ABENA FREMAAH FREMPONG
10558354
2ND NOVEMBER, 2018
ISOLATION OF DNA FROM FUNGI AND ACCESSING THE FACTORS THAT AFFECT
DNA STABILITY.
GROUP 2 MEMEBERS
MIRIAM EYRAM GAKPEY LAWSON
KELVIN AGYAPONG
AARON ADOM MANU
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TABLE OF CONTENTS
Abstract
3
Introduction
4
Specific objectives
7
Materials and method
8
Results
13
Discussion
14
Conclusion
18
References
19
Appendix
20
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ABSTACT
DNA can be found in living organisms such as bacteria, fungi, virus and other eukaryotic cells.
Nucleic acids are located in the nucleus of the cell. However, molecular biology techniques
such as the polymerase chain reaction requires the isolation of these nucleic acids before the
process can proceed. Several methods have been designed to isolate DNA from its natural
source. The CTAB extraction method has been proven to be very effective and efficient in
producing high yield of pure DNA when applied in the extraction of DNA from filamentous
fungi. Electrophoresis is a separation technique that can be applied to confirm successful DNA
isolation as well as the size of the DNA fragments produced. Southern blotting can further be
used to identity a DNA of interest.
DNA, unlike RNA is very stable in its native form. However, there are certain conditions that
affect the melting temperature of the double stranded DNA. These conditions can denature,
degrade or fragment the DNA helix by disrupting the bonds that contributes to its stability.
Such conditions include changes in pH, elevated temperature and agitation.
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Introduction
Fungi are organisms containing cells that have membrane bound organelles and defined nuclei.
They are eukaryotic organisms that generally develop from spores. They are widely distributed
in the environment with some being free living and others causing diseases. The significant
feature that distinguishes plants from fungi is the lack of chlorophyll in the latter. Thus, fungi
possess both cell wall and cell membrane. The cell wall of fungi is made up of chitin. They
also develop from hyphae that come together to form the mycelia.
The main purpose of optimization of methods used in extracting DNA is to increase yield and
co-purification of unwanted compounds as well as prevent sample degradation. Several
methods have been devised to extract DNA from organisms. For fungi, methods used in
extraction include the cetyl trimethyl ammonium bromide (CTAB) method. One limitation
involved in the extraction of high molecular weight DNA from filamentous fungi is the
presence of polysaccharides and phenols in many species. These compounds co-purify with the
nucleic acid leading to low yield. The CTAB method was originally designed for the extraction
of plant DNA. However, it was proven to have the ability to remove polysaccharides and
phenols present in the cells especially in oomycetes (Talbot, 2001).
Isolation of DNA from eukaryotic cells begins with collection of the organism from its natural
source or the organism can be grown in the lab. For fungi, it is collected from different kinds
of substrates including infected species (Weising, Hlide, & Markus, 1994). The fungi is
propagated and grown in the laboratory in liquid broth or agar plates. These media contain
sources of carbon, nitrogen and other important elements required for the growth of the fungi
at the log phase. Since the substrates for most fungi is semisolid or solid, the agar media is
considered as natural. Antibiotics are substances that are known to destroy microorganisms by
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inhibiting bacteria growth as in the case of bacteriostatic antibiotics or by killing them as in the
case of bactericidal antibiotics (Hanson, 2008).
DNA is mostly located in the nucleus hence the need to break open the cells. Cell lysis can be
done by sonication, mechanically, enzymatically or chemically. Sonication involves the use of
sonic waves to disrupt the cell wall. Enzymatic lysis method involves the use of lysozymes for
lysis of cell wall. Mechanically lysis involves the use of mortar and pestle or other tools for
grinding by hand in the presence of lysis buffer (Swapan et al, 2017).
The isolation of DNA involves the use of several chemicals that play important roles in the
production of high amount of pure DNA. CTAB is a cationic detergent that solubilizes the cell
wall and lipid membrane. It also denatures enzymes and inhibits restriction enzyme digestion
during polymerase chain reaction, PCR and electrophoresis. Together with 2-mercaptoethanol
which acts as a strong reducing agent, CTAB removes polysaccharides and phenols contained
in the fungi. Tris is a strong base that can complex with HCl to form Tris -HCl. It permeates
the outer cell wall and thus maintains a constant pH in solution. EDTA serves as a chelating
agent by removing ions that are required by DNase to degrade the DNA. DNA can be separated
from proteins and hydrophilic components of the cell by use of chloroform:phenol:isoamyl
alcohol. The aqueous phase containing the DNA components is separated from the organic
phase. Isoamyl alcohol also prevents foaming during extraction. DNA is precipitated from the
supernatant on addition of 70 % ethanol and 100 % ethanol or isopropanol at high salt
concentration. Sodium acetate can be used as the salt. The salt is added prior to centrifugation
as it precipitates the polysaccharides and protein (Swapan et al, 2017).
Chromosomal DNA from eukaryotic cells produces large aggregates after ethanol precipitation
due to its large size. DNA from small circular species such as plasmid and organelle DNA
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produce smaller DNA fragments after alcohol precipitation and resuspension (Wink &
FitzRoy, 2006).
Electrophoresis is a separation technique that separates nucleic acids and proteins based on size
and charge. The principle underlying this technique is that under the influence of an electric
ions, particles migrate in the electric field in the direction opposite the net charge it carries.
Positively charged particles move toward the cathode (negatively charged pole) and negatively
charged particles move towards the anode (positively charged pole). Due to varying charges
and sizes of the proteins or nucleic acids, the migration in the field is at different velocities.
The bigger particles move slowly in the medium as compared to the smaller ones. The relative
mobility of the particles can be monitored by staining the DNA with a tracking dye such as
bromophenol blue. Before loading the gel, the DNA is mixed with ethidium bromide to allow
for easy visualization after electrophoresis since DNA in itself is colourless. To know the size
of the DNA, molecular markers are used. Molecular markers are particles of known sizes.
(Westermeier, 2006)
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Specific Objectives
To isolate DNA from non- pathogenic fungi obtained from a leaf substrate using the CTAB
method.
To determine the size of the DNA fragments obtained from the isolation using electrophoresis.
To determine the effect of acid, alkaline, mechanical and heat treatment on DNA stability.
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Materials
Tetracycline stock, chloramphenicol, source of fungi, agar, sabourand dextrose agar, bleach,
ethanol, tissue paper, sterile petri dish, yeast extract, peptone, sucrose, sodium acetate, 1.4
M sodium chloride, 20 mM EDTA pH 8, tris-HCl, tris base, NaOH, pH metre, eppendorf
tubes, chloroform : isoamyl alcohol (24:1), 100 mM tris-Cl, 2 % CTAB, 100 % isopropanol,
Tris EDTA buffer, Tris borate buffer, bromophenol blue, ethidium bromide, electrophoretic
unit, proteinase K, 1 % SDS.
Method
Isolation and sterilization of fungi from a natural source and preparation of media.
Several petri dishes were washed and rinsed thoroughly and dried in the oven. After, a thick
layer of tissue paper was cut into shapes to fit the petri dish. The shaped tissue layer was used
to line every petri dish
Media preparation.
In preparing the agar media, 65 g of dextrose sugar was weighed and 5 g of agar was added.
The mixture was dissolved in 1000 ml of distilled water and mixed thoroughly. The media was
autoclaved and left to cool before storing.
The liquid broth was prepared by 8 g of peptone, 0.8 g of yeast extract and 8 g of sucrose into
400 ml of water. The solution was autoclaved at 121 °C for 2 hours.
To the liquid broth and the agar media, 150 µg/ml of tetracycline and chloramphenicol was
added to kill any bacteria present.
Preparation of bleach and 70 % ethanol.
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In preparing the bleach solution, 50 ml of the stock bleach was dissolved in 100 ml of distilled
water.
For the 70 % ethanol, 70 ml of absolute ethanol was diluted with 30 ml of sterile water.
Antibiotic preparation.
The antibiotic was prepared by weighing 0.45 g of the solid antibiotic into a conical flask. The
antibiotic was dissolved in 3 ml of 70 % ethanol and mixed thoroughly. Using Whatman's filter
and syringe, the solution was surface filtered into a falcon tube. Only 1/3 of the 0.15 mg/ml of
the resultant solution was added to the media the rest stored. The same procedure was used in
the preparation of chloramphenicol which was also added to the media.
Surface sterilization of specimen
The leaf (specimen) was cut into squares around the infected area. The cut leaves were placed
in bleach and agitated for few seconds. The leaf partitions are then rinsed with sterile water and
placed in 70 % ethanol to enhance sterility. The specimen was immediately washed with water
and placed in the tissue-lined petri dish and left for a week.
Inoculation of fungi
After a week, the leaf lesion is observed under a hand lens and stereoscope to view growth.
The agar plate was labelled and divide into three partition. Sterile toothpicks were moistened
with distilled water to inoculate the fungal growth. The growth was streaked gently on each
portion of the agar. The plates were kept in a dark drawer at about 25 °C for a week. Some of
the fungi was cultured in the liquid broth.
The procedures for the preparation of media and antibiotic above were used to prepare 500 ml
media and 1.5 ml of 0.15 mg/ml tetracycline respectively.
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Isolation of DNA
The fungal growth was obtained from the agar plate or the liquid broth to fill about one-third
of the eppendorf tube and 500 µl of the CTAB buffer was added. The cells were lysed
mechanically by continuous grinding using a pen cover until a fine solution was obtained.
Afterwards, the tube was placed in a water bath at 65 °C for 5 minutes. The process of grinding
and incubation was repeated several times until the final incubation time was 2 hours. The tube
was taken out of the water bath and chloroform: isoamyl alcohol in the ratio 24:1 was added to
the solution. The solution was vortexed for about 30 seconds for centrifugation at maximum
speed for 10 minutes. The supernatant was carefully collected using a syringe into another tube.
A volume of 0.04 µl of 7.5 M sodium acetate was added to the supernatant and mixed
thoroughly. To the mixture, 0.2916 µl of 100 % isopropanol was added, mixed thoroughly and
allowed to stand for 15 minutes. The resultant solution was centrifuged at same speed for 10
minutes to obtain DNA pellets. The supernatant was discarded and the pellet was rinsed in 500
µl of 70 % ethanol. The pellet was rinsed again in 100 % ethanol. The absolute alcohol was
completely dried in the tube after which TE buffer added to suspend the DNA pellet. The tube
was placed in a water bath for 5 minutes and opened for a minute to allow any alcohol residue
to evaporate. This process was done thrice.
ELECTROPHORESIS
Preparation of agarose gel
In conducting the electrophoresis, 200 ml of 5x stock of Tris-borate-EDTA buffer was prepared
by dissolving 10.8 g of Tris base and 5.5 g of Boric acid in 196 ml of water and 4ml of EDTA.
Afterwards, 1 g of powdered agarose was added to 100 ml Tris Borate EDTA (TBE) buffer at
pH 8. The resultant solution was boiled for 3 minutes to completely dissolve agarose and
allowed to cool. The buffer was poured into the casting tray and a comb was inserted into it. It
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was allowed to stand at room temperature for 30 minutes for the gel to solidify as well as form
distinct wells in them.
After the gel preparation, the running buffer (1X TAE; Tris-Acetate-EDTA) was gradually
added to the unit until it just covered the top of the gel. The isolated DNA was mixed with 6x
bromophenol blue and loaded into one of the wells. The lid was then attached, the cords were
correctly connected, the power supply was plugged in and turned on. The voltage was set to 80
volts. The gel was then run for 2 hours. After the run, the gel was stained with Ethidium
bromide and viewed under UV light to visualize the band resolution.
Extraction of E.coli DNA
The E.coli cells were cultured in 50 ml liquid broth at 31° C overnight. An aliquot of 10 ml of
the overnight culture was put into 2 falcon tubes and spin at 1000 x g for 5 minutes. The
supernatant was discarded and 2 ml of Crombad buffer was added. A volume of 20 µl of
proteinase K was added and the solution was incubated at 55 °C for 30 minutes. A solution of
1 % SDS was added and incubate at 65 °C. The solution was deproteinased with chloroform:
isoamyl alcohol of ratio 24:1 and precipitate with 1 ml of ice -cold isopropanol. The solution
was spun at maximum speed for 5 minutes. The supernatant was discarded and the pellet was
washed with 70 % ethanol. The rinsing was repeated twice with absolute ethanol to obtain dry
pellet. TE buffer was added as in the previous extraction. A gel was run to obtain the sizes of
DNA extracted.
Treatment of DNA
The isolated DNA was subjected to several treatments. The total volume of the solution
containing the 300 µl DNA was 15 ml. For each treatment condition, 5 erppendorf tube were
set in duplicates. Each tube contained 50 µl of DNA. For the acid treatment, 50 µl of 1 M HCl
at pH 1.05 was added to sample 1 and the solution was left stand for 12 hours. For the alkaline
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treatment, 1 M of NaOH at pH 12.1 was added to sample 2 and subjected to same condition as
sample 1. To samples 3, 4 and 5, 50 µl of water was added. Sample 4 was agitated by vortexing
for 2 hours. Sample 5 was heated at 95 °C for 2 to 3 hours. Sample 3 was the control. The
effect of each treatment was analysed by running a gel.
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RESULTS
Mw
1
2
3
4
5
6
7
8
9
10
11
figure 1. An electrophoregram showing the band resolution of the isolated fungal DNA. The
separation produced unclear bands and diffused smears.
Figure 2. an electrophoregram confirming the isolation of DNA from E.coli. The separation
produced distinct bands of the same molecular weights.
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Mw
3
5
4
2
1
3
5
4
2
1
figure 3. an electrophoregram showing the effect of several treatment conditions on the
extracted DNA. Well 3 that contained the control gave the same band as well 4. Well 1 had no
bands and well 2 produced a smear. Well 5 produced thick band at the bottom of the gel.
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DISCUSSION
In the experiment, the liquid broth and agarose agar media were successful in culturing the
fungi in the lab. This is an indication that these media contained the necessary nutrient supplies
required by the fungi to proliferate at the log phase (Hanson, 2008).
The DNA was successfully isolated from the cultured fungi. The precipitation was enhanced
by the addition of the right amount of isopropanol. The presence of CTAB and mercaptoethanol
in the extraction solution inhibited co-purification of polysaccharides and phenols during the
extraction. However, the extraction produced low yield and short fragments. This could have
been to inadequate lysing of the cell during grinding (Swapan et al.,2017)
In the separation of nucleic acids after extraction, electrophoresis is applied. This technique
requires the use of the same buffer throughout in order for the runs to take the same time to
produce better resolutions at constant voltage. The use of constant voltage produces the
appropriate temperature for good separation. The use of constant power supply allows for
adjustment of voltage and current during the run in order to maintain the raised temperature
required for good band resolutions. High or low voltage supply causes suboptimal resolution
during DNA separation. The duration of the run can also have an effect on the resolution.
Inappropriate run time and can produce smears and unclear bands. During the electrophoretic
run in figure 1, there were power fluctuations. The irregular power supply led to the formation
of indistinct bands and smears. The resolution of the extracted fungal DNA based on the
(Gerstein, 2004).
Electrophoresis is a separation technique that can be used after DNA extraction to confirm the
success of the isolation and the size of the fragments obtained. Figure 2 shows the results
obtained after running a gel after isolating from the E.coli. The production of distinct bands is
an indication of successful isolation of DNA.
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DNA in the double stranded form is stable due to the presence of hydrogen bonding and base
stacking interactions. The DNA helix is also surrounded by water molecules leading to the
formation of shell of hydration around the DNA which stabilizes it. At pH greater than 12 or
lesser than 2, the bases present in the DNA are ionized causing denaturation. There is an
alteration in the H-bonding donor and acceptor properties of bases which disturbs the normal
Watson and Crick base pairing and the hydrogen bond formed. Base stacking interactions are
destabilized in the DNA by destroying the shell of hydration around the helix. Acid treatment
of DNA also causes depyrimidination and depurination. Loss of bases is caused by cleavage of
glycosidic bonds (Sinden, 1994).
Unlike acidic treatment that degrades the DNA, alkaline treatment denatures the DNA. It
separates the DNA helix into single strands. Depending on the duration the alkaline treatment,
the DNA can be completely loss in solution (Sinden, 1994).
Temperature destabilizes the double helix structure of the DNA by destroying the hydrogen
bonds and shell of hydration. There is melting of the DNA leading to denaturation. Thermal
stability of the DNA is a function of the base stacking as well (Sinden, 1994).
Subjecting DNA to harsh treatment such as agitation by vortexing leads to mechanical shearing
of the DNA into smaller fragments. Vortexing is required during DNA isolation. However, the
ability of the agitation to shear the DNA is dependent on the length of the DNA isolated and
the vortexing time (Sinden, 1994).
From the results obtained in figure 3, sample 1 showed no bands this is an indication that the 1
M HCl used completely degraded the DNA present in the solution. Sample 2 produced a smear.
This is an indication that the duration of the treatment in 1 M NaOH was long enough to
degrade the nucleic acid partially leaving behind bits of low molecular weight molecules in the
solution. After vortexing as with sample 4, there was no effect comparing the results to that of
the control in sample 3. This is an indication that the length of the DNA isolated was short
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making it less prone to fragmentation. Also, it is possible that the duration of the treatment was
no long enough to shear the DNA isolated. Heating the DNA at 95 0C in sample 5 produced
single stranded DNA which due to their smaller size is able to fold on itself and thus move
faster through the gel. The control sample produced a band with high molecular weight as
compared to sample 5. This means that the treatment led to the denaturation of the DNA to
produce low molecular weight molecules and a band at the far bottom.
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CONCLUSION
The lysis step plays an important role in determining the yield of the isolated DNA. The cells
must be properly grinded to properly to break open the cell completely. The rinsing step is also
important in generating clean DNA pellet.
Electrophoresis requires a constant supply of power to give better band resolutions.
The stability of DNA can be disturbed by changes in pH. Very low or high pH denatures or
degrades the DNA. Heating DNA melts it and separates it into two single strands.
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REFERENCES
Gerstein, A. S. (2004). Molecular Biology Problem Solver: a Laboratory Guide. pp.105.
Weising, K., Hlide, N., & Markus, P. (1994). DNA Fingerprinting in Plants and Fungi. pp.61.
Westermeier, R. (2006). Electrophoresis in Practice: a Guide to Method and Applications.
pp.14-15.
Wink, M., & FitzRoy, R. (2006). An Introduction to Molecular Biotechnology.
Swapan, K. T. et al. (2017). Exploring Rapid and Efficient Protocol for Isolation of Fungal
DNA. pp.951-955.
Hanson, J. R. (2008). Chemistry of Fungi. pp.18-20
Sinden, R. R. (1994). DNA Structure and Function. pp.34-36
Talbot, N. J. (2001). Molecular and Cellular Biology of Filamentous Fungi. pp.23-26.
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APPENDIX
Figure 1: Isolation of non-pathogenic fungi
Leaves suspected to contain non-pathogenic fungi were selected based on the presence of
lesions, appearance of brown patches and yellow colouration on the leaves.
Figure 2: View of fungi under the stereoscope
The view of fungi on the leaves under the stereoscope revealed the structure and morphology
of the mycelia mat formed on the surface of the sterilized leaves 7 days after incubation.
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Fig 3: Agar plates showing growth of fungi
Cultured fungi of Sabouraud dextrose agar for a week showed massive growth of fungi
containing spores. Some plates contained mixed cultures of different types of fungi as shown
in the figure below.
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