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Will Rosetti

Dr Dimos

Biotechnology Lab Report


Introduction:
Biotechnology in its simplest form is the bridging of biology and technology to
utilize cellular and molecular processes to develop technologies and new products
to improve life on this planet. This practice is not necessarily new, rather dating
back six-thousand years to the first times homo-sapiens utilized microorganisms for
the development of breads, cheeses and fermented beverages. More modern
biotechnologies are providing new products and technologies to battle diseases &
environmental issues, while also helping to increase agricultural yields and
industrial efficiency. (How Can Biotechnology Benefit You?)
More than 13.3 million farmers worldwide are currently utilizing biotechnology
to reduce their footprint on the environment and losses from pests, while still
increasing the yield each harvest. Fifty plus bio-refineries have been constructed in
North America to pioneer new technologies to produce biofuels and chemicals from
renewable resources in hopes of reducing greenhouse gas emissions. These same
types of technologies are pioneering the future of sciences with hopes for a greater
tomorrow for our world. (What is Biotechnology?)
One type of biotechnology in use today is DNA Fingerprinting. DNA
Fingerprinting, also known as DNA typing, is a practice in which DNA is isolated,
then images are made from the portions of the DNA. The key to this process is in
the isolation of a DNA sample. Firstly, a sample containing cells must be collected.
From here the DNA must be extracted then purified. If not enough DNA is present, a
polymerase chain reaction (PCR) may be utilized to copy the DNA segment. Once
enough DNA has been purified, restriction enzymes are utilized to cut the DNA
sample into segments of varying length. The segmented DNA sample is then placed
into the well of a gel plate, and undergoes Electrophoresis. (Editors of Encyclopedia
Britannica)
Electrophoresis is the process in which DNA molecules are separated
according to size via the application of an electric field. The electric field causes the
molecules to migrate from a negatively charged position, towards a positively
charged end in a closed chamber, with the size of the molecules dictating the rate
and distance in which they travel. Smaller molecules will travel faster and farther
than larger sized molecules. In this process a porous gel matrix is used, most
commonly agarose gel, while being suspended in a buffer solution to encourage
that electricity is conducted more efficiently than in mere water. The buffer controls
the pH of the solution to encourage this process. Once the DNA sample has
undergone electrophoresis, visual bands appear on the gel allowing for
measurement of length and to create the fingerprint. Once the fingerprint is
created, it allows for comparison and identification of the DNA to be utilized in

determining base pairs, identify genetic trends, and provide biological evidence.
(Forensic Science Central)
The purpose of this experiment was to determine if the Missouri virus
samples DNA fingerprint more closely resembled the DNA fingerprint of either the
Pennsylvania or Alabama samples in order to determine if the virus was a deadly
strain via the use of DNA fingerprinting by Electrophoresis.
Hypothesis:
The DNA fingerprint of the Missouri virus sample will have the same band
placement as the Pennsylvania virus sample indicating that the Missouri virus is not
the same deadly strain as the Alabama virus.
Methods:
Cast Agarose Gel
The gel-casting tray pieces were gathered and assembled. Tape was used to
seal the edges of the trays, and the well-forming comb was put into place to forms
the wells to place the DNA samples in. Agarose gel was then poured into the mold
until it reached a height of about 1/3 of the way up the teeth of the well-forming
comb. The gel was then allowed to rest until it became solid, which took about 15
minutes. Once the gel was set, the agarose tray was played on the platform of a gel
box so that the combed end was nearest the black lines on the tray.
Loading the Samples
Three micro-centrifuge tubes were labeled Alabama, Missouri and
Pennsylvania, and then loaded with small samples of each of the respective virus
samples that were already processed with a restrictive enzyme. The same enzyme
was used for all three samples in preparation so that the DNA was clipped in all of
the same placed for each virus respectively. The viral DNA samples were then mixed
with bromophenol blue dye to make the DNA fragments more visible when passing
through the Agarose gel, but also to cause the DNA to sink to the bottom of the
wells and not diffuse into the buffer solution. A fresh micro pipet tip was place on a
micropipeter and 200 L of the Pennsylvania sample was taken from the microcentrifuge tube and placed in well 3 of the agarose gel tray. The tip was then
discarded, and a fresh one was reapplied to the device. The following was then
repeated for the Alabama sample being placed in well 4 and finally for the Missouri
sample that was placed in well 5.
Electrophorese
The tray was then placed inside of the electrophorese chamber with the well
closest to the black (negative) terminals so that when the device is turned on the
DNA fragments will move with the charge from the negative side down the gel tray
towards the positively charged side. The box was then slowly filled with Tris-BorateEDTA buffer which conducts electricity better than water does. The buffer was used
in the same concentration as the Agarose tray so that there would be not negative
effects on the conduction of electricity across the Agarose gel. Once the chamber

was filled to a level slightly above the top of the Agarose tray, the lid was placed on
the chamber with the black leads meeting and the red leads meeting respectively.
The power supply was then set to a voltage of 90 volts and then turned on. The tray
was allowed to run for one hour then was turned off, and the tray was removed then
allowed to dry for observations.
Results:

Figure 1: Fingerprint of Virus DNA for Pennsylvania, Alabama & Missouri strains on a
Agarose plate after undergoing electrophorese for one hour at 90 Volts.
Viral DNA Fingerprint
Pennsylvani
Alabama
Missouri
a

Figure 2: Drawing of Virus DNA fingerprints for Pennsylvania, Alabama & Missouri
strains on a Agarose plate after undergoing electrophorese for one hour at 90 Volts.

The DNA fingerprint of the Pennsylvania sample has a large gap between the
origin at the well until the first band is seen. Following it is a 1 cm gap and another
dark thick band. From here, there is another band 1.5 cm down that is very thin. It is
followed closely by three very thin and faint bands every cm until the fingerprint
ends.
For the Alabama strain there is a faint band very near to the well where the
sample was originating from. Slightly below it is another very faint band at a
distance of about 1/8 cm. Two cm down from this band there is an extremely faint
thin band towards the middle of the length of the agarose gel.
The DNA fingerprint of the Missouri sample has a large gap between the
origin at the well until the first band is seen. Following it is a 1 cm gap and another
dark thick band. From here, there is another band 1.5 cm down that is very thin. It is
followed closely by three very thin and faint bands every cm until the fingerprint
ends. These final three bands are much fainter than those of the Pennsylvania
sample. It also appears that there may be one thick band 2 cm down from these
three, but with the imaging the results were not definitive that this was the case.
Conclusion:
The DNA fingerprint of the Pennsylvania virus sample contained six DNA
bands, with three of those bands in a very close formation to the bottom of the
agarose gel. The Alabama sample only contained three bands of DNA, two of which
were very close to the well unlike that of the Pennsylvania strand. This difference in
the numbers of bands is significant to show that these samples differ on the genetic
level. The Missouri sample contained six DNA bands, similarly to the Pennsylvania
sample, and also had an arrangement of three thick bands towards the middle of
the agarose gel plate. There was also another similar formation of three thin bands
at the bottom of the gel, nearly mirroring that of the Pennsylvania fingerprint.
By having the same quantity of bands, and very similar placements, it
indicates that the Pennsylvania virus and Missouri virus are closely related, and thus
this virus is not the deadly strain that was seen in the Alabama outbreak. We came
to this conclusion due to the same restriction enzymes being utilized to cut the DNA
of all of the samples at the same base pairs, leading to the assumption that if two
genomes are similar then the size of the molecule fragments created by this cutting
would also be similar. Once the DNA fingerprint of each virus was created, two
similar viruses would have the same molecule size, indicating that if their
fingerprints matched; hence that the Pennsylvania and Missouri viruses are the
same.
Our original hypothesis that the Missouri virus sample would have the same
DNA band size and placement as the Pennsylvania sample proved to be valid, and
thus confirmed that the Missouri virus was not a deadly strain.
DNA fingerprinting is commonly used in forensic sciences. Single-Cell DNA
profiling is particularly utilized in the identification of perpetrators in rape cases.
This technique is also used in the formation of DNA profiles to compile databases for

the identification of individuals and organisms, such as the FBI Combined DNA Index
System, to be utilized to compare collected samples for quick identification. DNA
fingerprinting is also utilized in paternity tests, and in the identification of a body if
there is too severe of damage to it for alternative identification. Another manner in
which this technology is used is in the diagnosis of inherited disorders (cystic
fibrosis, hemophilia, Huntingtons disease, etc.) between parents and offspring,
where DNA may be analyzed to see if the genetic traits are present for early
detection and treatment. (Betsch)

Works Cited:
Betsch, David F. "DNA Fingerprinting in Human Health and Society." DNA Fingerprinting in Human
Health and Society. National Health Museum, n.d. Web. 14 Nov. 2014.
<http://www.accessexcellence.org/RC/AB/BA/DNA_Fingerprinting_Basics.php>.
"Forensic Science Central." DNA Analysis. Forensic Science Central, n.d. Web. 14 Nov. 2014.
<http://forensicsciencecentral.co.uk/dna.shtml>.
"How Can Biotechnology Benefit You?" Europabio. Europa Bio, n.d. Web. 16 Nov. 2014.
<http://www.europabio.org/how-can-biotechnology-benefit-you>.
The Editors of Encyclopdia Britannica. "DNA Fingerprinting." Encyclopedia Britannica Online.
Encyclopedia Britannica, 17 Feb. 2014. Web. 16 Nov. 2014.
<http://www.britannica.com/EBchecked/topic/167155/DNA-fingerprinting>.

"What Is Biotechnology?" Biotechnology Industry Organization, n.d. Web. 16 Nov. 2014.


<http://www.bio.org/articles/what-biotechnology>.