TEACHING DEMO (MOLECULAR BIO)

-DANI
Key points:
 Gel electrophoresis is a technique used to separate DNA fragments
according to their size.

 DNA samples are loaded into wells (indentations) at one end of a gel, and
an electric current is applied to pull them through the gel.

 DNA fragments are negatively charged, so they move towards the

positive electrode. Because all DNA fragments have the same amount of
charge per mass, small fragments move through the gel faster than large
ones.

 When a gel is stained with a DNA-binding dye, the DNA fragments can be
seen as bands, each representing a group of same-sized DNA fragments.

Introduction
Suppose you have just done a PCR reaction, making many copies of a
target DNA region. Or perhaps you’ve done some DNA cloning, trying to
"paste" a gene into a circular DNA plasmid.

Now, you want to check and see whether your PCR worked, or whether
your plasmid has the right gene in it. What technique can you use to
visualize (directly observe) the fragments of DNA?

DNA gel electrophoresis is commonly used in forensics to determine the

specific sequence of DNA to help find the leading suspect. It is also
commonly used in genetics and the fields of molecular biology and
biochemistry.

Southern blotting is a technique used in DNA gel electrophoresis to

determine the presence or absence of a specific nucleotide sequence in a
DNA molecule. Northern blotting is a different technique used to identify
gene expression in the presence of RNA, or ribonucleic acid. Eastern
blotting is used to determine the presence of lipids, proteins and
carbohydrates in a DNA sequence. To simply detect proteins in a DNA
sequence, the technique of western blotting is used in the
electrophoresis chamber through gel isolation.

Zymography is also an approach to detect certain biological functions

through DNA gel electrophoresis. It is mainly used to study extracellular
matrix-degrading enzymes, which catalyze the destruction of the matrix
outside the cell, as well as the basement membrane of a cell. The use of
polyacrylamide gel aids in separating the enzymes, which are proteins,
by using a protein substrate such as gelatin or casein. This technique is
used to measure the amount of degradation activity occurring outside
the cell.

DNA gel electrophoresis is used quite often in forensics. It is used to

separate blood proteins and DNA found at a crime scene to determine
correlations with the available suspects. Gel electrophoresis is also used
to determine a specific inheritance within genetics, as certain DNA and
proteins are associated with different races. It is also used to solve
paternity cases to determine the relative relationship between
individuals.
Gel electrophoresis
Gel electrophoresis is a technique used to separate DNA fragments (or
other macromolecules, such as RNA and proteins) based on their size
and charge. Electrophoresis involves running a current through a gel
containing the molecules of interest. Based on their size and charge, the
molecules will travel through the gel in different directions or at
different speeds, allowing them to be separated from one another.

The suffix phoresis means "migration" or "movement." The prefix electro tells
us that we are using electricity to make molecules migrate.
All DNA molecules have the same amount of charge per mass. Because of
this, gel electrophoresis of DNA fragments separates them based on size
only. Using electrophoresis, we can see how many different DNA
fragments are present in a sample and how large they are relative to one
another. We can also determine the absolute size of a piece of DNA by
examining it next to a standard "yardstick" made up of DNA fragments of
known sizes.

Separation of molecules in electrophoresis is based on charge (the thing

that gets pulled) and the effective cross-section of the molecule in
whatever state it finds itself – folded, unfolded, bound to other
molecules, etc.

A collection of DNA fragments separate by length because they are all the
same type of molecule. In general, the only meaningful difference
between the various fragments should be their length.
However, there are some exceptions to this rule. For instance, some DNA
molecules are circular (like bacterial plasmids), while others are linear.
Circular DNA molecules may run differently than linear ones through a
gel. Plasmids, for example, can exist in a form called "supercoiled," in
which they actually move faster through a gel than they should for their
size, because they have twisted into a skinny shape that can move
through the gel more easily.
plasmid is a small DNA molecule within a cell that is physically separated
from chromosomal DNA and can replicate independently. They are most
commonly found as small circular, double-stranded DNA molecules in
bacteria; however, plasmids are sometimes present in archaea and
eukaryotic organisms.

What is a gel?
As the name suggests, gel electrophoresis involves a gel: a slab of Jello-
like material. Gels for DNA separation are often made out of a
polysaccharide called agarose, which comes as dry, powdered flakes.
When the agarose is heated in a buffer (water with some salts in it) and
allowed to cool, it will form a solid, slightly squishy gel. At the molecular
level, the gel is a matrix of agarose molecules that are held together by
hydrogen bonds and form tiny pores.

At one end, the gel has pocket-like indentations called wells, which are
where the DNA samples will be placed:

Before the DNA samples are added, the gel must be placed in a gel box.
One end of the box is hooked to a positive electrode, while the other end
is hooked to a negative electrode. The main body of the box, where the
gel is placed, is filled with a salt-containing buffer solution that can
conduct current. Although you may not be able to see in the image above
(thanks to my amazing artistic skills), the buffer fills the gel box to a level
where it just barely covers the gel.

The end of the gel with the wells is positioned towards the negative
electrode. The end without wells (towards which the DNA fragments will
migrate) is positioned towards the positive electrode.

How do DNA fragments move through the

gel?
Once the gel is in the box, each of the DNA samples we want to examine
(for instance, each PCR reaction or each restriction-digested plasmid) is
carefully transferred into one of the wells. One well is reserved for a DNA
ladder, a standard reference that contains DNA fragments of known
lengths. Commercial DNA ladders come in different size ranges, so we
would want to pick one with good "coverage" of the size range of our
expected fragments.

Next, the power to the gel box is turned on, and current begins to flow
through the gel. The DNA molecules have a negative charge because of
the phosphate groups in their sugar-phosphate backbone, so they start
moving through the matrix of the gel towards the positive pole. When
the power is turned on and current is passing through the gel, the gel is
said to be running.
A typical voltage for running an agarose DNA gel would be in the range of
120V. A higher voltage will make the gel run faster, but may also melt it if
it runs for a long period of time. A lower voltage will make the gel run
more slowly (which can be convenient if you want it to finish up at a
particular time, say, after you get back from lunch)

As the gel runs, shorter pieces of DNA will travel through the pores of the
gel matrix faster than longer ones. After the gel has run for awhile, the
shortest pieces of DNA will be close to the positive end of the gel, while
the longest pieces of DNA will remain near the wells. Very short pieces of
DNA may have run right off the end of the gel if we left it on for too long
(something I've most definitely been guilty of!).

Visualizing the DNA fragments

Once the fragments have been separated, we can examine the gel and see
what sizes of bands are found on it. When a gel is stained with a DNA-
binding dye and placed under UV light, the DNA fragments will glow,
allowing us to see the DNA present at different locations along the length
of the gel.

A well-defined “line” of DNA on a gel is called a band. Each band contains

a large number of DNA fragments of the same size that have all traveled
as a group to the same position. A single DNA fragment (or even a small
group of DNA fragments) would not be visible by itself on a gel.

By comparing the bands in a sample to the DNA ladder, we can

determine their approximate sizes. For instance, the bright band on the
gel above is roughly 700700700700 base pairs (bp) in size.