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.
