Page 19 - An Identity Crisis

seaweed which is dissolved in a heated solution, reverse direction and move toward the negative
form microscopic pores when the solution cools pole.
that act as a molecular sieve through which DNA
molecules pass. Following separation by electrophoresis, DNA
fragments may be visualized using several
To illustrate this concept, the separation matrix methods that directly bind to the DNA molecule.
may be pictured as a kelp forest, dense with The most common method in an educational
fronds, but having pockets of space in between. setting is to stain with methylene blue. After
These spaces are much like the pores in the gel. electrophoresis, the gel is soaked in a stain solution
Due to their size, small fish have an easier time allowing the stain to adhere to the DNA molecules,
navigating through the plants than larger fish. however, the stain is also absorbed into the pores
Both eventually get through, but the small fish of the gel. Because of this, the entire gel is blue
travels faster. Applying this analogy to a gel matrix, until allowed to soak in clear water, which dilutes
the DNA travels through a gel and, just as in the the stain absorbed into the gel in a process called
seaweed forest, smaller fragments travel faster de-staining. The dye bound to the DNA molecules is
than the larger. When, after a certain period of time, not removed during de-staining, and may be seen
the electrical force that moves the DNA through with the naked eye. In commercial and research
the matrix is removed, the newly separated DNA settings, ethidium bromide is the most common
fragments cease their migration. Their position nucleic acid stain, however, it can only be visualized
in the gel is a direct relation to their size, and under UV light and is mutagenic, which makes it
these fragments may be stained and their sizes unsuitable for novices. In this lab, a proprietary
compared to one another. formula called BlueVis is utilized. BlueVis has no
special safety concerns and stains the DNA while
The figure at the top of the page is a schematic of a it migrates on the gel, reducing the need for de-
stained gel of each of the four possible genotypes staining. Other visualization methods include
of children from the mother and father in the figure radioactive labels and commercially available
on the previous page. Note that there is only one fluorescent dyes which can be incorporated during
band for the homozygous genotype, C, because the PCR process.
both the mother and father contributed the same
allele. When visualized, this band should be darker To confirm the size of each PCR product, DNA
than the other bands as there is twice as much size ladders are run on the same gel as unknown
DNA in that area. samples. These ladders contain a series of known
purified DNA fragments corresponding to most of
In electrophoresis, the solution used to create the known variant forms of each portion of target
an agarose gel contains a salt dissolved in water, DNA. As each of these variant forms is called an
commonly either Tris-acetate-EDTA (TAE) or allele, these allelic ladders provide a means for
Tris-borate-EDTA (TBE), that promotes electrical determining the length, and, thus, the specific
current and stabilizes the gel’s ph. This same variant or allele type, of each amplified product
solution is also used to submerge the gel, providing for a given sample. Theoretically, a large number
an electrically and chemically uniform medium of alleles are possible for any given loci, however,
for the electrophoresis process. As the phosphate generally, only four to ten alleles are common
backbone of DNA is negatively charged in neutral within a population. The alleles present on the
to basic solutions, the solution is pH buffered to ladder are common alleles, and those rare alleles in
prevent acidification that would cause the DNA to the population are collectively known as ‘off ladder’

THE MYSTERY OF LYLE AND LOUISE 19