretty laborious and used to take a long
time for us to get these small sets of sequence
and then overlap the fragments and align them
and figure out what the whole genome sequence
is, but things became automated. That was
really a pivotal point. We could do automated
DNA sequencing and in this case, it works
in just the same way. We have DNA replication
occurring and we have dideoxynucleotides,
but they are just done in one vial. We mix it all
together. We have got one reaction. We have
got dideoxy-A, dideoxy-G, dideoxy-C, dideoxy-T.
And we have all of the other regular nucleotides.
Synthesis is going to occur as normal. Most
of the time DNA polymerase is going to pick
up the right nucleotides and then every once
in a while you will pick up a dideoxy, no
three prime OH handle, termination of synthesis.
So we have varying fragment lengths. Exactly
the same philosophy going on in this automated
sequencing. How it comes automated then is that
we can label those dideoxynucleotides with
a glowing or fluorescing label. And we have
them in four different colors to match the
four different letters. And the core part now
is that we can run electrophoresis in a capillary
tube, a very small tube and as the nucleotide
fragments move across the gel, there is a
laser that essentially reads them at the end
of the capillary tube as they fall off the
end of the gel and then a computer puts together
that sequence and spits out precisely what
that DNA sequence is. A lot less laborious,
much quicker and this is one of the ways that
allowed us to sequence the human genome much
more rapidly than we would have if we were
using the enzymatic method being much slower.
So automated still using enzymes, but it is
an automated method. That is the way that
we sequence genomes these days.