THE USES AND PITFALLS OF SOUTHERN BLOTTING: what you would have seen previously is how we set up a Southern blot. So we digest the DNA with enzymes. We run it through an agrose gel. The gel is then
positioned on top of a certain platform with a buffer, which enables the buffer
to transfer through the gel which pulls the DNA with it onto a membrane.
That takes a good few hours. And then eventually you end up with most of the
DNA transferred to the membrane, we hope. It's then stabilised or fixed to the
membrane by baking the membrane at about 80 degrees for a couple of hours,
after which we then hybridise it with a probe which is a small fragment of DNA
which contains a known sequence. And that's the sequence that we're looking
for when we actually then come to develop the blot, which used to be done by
1
FutureLearn
autoradiography or x-ray film. But nowadays more commonly done by
fluorescence.
Nowadays, we use Southern blotting occasionally. Our lab is one of the few
left in the UK which is doing it and we are doing it an awful lot less than we
were before. So back in the early 2000s, late '90s, we were doing two, possibly
three sometimes a week, for Fragile X and myotonic dystrophy, normally.
Nowadays, it's been superseded by PCR-based techniques. But PCR is still a bit
limited. So we can detect the presence of large expansions in Fragile X and
myotonic dystrophy, but we can't tell what size they are.
So occasionally, mainly now for myotonic dystrophy pre-natals, we are asked
to run blots. Myotonic dystrophy is a bit of a tricky one because what the
Southern blot allows us to do is it allows us to get a size of the expansion. So
in Fragile X we can use the PCR technique to say that there is an expansion
there. And clinically, that's what they need to know. The size of the expansion
is kind of not as relevant, clinically. They've just got an expansion, therefore
that confirms the diagnosis of Fragile X.
But with myotonic dystrophy, it exhibits a phenomenon called anticipation,
which is where in each successive generation, if the expansion increases in
size, the severity of the phenotype changes and gets more severe and also the
onset of the disease is sooner in life. And what we're most interested in is
being able to pick up congenital myotonic dystrophy, which is where an
expansion can be passed from the mother to a child. And in that process, the
expansion becomes very large.
Ещё видео!