Professor Dave here, let’s hit the lab.
Now that we have learned a lot about some major breakthroughs in molecular biology in
the 20th century, it’s time to take a look at just a couple of lab techniques that have
been tremendously important in the development of this knowledge.
The first of these is called gel electrophoresis, which is a method of separating large molecules
like segments of DNA.
At first glance it will look a little bit similar to thin layer chromatography, which
we learned about in the organic chemistry series, but the principles involved are a
little bit different, so let’s get a closer look at this technique now.
When we introduced some concepts in biotechnology, we talked about how we can make recombinant
DNA plasmids and insert them in bacteria to produce many copies of a gene, or many copies
of the protein produced when a gene is expressed.
Say that we want to cut up the plasmid and analyze it, to make sure it is being copied
as desired.
Or imagine any other scenario where we have a mixture of DNA fragments that we want to
separate and visualize.
Gel electrophoresis enables us to do this, and it works as follows.
In this tray there sits a slab of either polyacrylamide or agarose gel, which is immersed in an aqueous
buffer solution.
At one end of the gel there are a series of wells, and a number of samples can be loaded
into these wells, each of which is a mixture of some DNA molecules of varying length.
This apparatus is also equipped with electrodes at each end, with the cathode, or negatively
charged electrode, at the end where the wells sit, and the anode, or positively charged
electrode, at the other end.
Once everything is loaded and ready to go, the current is turned on.
Now recall that phosphate groups line the DNA backbone, and that each phosphate group
contains one oxyanion, and thus carries a formal negative charge.
We can therefore say that DNA molecules are negatively charged.
This means that the negatively charged cathode will repel the DNA molecules, and they will
begin to travel along the gel, towards the positively charged anode, to which they are attracted.
The thing is, this gel is a sticky, porous substance, and the molecules have to migrate
through the pores to move along the gel, in a process called sieving.
The larger the DNA molecule, the more difficulty it will have in navigating through the pores,
which means that smaller DNA molecules will travel greater distances through the gel,
while larger ones will travel shorter distances through the gel, in the same time interval.
This process is so reliable and quantifiable, that we can plot the approximate number of
base pairs in a DNA molecule as a function of the precise distance it travels during
gel electrophoresis.
Once separation is complete, the current is turned off, and a DNA-binding dye is added
to the system that glows a fluorescent pink in UV light.
This is how the data is gathered, which will show up as thin bands that sometimes resemble
a ladder, if many different DNA molecules were present in the sample, and each band
contains thousands of identical DNA molecules of that particular length.
Remember, the farther away from the well a band shows up, the shorter the DNA molecule
is that has produced that band.
As we said, this technique may be used to chop up plasmids with restriction enzymes
and analyze the results.
It can be used to assess the products of gene amplification using the polymerase chain reaction.
It can be used to isolate a specific DNA molecule of a mixture for sequencing or further characterization
via a technique called Southern blotting.
In addition to separating mixtures of DNA according to length, this technique can also
be used to separate mixtures of proteins according to electrical charge, which offers information
about the identity of the side chains, or a number of other very useful applications.
The simplicity and immense utility of gel electrophoresis make it a very important part
of any molecular biology laboratory today.