Extraction of DNA from Live Organism and Agarose Gel

Electrophoresis of DNA
Natalie B. Nisce
Department of Food Science and Nutrition, College of Home Economics, University of the Philippines Diliman,
Quezon City, Philippines
ABSTRACT The following experiment describes the process and principles behind extracting DNA from live shrimps
and assessing the DNA extracts concentration and purity, based on the detection of its basic unit, nucleic acid. This
experiment aims to obtain an extract with high nucleic acid purity and concentration. The extraction process involves
cell lysis, the denaturing of proteins and destruction of protein-DNA complexes. The purity of the nucleic acid obtained
was assessed using a double beam UV-vis spectrophotometer at wavelengths 260nm and 280nm. The recorded
absorbance values were 0.6598 and 0.4757 for 260nm and 280nm, respectively. The % nucleic acid of the extract was
10% and the concentration of DNA was computed to be 3.299
. To further evaluate the purity of the extracted
DNA, the obtained sample was subjected to agarose gel electrophoresis. However, due to possible old reagents, the
gel did not impart clear bands. Apart from the agarose gel electrophresis, it can be concluded that the methods used in
this experiment are significant in the extraction and overall study of DNA.

INTRODUCTION
All living organisms contain the genetic carriers,
DNA. With the exception of identical siblings, each
organism carries a different set of DNA, making each
one unique from the other. This is because DNA is
responsible for coding most of the genetic information
in an organism and is expressed in an organisms
physical appearance, personality, and behavior. With
the enormous amount of information that completes an
organisms genetic make up, it is astounding to
discover how it all fits within the DNA molecule. The
answer lies within the DNAs double-stranded helix
structure.
The basic units of DNA are nucleotides.
Nucleotides are made up of three covalently bonded
parts, a nitrogenous base (purines: adenine and
guanine, pyrimidines: thymine and cytosine), a
deoxyribose sugar, and a phosphoric acid residue. An
N-glycosidic bond links the C-1 carbon of the
deoxyribose sugar to the N-9 nitrogen in purines or the
N-1 nitrogen in pyrimidines.

Figure 8.1 DNA Nucleotide structure

Nucleotides form nucleic acids through

polymerization. The repeating 3-5 phosphodiester
bonds form in the DNA backbone between phosphoric

acid and the 3 and 5 carbons of adjacent deoxyribose

sugars.

Figure 8.2 Phosphodiester bond connecting two

nucleotides
The four nucleotide bases combine in different
sequences to form the essential information of genes.
The nucleotide bases from a single DNA strand form
hydrogen bonds with their complementary nucleotide
bases from the second strand to form the doublestranded structure. Adenine forms two hydrogen bonds
with thymine while guanine forms three hydrogen
bonds with cytosine.

The goal of this experiment is to extract DNA

from the muscle tissue of live shrimp and to rationalize
the procedure employed in the extraction. The
characterization of the DNA extract will be determined
by estimating the concentration and purity through UV
spectroscopy and agarose gel elctrophoresis.
EXPERIMENTAL DETAILS
The experiment was divided into two parts, the
first part was where the DNA was extracted from the
live shrimp and purified and the second part was the
analysis of the purified DNA sample.
Figure 8.3 Nucleotide base pairing between the
DNA double helix
The mentioned structure can be found in DNAs
ideal functioning form. Unfortunately, genes may
mutate and cause severe and even fatal diseases.
Unlocking the secrets behind DNA codes would be
beneficial to understanding the causes and functions of
many diseases caused by genetic mutations. Human
genetic engineering and gene therapy may be the key
to creating cures and more importantly, preventive
medicine. To study DNA, first it must be extracted and
isolated.

In the first part, the DNA sample was acquired

from fresh shrimp. The shrimp head, tail, legs, and
exoskeleton were removed and discarded. The
muscular meat was obtained and cut into smaller
pieces over ice. The sample, along with liquid nitrogen,
was pulverized through grinding in circular motion.

DNA molecules are large and fragile thus,

extracting a pure, undamaged, large yield needs to
follow a carefully calculated procedure set within ideal
conditions to keep extracted DNA stable. The ideal
conditions that must be considered include pH,
temperature, ionic strength, cellular conditions, and
mechanical stress.
Upon extraction and isolation, double-beam
UV-vis spectrophotometry can be used to determine
the concentration and purity of the DNA extract. The
aromatic nucleotide bases found in DNA absorb UV
light, allowing this method to detect the presence of
DNA.
Aside from UV light absorption, further
assessment of the purity of the DNA extract was
performed through electrophoretic methods.
Electrophoresis is an experimental method based on
the differential movement of charged molecules in an
electric field and can be used to purify and analyze
many biomolecules. The movement of molecules takes
place in a polymerized gel matrix, which is used
because it is more stable as compared to a free
solution. Applying electric current causes migration of
the different molecules across the rigid gel matrix. The
difference in movement of molecules is influenced by
molecule size, shape, charge, and chemical
composition.
Agarose was chosen 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.

Figure 8.4 Grinding of shrimp sample with liquid

nitrogen
The pulverized sample was placed inside two
centrifuge tubes, one tube containing 0.34g and the
other containing 0.31g of the sample. Then, the
samples were suspended in 10mL of pre-heated 0.05
M Tris-HCl buffer, pH 8.0 at 55 . Next, around 1mL 10%
SDS was added dropwise to the sides of the tube to
get a final concentration of 1% SDS.
The centrifuge tubes were incubated by
submerging the tubes in a heated water bath at 55
for 45 minutes, while gently shaking the tubes every 10
minutes. Chloroform was added slowly, dropwise to the
sides of the tubes. The tubes were shaken then,
centrifuged twice, five minutes each time.
A wide-tipped Pasteur pipette was used to
collect the aqueous layer and transferred to a small
beaker. 5 M NaCl was then added to the collected
aqueous layer. 95% ethanol was added to the beaker.
The solution was put inside tubes and centrifuged for 5
minutes. DNA appeared as fibrous white precipitate.
The solution was decanted and the precipitate was airdried.

In preparing the sample for DNA extraction, the

sample was cut into small pieces over ice. Ice was
needed to prevent DNA degradation by nuclease
enzymes. The sample was cut into small pieces to aid
in cell lysis to release the DNA from the cell. Liquid
nitrogen was also used for the purpose of destroying
the cell membrane and keeping the sample at a very
cold temperature. Upon adding liquid nitrogen, the
shrimp sample hardened up and froze, making it easier
to grind the sample.

Figure 8.5 Isolated DNA extract

Lastly, the DNA was dissolved using 10 mL
0.05 M Tris-EDTA buffer at pH 8.0. The concentration
in % (w/v) of the stock solution was obtained.
For the second part of the experiment, 40 L of
the sample was pipetted out and diluted to 5.0 mL
using the Tris-EDTA buffer at pH 8.0. The absorbance
of the solution was read at 260 and 280 nm against the
Tris-EDTA buffer pH 8.0 as blank. From this, the ratio
of A260 and A280 was calculated and the % DNA purity
was obtained. The DNA concentration was also
estimated by following a specific formula.
Agarose gel electrophoresis was employed to
further analyze the DNA extracts purity. The gel was
prepared from 0.32 g gel powder, which was mixed in
32 mL of 1X TAE buffer. The mixture was heated and
mixed constantly but prevented from boiling. The
agarose solution was removed from heat once it
became completely transparent. It was allowed to cool
to 37 C. 300 L of ethidium bromide was added and
swirled to mix.
The solution was poured carefully and smoothly
into the gel tray to prevent air bubbles from forming.
The comb was placed over the gel but was not allowed
to touch the bottom of the gel. Once the gel solidified
after 20-30 minutes at room temperature, the comb
was removed from the gel in one fluid motion.

Tris-HCl buffer pH 8.0 was added to the

sample to set the proper pH conditions for DNA. The
bonds stabilizing the DNA molecule are all stable at pH
8.0. H-bonds are stable within pH 4-10, phosphodiester
linkages are stable within pH 3-12, and glycosidic
bonds are stable at pH 4 and up. Aside from stabilizing
DNA, higher pH levels also deactivate nucleases,
which in turn prevent DNA from being degraded. The
buffer was also heated to 55
At this temperature,
most proteins will denature whereas DNA can remain
intact up to 80 .
Like proteins, DNA structure can be viewed in
levels. The primary structure is the order of the bases
in the polynucleotide sequence. The secondary
structure is the 3D conformation of the backbone. The
tertiary structure is the supercoiling of the double
strands into a helix. Supercoiling occurs to make all the
information found in DNA fit into a very compact space.
In eukaryotic DNA, supercoiling occurs by
formation of protein-DNA complexes called chromatin.
These complexes form through electrostatic interaction
between negatively charged phosphate groups found
in the DNA backbone and positively charged histone
proteins. When isolating DNA, DNA must be freed from
these complexes.
At pH 8.0 electrostatic interactions between
DNA and histones are reduced. Sodium dodecyl
sulfate (SDS), an anionic detergent further breaks
protein-DNA complexes by reducing the positive
character of histones. SDS disrupts ionic interactions
between the positively charged histones and the
negatively charged phosphates on the backbone of
DNA. SDS also denatures deoxyribonucleases and
other proteins.

The wells were flushed with 1X TAE buffer

before the samples were added. The sample was
prepared by adding 30 L loading buffer into an
eppendorf tube before pipetting 70 L DNA sample into
the same tube. A pipette tip was used to mix the
solutions. 20 L of the solution was loaded into the well.
Care was made not to puncture the bottom of the gel
with the pipette tip or spill sample out of the wells.

Denatured proteins were removed from the

sample solution by adding chloroform. Chloroform
promotes separation of organic and aqueous phases.
Denatured proteins stay in the organic phase while
DNA remains in the aqueous phase. To further initiate
separation of layers, the sample solutions were
centrifuged.

The gel chamber was filled with running buffer

until the gel containing the sample was completely
immersed. The power supply was set to 60 V. The
apparatus was turned off once the tracking dye
reached 80% of the gel length.

After centrifugation, the aqueous layer

containing the DNA was collected. 5M NaCl, a high
concentration salt, was added to remove bound
cationic amines and to dissociate any leftover proteins.
The salt weakens ionic interactions between DNA and
cations.

RESULTS AND DISCUSSION

Lastly, ethanol, an organic solvent, was added

to precipitate DNA. Ethanol works to precipitate DNA
by making the aqueous medium less polar. DNA
precipitate is threadlike in appearance due to its long
standed supercoiled double helix structure. The DNA
obtained was resuspended in 0.05M Tris-EDTA buffer
pH 8.0 to keep the sample in stable conditions.
UV spectroscopy was used to determine the
nucleic acid concentration and purity of the extracted
and isolated DNA sample. Nucleic acids contain
aromatic nucleotide bases adenine, guanine, thymine
and cytosine. These nucleotide bases can absorb UV
light due to the rich amount of electrons found in their
aromatic rings, carbonyl groups, and nitrogen and
oxygen atoms. Nucleic acids absorb UV light at a
maximum of 260nm. UV absorption was also
measured at 280nm to account for the possible
absorption of proteins still present in the DNA solution.
Table 8.1 UV absorbance readings of shrimp DNA
sample
Wavelength
UV absorbance
260nm
0.6598
280nm
0.4757

Besides direct measure of UV spectroscopy,

thermal denaturation is another method that can be
used to characterize DNA. In thermal denaturation, the
DNA solution is treated with denaturing agents and
then UV absorbance is measured. The UV absorbance
shows notable increase after addition of denaturing
agents and increase in temperature. In denatured DNA,
there is minimal base-to-base interaction, which alters
the resonance behavior of the aromatic rings found in
the bases and thus, absorption increases. The
midpoint in the absorption increase is called melting
temperature. Each DNA has a characteristic melting
temperature value. The advantage of this method is
that it can identify unknown DNA samples by matching
it with the known meting temperature values. A
disadvantage is that the DNA samples will be
denatured and cannot be recovered in its native form.

DNA molecules are negatively charged at

neutral pH due to the presence of phosphate groups.
The negative charge of the DNA molecules cause them
to move toward the positive electrode at the opposite
end of the gel. Each nucleotide residue contributes to
the overall negative charge of the molecule due to the
amount of phosphate groups. More phosphate groups
means more negative charges but this also means that
the molecule is larger and heavier. This makes the
charge to mass ratio nearly the same for each
molecule thus, without the charge playing a role in
separation, the molecule size and shape are the only
separating factors. The smaller the molecule, the
easier for it to navigate through the cross-linked
agarose gel.
The concentration of the agarose gel also
influences the mobility of the DNA molecules. Agarose
polymers form a network of bundles whose pore sizes
depend on the agarose concentration. A lower
concentration of agarose, around 0.3%, allows for the
sieving of DNA molecules within 5-60 kilobase pairs.
Tris-Acetate-EDTA (TAE) was the
electrophoretic buffer used because of its near neutral
pH, which allows for the negative DNA molecules to
migrate to the anode at the opposite end of the gel.
TAE buffer is commonly used to separate large DNA
because it interacts with the agarose gel resulting in
larger pore size and lower field strength. These
interactions lead to a decrease in gel smearing.
After obtaining the gel after electrophoresis
proper, ethidium bromide was used to detect the bands
formed. Ethidium bromide is a fluorescent assay
commonly used because of its convenience, sensitivity,
and versatility. However, ethidium bromide is highly
toxic and must be handled with utmost precaution. To
limit the amount of ethidium bromide used and its
contact to apparatus, it was administered in-gel,
before the gel solidified. Purines and pyrimidines have
weak fluorescence spectra. Ethidium bromide inserts
between stacked base pairs in nucleic acid and
enhances fluorescence twenty-five fold, making bands
in the gel visible.
CONCLUSION AND RECOMMENDATION

Another method used to assess the purity of

extracted DNA is agarose gel electrophoresis. Agarose
gel electrophoresis is chosen as a gel medium to
analyze larger fragments, such as DNA.

Figure 8.6 Agarose gel electrophoresis

The resulting shrimp DNA solution was

calculated to be 3.4% (w/v). There may have been
some product loss due to an error in the execution of
the procedure. Instead of slowly adding ethanol, a
large amount was added all at once thus, the solution
needed to be centrifuged for product to be collected.
For future experiments of the same nature, it is
recommended that the procedures be followed
carefully and strictly as to not commit careless
mistakes that may lead to product loss and worse,
laboratory accidents.
Based of the UV absorbance readings, the
percent purity and estimated DNA concentration of the
sample was calculated to be 10% and 3.299 g/mL
respectively. 10% nucleic acid is a relatively low
percentage considering the steps performed to isolate

and purify the DNA sample. This may be due to the

error in procedure mentioned. This percentage is only
based on the nucleic acid to protein ratio and does not
include other possible contaminants thus, the true
percent nucleic acid may be even less if the other
contaminants were put into consideration. In
calculating for percent nucleic acid, a formula, which
takes other contaminants into consideration, should be
included in future experiments that require the same
method so that the most accurate amount of nucleic
acid present can be calculated.
Agarose gel electrophoresis is a method often
used in DNA separation and assessment due to its
rapid and simple process, ease of separation, sensitive
staining procedures, high resolution, and ability to
analyze a wide range of molecular weights. However,
when performed in this experiment, the resulting gel
was not successful. This is probably due to old agarose
gel powder and reagents. New reagents and materials
should be used when performing agarose gel
electrophoresis so that a gel with visible bands may be
produced.
Other possible sources of error in DNA
extraction include improper handling of reagents, and
unwanted cleavage of DNA fibers by nucleases that
may not have been denatured.
Further study about the structure of the DNA
extracted may also be added to the experiment such
as the determination of the conformation and structure
of the DNA sample.
REFERENCES
(1) Boyer, R. (2012). Biochemistry Laboratory
Modern Techniques and Theories. New
Jersey: Pearson Education, Inc.
(2) Campbell, M. K., Farrell, S.O. (2012).
Biochemistry 7th Edition. USA: Cengage
Learning.
(3) Lee, P.Y., Costumbrado, J, Hsu, C.Y., Kim, Y.H.
Agarose gel electrophoresis for the
separation of DNA fragments. Journal of
visualized experiments 10.62 (2012): 37913923. 4 Nov 2014.

APPENDIX
Weight centrifuge tube: 6.68g
Weight centrifuge with sample: a.) 7.02g
Weight sample: a.) 0.34g b.) 0.31g

b.) 6.98

Conc.
(10%)(x)=(1%)(10mL+x)
x=0.909mL
x100= 3.4% w/v

=1.3870 -> 10% nucleic acid

dsDNA concentration=50

x dilution
factor

=50

x0.6598x

= 3.299