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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 Agarose was used as the gel medium for the analysis
of DNA. In agarose gel electrophoresis, the DNA
DNA carries genetic instructions essential in growth, molecules move through the electric field due to charge
development and functioning for all known living but because the molecules have an equal charge to
organisms. DNA is responsible for coding most of the mass ratio, the basis of separation depends on the size
genetic information in an organism and is expressed in and shape of the DNA molecules. Nucleic acids
an organisms physical appearance, personality, and migrate at a rate inversely proportional to its size.
behavior. In 1969, the first isolation of DNA was done
by Friedrich Miescher. Combinations of mechanical The objective of this experiment was to understand the
and chemical procedures are done to extract DNA from principles behind agarose gel electrophoresis and
a sample. apply these concepts to analyze DNA. Specifically, the
goal of this experiment is to extract DNA from muscle
In this experiment, the characterization technique used tissues of the duck embryo and to estimate and assess
to analyze the DNA sample from the fertilized duck egg the purity and concentration using UV spectroscopy
was UV spectroscopy and agarose gel electrophoresis. and estimate the molar weight of the extracted DNA.
For sample loading and preparation, 30 L of loading The isolated DNA sample contains aromatic nucleotide
buffer was pipetted out to two pieces of parafilm. 70 L bases such as adenine, guanine, thymine and
of the sample was added and mixed to the loading cytosine. In order to determine the nucleic acid
buffer. 20 L of the mixture was loaded on the well. concentration of the DNA, UV spectroscopy was
Puncturing the bottom of the well with the pipette tip or employed because these nucleotide bases have the
spillage of excess sample was avoided so that other ability to absorb UV light due to the rich amount of
samples in the well will not be contaminated. electrons found in their aromatic rings, carbonyl
groups, and nitrogen and oxygen atoms. The
The gel chamber was then filled with running buffer
absorbance was measured at 260 nm and 280 nm
with the gel immersed completely. The power supply
wavelengths. 260 nm is the maximum wavelength
was set to 40-60 V. The running of the gel took place
where nucleic acids can absorb UV light.
for 30-45 mins. The resulting gel was visualized using a
lightbox. Table 1. UV absorbance readings of balut DNA
sample
RESULTS AND DISCUSSION
Wavelength UV Absorbance
260 nm 4.610046667
It was made sure that the balut sample was alive and
280 nm 4.111303333
fresh by observing movements in the egg. After cell
death, enzymes break the bond that makes up the
DNA backbone. Other factors, such as Aside from UV spectroscopy, thermal denaturation is
microorganisms, help in the decay of the DNA also used to analyze DNA. In this technique, the DNA
backbone that causes destruction of the nucleic acid solution is treated with denaturing agents and then UV
(Kaplan, 2012). Extraneous tissues such as eyes, absorbance is measured. The resulting reading in the
innards, appendages, and feathers were removed from UV absorbance shows a spike upon addition of
the sample so the yield will not be lowered. Cutting the denaturing agents and increase in temperature.
sample in ice prevented DNA degradation by nuclease Absorbance increases due to alterations in the
enzymes. resonance behavior of the aromatic rings found in the
bases. This method can identify unknown DNA for visualization. Addition of Ethidium bromide enables
samples by matching it with the known meting band detection. It complexes with the nucleic acid to
temperature values. However, DNA samples will be emit visible orange light when absorbing invisible UV
denatured and cannot be recovered in its native form. light (Odgens & Adams, 1987) and has a fluorescence
which is 10 times moe intense than free EtBr (Cheng &
Another method is a flourometric procedure that uses Zhang, 2010).
the dye Bisbenzmide (Hoechst Dye-H33258) which is
non-intercalating and binds to the minor groove of DNA Figure 2. DNA Band Profile (AGE)
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.
In the experiment, Tris-Acetate-EDTA (TAE) buffer at Agarose gel electrophoresis is widely used nowadays
pH 8.0 was used. It is a running buffer used for large for DNA testing since it is relatively easy and definitive
fragments of DNA. It is prepared at pH 8 with Tris base way to test and compare DNA samples. It is also used
which inhibits buffer depletion because of its movement in forensics to obtain link to possible suspect. This
slower than small ions made possible by its low charge technique is also used in paternity testing.
to mass ratio (National Diagnostics, 2011); glacial
acetic acid which allows migration through the electric CONCLUSION AND RECOMMENDATION
field (Zoski, 2007); and EDTA which chelates divalent
The resulting duck embryo DNA solution was
cations needed by DNAses and prevents degradation
calculated to be 6.6% (w/v). There might have been a
of the sample in the gel (Burden & Whitney, 1995).
low yield due to errors in the execution of the
Another one used was the loading buffer composed of
procedure or inability to use the suggested reagents in
glycerol. The loading buffer makes the solution denser
the manual such as liquid nitrogen and proteinase K.
sink deeper to the well and contains bromophenol blue
For future experiments of the same nature, it is
recommended that the procedures be followed Cheng, L. and David, Z. (2010). Molecular Genetic
carefully and strictly and use the products suggested in Pathology. USA: Springer Science & Business Media
the manual as to increase the product yield.
Gad, S. (2007). Handbook of pharmaceutical
The calculated percent purity and estimated DNA biotechnology. Hoboken, N.J.: Wiley-Interscience.
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 Guo-qing, G. and Douches, D. (n.d). Agarose Gel
procedures performed to isolate and purify the DNA Electrophoresis (1st ed.). Michigan: Plant
sample. This percentage does not include other Biotechnology Resource and Outreach Center.
possible contaminants and is only based on the nucleic Retrieved from:
acid to protein ratio thus, the actual percent nucleic https://www.msu.edu/course/css/451/Lecture/PT-
acid may be even less if contaminants were put into electrophoresis%20(2009).pdf
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
Mahato, R. (2005). Biomaterials for delivery and
calculated.
targeting of proteins and nucleic acids. Boca Raton:
Agarose gel electrophoresis was the method employed CRC Press.
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. Odgen, RC and Adams DA (1987). Electrophoresis in
However, too much amount of current applied can melt Agarose and Acrylamide Gels Guide to Molecular
either the DNA fragment or the gel itself and produce Cloning Techniques. 152:61-89
inaccurate results or no results at all.
Agarose Gel Electrophoresis of DNA.. (2000). Weising, K. (2005). DNA fingerprinting in plants. Boca
Retrieved from: Raton, FL: Taylor & Francis Group.
http://arbl.cvmbs.colostate.edu/hbooks/genetics/biotec
h/gels/agardna.html Zoski, CG (2007). Handbook of Electrochemistry.
Oxford: Elsevier B.V.
Value 1 2 3
A 3.90803 3.92845 3.91837
B 4.38836 4.38805 4.42574
C 4.61486 4.70054 4.51474
D 4.24429 4.24641 4.2403
E 3.79941 3.783 3.79224
Plate 1: 1 - Wavelength: 280
nm
Value 1 2 3
A 2.73064 2.70431 2.70785
B 3.84675 3.8682 3.88162
C 4.10046 4.21225 4.0212
D 3.74077 3.74598 3.75354
E 3.27303 3.24616 3.26792
50ug 1
dsDNA concentration= 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