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DNA Extraction from Live Organism and Analysis Using

Agarose Gel Electrophoresis


1

Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman,
Quezon City, Philippines
2
Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman,
Quezon City, Philippines
ABSTRACT:
This experiment aimed to understand the underlying principles behind the extraction and characterization of DNA
sample from duck egg embryo. Using the DNAs basic unit, nucleic acid, the purity and concentration was assessed
through UV-Vis Spectrophotometry at wavelengths 260 nm and 280 nm. For further analysis if the DNA sample,
agarose gel electrophoresis was employed. This technique separates DNA molecules based on its size and shape.
DNA was successfully extracted from the fertilized duck embryo which was calculated to have a low yield of 6.6% (w/v),
an estimated concentration of 23.05023334 g/mL and 10% purity.

INTRODUCTION
DNA carries genetic instructions essential in growth,
development and functioning for all known living
organisms. DNA is responsible for coding most of the
genetic information in an organism and is expressed in
an organisms physical appearance, personality, and
behavior. In 1969, the first isolation of DNA was done
by Friedrich Miescher. Combinations of mechanical
and chemical procedures are done to extract DNA from
a sample.
In this experiment, the characterization technique used
to analyze the DNA sample from the fertilized duck egg
was UV spectroscopy and agarose gel electrophoresis.
Double beam UV-Vis spectrophotometry can be used
to analyze DNA and its concentration and purity. The
aromatic nucleotide bases found in DNA absorb UV
light that allows detection of the presence DNA.
Besides UV light absorption, further analysis of the
purity of the DNA extract was done using
electrophoretic methods. Electrophoresis is an
experimental method based on the differential
movement of charged molecules in an electric field. It
can also be used to purify and analyze many other
biomolecules. The movement of molecules takes place
in a polymerized gel matrix, which was utilized because
it is more stable as compared to a free solution.
Application of electric current administers migration of
the different molecules across the rigid gel matrix. The
difference in movement of molecules was influenced by
molecule size, shape, charge, and chemical
composition.

Agarose was used as the gel medium for the analysis


of DNA. In agarose gel electrophoresis, the DNA
molecules move through the electric field due to charge
but because the molecules have an equal charge to
mass ratio, the basis of separation depends on the size
and shape of the DNA molecules. Nucleic acids
migrate at a rate inversely proportional to its size.
The objective of this experiment was to understand the
principles behind agarose gel electrophoresis and
apply these concepts to analyze DNA. Specifically, the
goal of this experiment is to extract DNA from muscle
tissues of the duck embryo and to estimate and assess
the purity and concentration using UV spectroscopy
and estimate the molar weight of the extracted DNA.
EXPERIMENTAL DETAILS
For the extraction and purification of DNA, the following
equipment were utilized: top-loading balance, J-tube,
quartz
cuvette,
and
double
beam
UV-Vis
spectrophotometer.
DNA sample was extracted from the duck egg embryo.
2.0 g sample was weighed and cut into smaller pieces
over ice. Extraneous tissues such as eyes, innards,
appendages, and feathers were removed from the
sample.
The sample was suspended in 10.0 mL 0.05 Tris-HCl
buffer pH 8.0, which is pre-heated in at 555C. 10% SDS
was slowly added to the solution to make a solution
with final 1% SDS concentration. The solution was
incubated in a water bath at 555C for 45 mins. The
viscosity of the sample was observed every 10
minutes. Ethyl acetate was added instead of
chloroform to remove proteins and RNA in the sample.

The sample was then centrifuged twice for five


minutes. To the aqueous layer, 5.0 M NaCl was added.
Addition of 2.5 equivalent volume of cooled 95%
ethanol completes alcohol precipitation together with
the salt resulting to fibrous white precipitates at the
surface which are clumped DNA. The clumps were
then removed using a J-tube and air-dried. The sample
was then resuspended in 10.0 mL 0.05 M Tris-EDTA
buffer pH 8.0.
To determine the nucleic acid concentration and purity,
500 L DNA solution was pipetted out from the solution
that was diluted to 5.0 mL 0.05 M Tris-EDTA buffer pH
8.0. The absorbance was read at 260 and 280 nm with
0.05 M Tris-EDTA buffer pH 8.0 as blank.
For further analysis of the DNA sample, agarose gel
electrophoresis was employed. AGE apparatus and
accessories were used in this experiment.
First, the gel was prepared from 0.175 g gel powder
dissolved in 25 mL 1X TAE buffer. The mixture was
heated until transparent but was not allowed to boil.
The solution was cooled to 375C. 300 L EtBr was
added and swirled to mix. The solution was then
poured carefully and smoothly to the tray to prevent air
bubbles from forming. The comb was placed into the
gel. Solidification of the gel took place for 20-30 mins.
The comb was removed carefully and the wells were
flushed with buffer before the sample was loaded.
For sample loading and preparation, 30 L of loading
buffer was pipetted out to two pieces of parafilm. 70 L
of the sample was added and mixed to the loading
buffer. 20 L of the mixture was loaded on the well.
Puncturing the bottom of the well with the pipette tip or
spillage of excess sample was avoided so that other
samples in the well will not be contaminated.
The gel chamber was then filled with running buffer
with the gel immersed completely. The power supply
was set to 40-60 V. The running of the gel took place
for 30-45 mins. The resulting gel was visualized using a
lightbox.
RESULTS AND DISCUSSION
It was made sure that the balut sample was alive and
fresh by observing movements in the egg. After cell
death, enzymes break the bond that makes up the
DNA
backbone.
Other
factors,
such
as
microorganisms, help in the decay of the DNA
backbone that causes destruction of the nucleic acid
(Kaplan, 2012). Extraneous tissues such as eyes,
innards, appendages, and feathers were removed from
the sample so the yield will not be lowered. Cutting the
sample in ice prevented DNA degradation by nuclease
enzymes.

Tris-HCl buffer pH 8.0, which is pre-heated in at 5 55C


was added to the sample to set proper pH conditions
for DNA. The optimal activity of degrading enzymes
was avoided by maintaining pH through the buffer
systems (Weising, 2005). Aside from stabilizing the pH,
the buffer also prevents DNA degradation by
deactivating nucleases. The temperature enables the
proteins to denature while keeping the DNA intact.
10% SDS was added to the solution to make a solution
with final 1% SDS concentration. Membranes were
destroyed and proteins were denatured until it is
dissociated from the DNA by adding detergents
(Weising, 2005).
Ethyl acetate was added instead of chloroform to
remove proteins and RNA in the sample. To the
aqueous layer, 5.0 M NaCl was added to neutralize the
negative charges the DNA contains so the molecules
can aggregate together and so the nucleic acids
precipitate. Addition of 2.5 equivalent volume of cooled
95% ethanol completes alcohol precipitation together
with the salt resulting to fibrous white precipitates at the
surface which are clumped DNA. Tris-EDTA buffer pH
8.0 prevents degradation of DNA. DNA degradation
happens when EDTA, a chelating agent, divalent ions
(Gad, 2007). Depurination is an acid catalyzed process
which also leads to DNA degradation (Mahato, 2005).
These processes were prevented by maintaining an
alkali environment.
The isolated DNA sample contains aromatic nucleotide
bases such as adenine, guanine, thymine and
cytosine. In order to determine the nucleic acid
concentration of the DNA, UV spectroscopy was
employed because these nucleotide bases have the
ability to absorb UV light due to the rich amount of
electrons found in their aromatic rings, carbonyl
groups, and nitrogen and oxygen atoms. The
absorbance was measured at 260 nm and 280 nm
wavelengths. 260 nm is the maximum wavelength
where nucleic acids can absorb UV light.
Table 1. UV absorbance readings of balut DNA
sample
Wavelength
UV Absorbance
260 nm
4.610046667
280 nm
4.111303333
Aside from UV spectroscopy, thermal denaturation is
also used to analyze DNA. In this technique, the DNA
solution is treated with denaturing agents and then UV
absorbance is measured. The resulting reading in the
UV absorbance shows a spike upon addition of
denaturing agents and increase in temperature.
Absorbance increases due to alterations in the
resonance behavior of the aromatic rings found in the

bases. This method can identify unknown DNA


samples by matching it with the known meting
temperature values. However, DNA samples will be
denatured and cannot be recovered in its native form.
Another method is a flourometric procedure that uses
the dye Bisbenzmide (Hoechst Dye-H33258) which is
non-intercalating and binds to the minor groove of DNA
which gives 458 nm. This technique is simple and
sensitive. Advantages are accurate DNA estimation
can be done due to the changes in fluorescence
characteristics exhibited by the dye in the presence of
DNA and it does not bind with RNA so the measured
fluorescence is a direct indicator of DNA concentration.
The disadvantage, however, is that it is limited to the
use of a clean DNA standard of known concentration.
Its sensitivity decreases as the nuclease degrades
which increases the denaturation of the DNA.

for visualization. Addition of Ethidium bromide enables


band detection. It complexes with the nucleic acid to
emit visible orange light when absorbing invisible UV
light (Odgens & Adams, 1987) and has a fluorescence
which is 10 times moe intense than free EtBr (Cheng &
Zhang, 2010).
Figure 2. DNA Band Profile (AGE)

Besides those mentioned, another technique which can


be used to characterize DNA extracts is Agarose Gel
Electrophoresis (AGE).
Figure 1. Agarose Gel Electrophoresis Diagram

The resulting gel for this experiment is shown in Figure


2. The bands are visible and the resolution of the gel is
high. The distance traveled by the sample is the same
for all groups which is 2.5 cm.

AGE uses a fluorescent dye which is ethidium bromide


to stain DNA and subject it to UV irradiation at 254 nm.
This technique is very rapid and sensitive in terms of
estimating the nucleic acid concentration of the extract.
Agarose gel electrophoresis can quantify large number
of samples including DNAs as small as 5 ng. It also
analyzes the quality of the DNA preparation which
gives it an edge in the quantification of DNA extracts. If
the sample is contaminated with RNA or sheared DNA,
it can be visually identified on the gel.
In the experiment, Tris-Acetate-EDTA (TAE) buffer at
pH 8.0 was used. It is a running buffer used for large
fragments of DNA. It is prepared at pH 8 with Tris base
which inhibits buffer depletion because of its movement
slower than small ions made possible by its low charge
to mass ratio (National Diagnostics, 2011); glacial
acetic acid which allows migration through the electric
field (Zoski, 2007); and EDTA which chelates divalent
cations needed by DNAses and prevents degradation
of the sample in the gel (Burden & Whitney, 1995).
Another one used was the loading buffer composed of
glycerol. The loading buffer makes the solution denser
sink deeper to the well and contains bromophenol blue

Good band profile depends on right agarose


concentration, type of dye used in visualizing the
bands, optimum voltage applied, the right buffer and
the amount of DNA used. For larger molecules, lower
agarose concentration is preferred while higher
concentration of agarose for smaller molecules (Gouqing & Douches, nd.). The optimum voltage for
fragments larger than 2kb is more or less 5 volts/cm
(Agarose Gel Electrophoresis of DNA, 2000). Too low
voltage reduces DNA mobility thereby broadening the
band. High voltage may result to overheating reducing
band resolution (Thermo Fisher Scientific Inc., 2014).
Agarose gel electrophoresis is widely used nowadays
for DNA testing since it is relatively easy and definitive
way to test and compare DNA samples. It is also used
in forensics to obtain link to possible suspect. This
technique is also used in paternity testing.
CONCLUSION AND RECOMMENDATION
The resulting duck embryo DNA solution was
calculated to be 6.6% (w/v). There might have been a
low yield due to errors in the execution of the
procedure or inability to use the suggested reagents in
the manual such as liquid nitrogen and proteinase K.
For future experiments of the same nature, it is

recommended that the procedures be followed


carefully and strictly and use the products suggested in
the manual as to increase the product yield.
The calculated percent purity and estimated DNA
concentration of the sample based on the UV
absorbance readings was calculated to be 10% and
23.05023334 g/mL respectively. 10% nucleic acid is a
relatively low percentage considering the series of
procedures performed to isolate and purify the DNA
sample. This percentage does not include other
possible contaminants and is only based on the nucleic
acid to protein ratio thus, the actual percent nucleic
acid may be even less if contaminants were put into
consideration. It is suggested that in calculating for
percent nucleic acid, the formula to be used should
take other contaminants into consideration so that the
most accurate quantity of nucleic acid present can be
calculated.
Agarose gel electrophoresis was the method employed
in DNA separation and assessment due to its rapid and
simple process, high resolution, ease of separation,
sensitive staining procedures, cost effective, and ability
to analyze a wide range of molecular weights.
However, too much amount of current applied can melt
either the DNA fragment or the gel itself and produce
inaccurate results or no results at all.
Other probable sources of error in the experiment
include human error, contamination of reagents and
unwanted cleavage of DNA fibers by nucleases that
may not have been denatured.

REFERENCES
Agarose Gel Electrophoresis of DNA.. (2000).
Retrieved
from:
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotec
h/gels/agardna.html

Burden,
D.W.
and
Whitney,
D.B.
(1995).
Biotechnology: Proteins to PCR (A Course in
Strategies and Lab Techniques). USA: Birkhauser
Boston

Carson, S. and Robertson, D. (2005). Manipulation


and Expression of Recombinant DNA. USA: Academic
Press.

Cheng, L. and David, Z. (2010). Molecular Genetic


Pathology. USA: Springer Science & Business Media
Gad, S. (2007). Handbook of pharmaceutical
biotechnology. Hoboken, N.J.: Wiley-Interscience.

Guo-qing, G. and Douches, D. (n.d). Agarose Gel


Electrophoresis
(1st
ed.).
Michigan:
Plant
Biotechnology Resource and Outreach Center.
Retrieved
from:
https://www.msu.edu/course/css/451/Lecture/PTelectrophoresis%20(2009).pdf

Mahato, R. (2005). Biomaterials for delivery and


targeting of proteins and nucleic acids. Boca Raton:
CRC Press.

Odgen, RC and Adams DA (1987). Electrophoresis in


Agarose and Acrylamide Gels Guide to Molecular
Cloning Techniques. 152:61-89

Thermo Fischer Scientific, Inc. (2014). Agarose Gel


Elelectrophoresis Tips and Tricks. Retrieved from Life
Technologies:
http://www.lifetechnologies.com/ph/en/home/lifescience/pcr/elevate-pcr-research/agarosecontent-withtips-and-tricks.html
Weising, K. (2005). DNA fingerprinting in plants. Boca
Raton, FL: Taylor & Francis Group.
Zoski, CG (2007). Handbook of Electrochemistry.
Oxford: Elsevier B.V.

APPENDIX
Weight sample: 0.66 g
___0.66 g balut DNA___ x 100 = 6.6% w/v
10 mL Tris-EDTA buffer
Raw Data for UV-Vis Spectrophotometry

Figure 2. DNA Band Profile (Age)

Plate 1: 1 - Wavelength: 260


nm
Value
1
2
A
3.90803 3.92845
B
4.38836 4.38805
C
4.61486 4.70054
D
4.24429 4.24641
E
3.79941
3.783
Plate 1: 1 - Wavelength: 280
nm

3
3.91837
4.42574
4.51474
4.2403
3.79224

Value
A
B
C
D
E

3
2.70785
3.88162
4.0212
3.75354
3.26792

1
2.73064
3.84675
4.10046
3.74077
3.27303

2
2.70431
3.8682
4.21225
3.74598
3.24616

dsDNA concentration=

50ug
1
x A 260 x df =50 x 4.610046667 x =23.05023334 ug / mL
mL
10

A 260 4.610046667
=
=1.1213 %Nucleic acid=10
A 280 4.111303333

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