Kwamina Nyame first report

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UNIVERSITY OF GHANA, LEGON
DEPARTMENT OF BIOCHEMISTRY, CELL AND
MOLECULAR BIOLOGY
FUNGI GENOMIC DNA
ISOLATION FROM A NATURAL
SOURCE
KWAMINA NYAME
ID: 10563324
02/11/2018
GROUP 5
CHRISTINE EWURAMA SAM
KEZIAH AKYAA AMOYAW
SELORM SABAH BLESS
TABLE OF CONTENTS
1.0 ABSTRACT .............................................................................................................................. 2
2.0 INTRODUCTION .................................................................................................................... 3
2.1 AIM ....................................................................................................................................... 6
2.1.1 SPECIFIC OBJECTIVES............................................................................................... 6
3.0 MATERIALS AND METHOD ................................................................................................ 7
3.1 MATERIALS ........................................................................................................................ 7
3.2 METHOD .............................................................................................................................. 7
3.2.1 PREPARATION OF MEDIA AND ANTIBIOTIC ....................................................... 7
3.2.2 SAMPLE COLLECTION AND SURFACE STERILIZATION ................................... 8
3.2.3 INOCULATION AND CULTURING OF FUNGI........................................................ 8
3.2.4 PREPARATION OF LIQUID BROTH AND CULTURING ....................................... 8
3.2.5 PREPARATION OF CTAB DNA EXTRACTION BUFFER, TE AND TBE
BUFFERS ................................................................................................................................ 9
3.2.6 DNA EXTRACTION ..................................................................................................... 9
3.2.7 AGAROSE GEL ELECTROPHORESIS ..................................................................... 10
3.2.8 DNA EXTRACTION FROM Escherichia coli............................................................ 11
3.2.9 DETERMINATION OF INFLUENCING FACTORS OF gDNA STABILITY ......... 12
3.2.10 PICTORIAL SUMMARY OF METHOD ................................................................. 13
4.0 RESULTS ............................................................................................................................... 15
5.0
DISCUSSION .................................................................................................................... 18
6.0 CONCLUSION ....................................................................................................................... 21
7.0 REFERENCES ....................................................................................................................... 22
1
1.0 ABSTRACT
DNA isolation with good quality is always been a necessity. Cetyltrimethylammonium bromide
(CTAB) buffer is used for DNA extraction from plants with various other extraction buffers.
CTAB buffer with beta-Mercaptoethanol was used in degrading polysaccharides and proteins to
get pure form of DNA. Extraction of genomic DNA extraction from filamentous fungi involving
isolation and surface sterilization followed by culturing in water agar and sabaroud agar plates,
profuse growing of fungus in liquid broth and extraction of DNA from fungi using various buffers.
Extracted DNA had low yield, hence DNA was extracted from E. coli and subjected to varying
conditions such as low and high pH, agitation and heat. The effect of heating, alkaline and acidic
conditions on the extracted DNA were noticeably observed while vortexing showed little
significant effect on the DNA.
2
2.0 INTRODUCTION
Fungi are spore-forming, non-chlorophytic, eukaryotic organisms and most of the true fungi are
filamentous and branched to form hyphae network called mycelia. Fungi are heterotrophs and
differ from plants mainly by their lack of chlorophyll and the presence of chitin in their cell walls
(Campbell, 1999).
Reproduction in fungi is by production of spores. Fungi are a large group of organisms including
yeasts, moulds and mushroom. Fungi are found in the air, water, soil, plants, dead woods and other
decaying matters (González-Mendoza et al., 2010).
The initial step in any research laboratory is to isolate DNA with good yield, efficiency and
intensity. DNA purification is very important because contaminants like tannins, proteins,
polysaccharides, and some secondary metabolites inhibits enzymes used for further analysis. The
major challenge for isolation of such DNA from fungi lies in breaking the rigid cell walls, as, they
are often resistant to traditional DNA extraction procedures (Delvin, 2011).
Generally there are two major steps involved in fungal genomic DNA extraction, which are
disruption of cell wall and extraction followed by purification of genomic DNA (Isleib, 2012). The
extraction of genomic DNA is usually done with cetyl trimethyl ammonium bromide (CTAB)
extraction buffer followed by purification through phenol/chloroform extraction and precipitation
with isopropanol or ethanol (Wilson and Walker, 2010).
YEPS or yeast extract peptone sucrose, also often abbreviated as YEPS, is a complete medium for
yeast growth. It contains yeast extract, peptone, distilled water and sucrose. It can be used as solid
medium by including agar. The yeast extract will typically contain all the amino acids necessary
3
for growth. By being a complete medium, YEPS cannot be used as a selection medium to test for
autotrophs. The broth version of YEPS typically contains 1 % yeast extract, 2 % peptone, 2 %
sucrose as well as distilled water and the agar version includes addition of 1.5 to 2 % of agar before
allowing mixture to cool and solidify according to Galewska et al, (2013).
Fungal DNA extraction is important molecular biology tool, which make use of variety of physical
and chemical method to disrupt the fungal cell wall, release the DNA in buffer containing
chemicals such as CTAB, Tris EDTA, NaCl and protein kinase K, all of which maintain the
integrity of the DNA after which it is precipitate out of solution for purification CTAB buffer is a
type of detergent that helps in lysis of cell membrane according to Wu et al. (2012).
Cetyltrimethylammonium bromide (CTAB) is one of the best method for problematic samples and
helps in separating polysaccharides and rescue of nucleic acids. Cetyltrimethylammonium
bromide (CTAB) buffer is mainly used for DNA extraction from plants species with various other
extraction buffers such as acetate buffer. Cetyltrimethylammonium bromide contains EDTA that
prevents DNA from degradation by chelating magnesium ions needed for enzymes that degrade
DNA according to Ageno et al. (1969).
Beta-Mercaptoethanol used along with CTAB aids in protein degradation which is the immediate
step after polysaccharides breakdown of cell and plasma membrane. CTAB inactivates some of
the proteins and also denatures others till some extend. CTAB is associated with high salts
concentration that act as reducing agents and some selective precipitants of nucleic acids. CTAB
has proven to be a widely used buffer to help in DNA isolation. TE buffer is a buffer solution
usually used in molecular biology, especially in procedures involving DNA, cDNA or RNA
(Ageno et al., 1969).
4
"TE" buffer is obtained from its constituents: Tris, a common pH buffer, and EDTA, a molecule
that chelates cations like Mg2+. The most common microorganisms used for microbiological work
is Escherichia coli. This is because of their ability to reproduce fast and its commercial availability
(Voet and Voet, 2011).
Electrophoresis is often classified according to the availability or absence of a solid supporting
medium or matrix through which the ionic molecules move in the electrophoretic system. Solution
electrophoresis systems uses aqueous buffers in the absence of a solid support medium. Most
practical applications of electrophoresis in biochemistry involve some form of zonal
electrophoresis, in which the aqueous ionic solution is carried in a solid support and samples are
applied as spots or bands of material according to Galewska et al, (2013).
In electrophoresis, the gel matrix through which the DNA samples will move is placed in a buffer
solution which will allows for electrical conductance and maintains a constant pH. Buffers with
higher concentration will allow the DNA molecules to migrate faster through the gel matrix
according to Galewska et al, (2013).
The migration properties and resolution of DNA molecules studied usually show that the migration
time and resolution decreases as temperature increases in an isoelectrostatic separation mode while
increasing temperature results in maximum migration. Temperature, pH and other factors affects
DNA migration on an electrophoretic run differently according to Aimar et al. (2015).
The buffers that are commonly employed in gel electrophoresis are Tris Acetate-EDTA (TAE) and
Tris Borate-EDTA (TBE). TAE Buffer is used efficient for separating fragments which are larger
than 4000 bp and is also used to separate super coiled DNA. Whilst TBE Buffer is effective for
5
the separation of fragments between 1 and 3000 bp in length. It provides an ionic solution in order
to allow the current to pass through the water (Aimar et al., 2015).
2.1 AIM
The aim of the experiment was to isolate and purify fungal DNA from fungal infected leaves found
on plants and to observe the influence of various factors such as pH, temperature and agitation on
DNA stability via electrophoresis.
2.1.1 SPECIFIC OBJECTIVES
1. To isolate the fungi from the plant.
2. To culture the isolates of fungi on the agar or yeast extract peptone and sucrose (YEPS)
liquid broth
3. To extract the DNA from the fungi
4. To purify the DNA using gel electrophoresis
5. To observe the influence of various factors such as pH, temperature, and agitation on DNA
stability via electrophoresis.
6
3.0 MATERIALS AND METHOD
3.1 MATERIALS
In this experiment, CTAB buffer (100 Mm Tris-HCl, 25 Mm EDTA, 1.5 M NaCl, 2 % CTAB,
distilled water, CIA: chloroform: isoamylalcohol (24:1), β- Mercaptoethanol), 70 % and 100 %
ethanol, HCl, NaOH TE buffer, TAE buffer. Agarose, sterile toothpick ethidium bromide (stock
concentration of 10 mg/ml), Electrophoretic tank, power unit, Centrifuge, Chemical balance,
Vortex, chloroform, petri dishes, autoclave, Eppendorf tube, Tetracycline, Chloramphenicol,
loading dye containing bromophenol blue, Sabarose Dextrose Agar (SDA), Deionized water and
UV illuminator were used.
3.2 METHOD
3.2.1 PREPARATION OF MEDIA AND ANTIBIOTIC
Exactly 65 grams of Sabarose Dextrose Agar (SDA) was weighed and added to 5 grams of Agar
in 1 litre of sterile water. The liquid media was poured into plates and allowed to harden before
autoclaving for 2 hours or sterilization. The antibiotics were prepared by adding 3 ml of 70 %
ethanol to 450 mg of each antibiotic namely, tetracycline and chloramphenicol.
The liquid media was mixed with the antibiotics tetracycline and chloramphenicol to a final
concentration of 150 µg/ml. Then, the antibiotic solutions were passed through a millipore filter
to rid them of contaminants. They were then autoclaved. When autoclaving of the media and
antibiotics were completed, 1 ml each of the prepared antibiotics were added to the molten media
and 15 ml aliquots of the resulting mixture were transferred into petri dishes.
7
3.2.2 SAMPLE COLLECTION AND SURFACE STERILIZATION
Leaves, from various plants, that showed signs of fungal growth were obtained from the University
of Ghana campus as shown in figure 1.0. The leaves were cut into small squares of 1 cm2 sizes
around the infected portion. The cut leaves were dropped in 0.5 % bleach (1:2) and washed for 20
seconds to remove other microorganisms except fungi.
Next, the cut pieces were removed and rinsed for 20 seconds in deionized water to rinse out bleach
and kill bacteria before being dropped in 70 % ethanol to complete the sterilization process. Water
was squirted onto the already sterilized plates to moisturize the tissue layers before the leaves were
placed on them. The plates were placed in a dark cabinet and closed for 1 week.
3.2.3 INOCULATION AND CULTURING OF FUNGI
The plates were inspected using hand lenses as well as stereoscope to check for fungal growth as
shown in figure 1.1. Sterilized toothpicks were used to pick growing fungi and used to inoculate
the plates (figure 1.2) containing the growth media. Portions of the fungal growth were streaked
onto the surface of the solidified Sabourand-Dextrose plate by rolling the toothpick to ensure all
spores are transferred onto the surface of the medium before incubating at 25 °C for 7 days.
3.2.4 PREPARATION OF LIQUID BROTH AND CULTURING
Liquid broth media (Yeast extract, peptone and sucrose YEPS) for the growing fungi was prepared
by the addition of 8 g of peptone, 0.8 g of yeast extract and 8 g of sucrose to deionized water to
make a final volume of 400 ml. This constituted the YEPS (yeast extract, peptone and sucrose)
liquid broth. The solution was then autoclaved and allowed to cool as shown in figure 1.3.
8
After the autoclaved had been completed, the fungi were cultured in the liquid broth streaking the
inoculum on the surface of the broth. After, the cultured fungi were incubated for some days to
allow the growth of the fungi.
3.2.5 PREPARATION OF CTAB DNA EXTRACTION BUFFER, TE AND TBE
BUFFERS
In preparing the CTAB DNA extraction buffer, 1 g of 2 % CTAB was weighed and transferred
into a solution containing 5 ml of 100 mM Tris-Cl, pH 8.0, 14 ml of 1.4 M NaCl, 2 ml of 20 mM
EDTA and 250 μl of 0.5 % ß-mercaptoethanol. This solution was then topped up with deionized
water to a final volume of 50 ml.
3.2.6 DNA EXTRACTION
A mass of the fungi was either obtained from the agar culture or the liquid broth into a 2 ml
Eppendorf tube after which 500 μl CTAB DNA extraction buffer was added. The resulting mixture
was then grounded with the aid of a sterile tooth pick before incubating the uniform mixture formed
in a water bath at 65 °C for 1 hour whiles remixing the content at a regular interval of 10 minutes.
After this, 500 μl of chloroform: Isoamylalcohol (24:1) was added and vortexed well for 30
seconds and the solution was subjected to centrifugation at maximum speed for 10 minutes.
The aqueous phase supernatant of the layered solutions formed was then transferred to another
Eppendorf tube doing well to estimate its volume in the new tube. Next, an equivalent volume of
0.08 volume of sodium acetate was added to the supernatant and mixed well.
This was followed by the addition of 0.54 equivalent of 100 % isopropanol and mixing well after.
The mixture was then left to stand at room temperature for 15 minutes and afterwards centrifuged
9
at maximum speed for 10 minutes to recover DNA pellet which was then rinsed with 70 % ethanol
and 100 % ethanol respectively. After, the pellet was allowed to dry and suspended in 50 μl TE
buffer made of 10 mM Tris and 1 mM EDTA.
3.2.7 AGAROSE GEL ELECTROPHORESIS
PREPARATION OF THE AGAROSE GEL
The casting tray was rinsed and dried with 95 % ethanol and the ends taped, after which it was set
on a level surface. One gram of the agarose was measured and mixed with 100 ml 1 X TAE buffer
in microwavable flask. The agarose was microwaved for 2 minutes until the agarose is completely
dissolved. The agarose solution was allowed to cool down to about 50 ᵒC for about 5 minutes and
then 3 µl of Midori Green was added to it.
The agarose was swirled to make sure there were no particles. The agarose was poured into a gel
tray with the well comb in place. The newly poured gel was allowed to sit at room temperature for
30 minutes until was completely solidified. The comb was put into it and its level was adjusted so
it rested evenly with a few millimeters of space between teeth and the tray to allow wells form in
the agarose. The comb and tape were then removed.
LOADING SAMPLES AND RUNNING AN AGAROSE GEL
Loading buffer was added to each of the DNA samples. To every 7 µl of each DNA sample, 2 µl
of 6 X loading dye was added. The solidified agarose gel was then placed into the electrophoresis
unit with the wells closest to the negative electrode. The electrophoresis unit was filled with 1 X
TBE until the buffer was just covered the top of the gel. A molecular weight marker was carefully
loaded into the first lane of the gel.
10
The DNA samples were then loaded into the addition wells. The samples were loaded into the
wells being cautious not to puncture the bottom of the wells. The lid was attached and the cords
correctly plugged into the power supply and running at 80 V electric potential until the dye line
was approximately 75 % to 80 % way down the gel. The electrophoresis was run until the
bromophenol blue in the loading dye had migrated to within three quarters of the positive electrode
end of the gel as shown in figure 1.7.
After the electrophoretic run, the power was then turn off with the electrodes disconnected from
the power source. The gel was carefully removed from the electrophoretic unit with the aid of
gloves. The gel was then placed into a container filled with 100 ml of TBE running buffer and 5
µl of ethidium bromide. After, the gel and destained and viewed under UV to visualize DNA
fragments.
3.2.8 DNA EXTRACTION FROM Escherichia coli
DNA was extracted from E. coli due to fungal DNA giving a low yield. E. coli was cultured in 50
ml Luria Bertani (LB) at 37 oC overnight. After that, 10 ml of the overnight culture was aliquoted
into two falcon tubes and span for 5 minutes at 1000 g. The supernatant was discarded and 2 ml
crombad buffer was added to the pellet, after which 20 μl proteinase K was added and the mixture
incubated at 55 oC for 30 minutes.
This was followed by the addition of 1 % SDS and incubation at 65 oC. The mixture was then
deproteinized with chloroform: isoamyl (24:1) and precipitated with 1ml ice-cold isopropanol. The
mixture was span for 5 minutes and the supernatant was discarded. The pellet was after washed
with 70 % ethanol followed by rinsing with 100 % ethanol. The rinsing was done twice and the
pellet was allowed to dry. T.E. buffer was then added to solubilize the pellet (DNA).
11
3.2.9 DETERMINATION OF INFLUENCING FACTORS OF gDNA STABILITY
The DNA obtained from the extraction process was then subjected to various treatment conditions
temperature, pH levels and agitation and the effects monitored using agarose gel electrophoresis.
to observe their effect on the macromolecule. HCl with pH below 4.0 of concentration 1 M was
prepared as well as 1 M NaOH with pH above 12.0 using 0.42 ml of concentrated HCl and 0.2 g
of NaOH all in 50 ml deionized water respectively.
Exactly 1 % of agarose gel was prepared again (5 μl of 10 mg/ml concentration of ethidium
bromide was added). The ethidium bromide, a fluorophore, used to prepare the gel facilitated DNA
for visualization, by intercalating into the DNA, giving off light in the process. Gel was submerged
in running buffer. First 50 μl of the DNA was aliquoted into five tubes.
For the first tube, 50 μl of HCl with pH 1.05 was aliquoted and added to it. For the second tube,
50 μl NaOH with pH 12.1 was also aliquoted and added to it. For the third tube, 50 μl of deionized
water was added and the three tubes with their contents were incubated for twelve hours. For the
fourth and fifth tubes 50 μl deionized water each were added vortexing and heating at 95 oC
respectively for 3 hours. The treated DNAs were then run on a gel to observe the effects of the
treatment conditions. This was done in duplicates.
12
3.2.10 PICTORIAL SUMMARY OF METHOD
Figure 1.0: Leaves sample showing some
fungal growth at University of Ghana
Figure 1.2: Plate Sabourand agarose
inoculated with fungal spores from leaves
Figure 1.1: Showing the Surfaced sterilized
leaf cuttings placed in moist Petri dishes to
promote fungal growth
Figure 1.3: An image of the YEPS solution
taken after the components were added to
water to form a uniform solution
13
Figure 1.4: Showing an image of the cut
leaves viewed with a hand lens
Figure 1.6: Fungal DNA pellet extracted
into tube.
Figure 1.5: Lysate obtained after addition of
chloroform followed by vortex. The aqueous
portion contains the DNA.
Figure 1.7: Image showing electrophoretic
unit and power supply during
electrophoretic run
14
4.0 RESULTS
After a week, fungal spores were observed on the inoculated plate as shown in figure 1.8 and in
YEPS as shown in figure 1.9. When the DNA from the fungus was run on a gel, the results showed
smeared bands, with some of the wells not showing bands at all. This implies the DNA extraction
and gel electrophoresis was not performed well. In addition, there was also contamination with
RNA as shown in figure 2.0. This represents a poor electrophoretic run with smearing bands due
to problems that arose during the electrophoresis process.
Due to the poor results from the fungi DNA, DNA was extracted from E. coli as a counter measure
using the protocol in section 3.2.8 and the product obtained was subjected to the various treatments
to observe their effects on DNA.
The DNA extracted was confirmed by running on a gel electrophoregram as shown in figure 2.1.
The observed electrophoretic run of the DNA after the various treatments was recorded as shown
in figure 2.2 with a molecular aligned to the sides.
Following the electrophoresis map, the untreated DNA or control banded at the top due to its huge
circular nature. Thus per this results, there was no effect on the DNA after vortexing in terms of
position but the size reduced slightly, whereas heating caused a reduction in the size as the band
was formed at a very low position. The were no observable bands for the DNA treatment with HCl
acid and NaOH alkali.
15
Figure 1.9: An image showing rapid fungal
Figure 1.8: An image showing the fungal
growth on the agar plate after a week of
inoculation. This represents spores of the
organism from which DNA was extracted
from.
growth in YEPS
Figure 2.0: An image showing the bands obtained after running the fungi DNA on a gel
electrophoresis. Different bands depicting both DNA and RNA with low yield
16
Figure 2.1: Image showing the gel electrophoresis confirmatory run of DNA isolated
L 1 2 3 4 5
1 2 3 4 5
L 1 2 3 4 5
1 2 3 4 5
Figure 2.2: Images showing the result of gel products of the treated DNA. control in lanes 1,
Heat treated in lanes 2, Vortexing treated in lanes 3, Base treated DNA in lanes 4 and Acid
treated DNA in lanes 5.
17
5.0 DISCUSSION
Extraction of DNA from fungi tissues usually vary depending on the reagents materials used and
other cautions taken during the process (Voet and Voet, 2011). To obtain an efficient and pure
DNA, aseptic techniques was employed. The lesions seen on the plugged leaves as shown in figure
1.1 obtained in the University of Ghana were due to fungal infection and the leaves were washed
in 0.5 % bleach in order to kill all bacteria and other microorganisms including spores present on
the surface of the leaves during surface sterilization according to Wu et al. (2012).
Most fungi grow well in moist regions due to the presence of water for their metabolic activities
to take place and hence the portions containing lesions on the leaves were incubated in a plate
lined with moisturized tissue as shown in figure 1.1. which facilitated their growth (GonzálezMendoza et al., 2010).
Fungi could not produce their own food unlike plants therefore required an external source of
nutrient supply for growth and survival. YEPS (yeast extract, peptone and sucrose) contained
carbohydrate from the yeast extract and sucrose, these served as carbon nutrient source for the
fungi incubated on the plate promoting growth as shown in figure 1.8 and figure 1.9 (Campbell,
1999). This indicated that the agar contained the appropriate nutrients for fungal growth. Also due
to the antibiotics such as tetracycline and chloramphenicol present in the media, there was no
bacteria growth and single colonies were obtained.
The nutrient broth also contained protein from the peptone which served as a nutrient source to
promote fungal growth. The CTAB buffer was used in order to facilitate the DNA extraction by
lysing the fungal cells to release the cell membrane. Cetyltrimethyl ammonium bromide (CTAB)
was used to inactivate all phenolic compounds and promoted the aggregation of carbohydrate out
18
of solution in the form of globules (Delvin, 2011). EDTA was used as a chelating agent which
served to denature nucleases which were likely to degrade the DNA whilst Tris buffer provided an
alkaline environment to prevent denaturation of the DNA in the buffer as well as the salt found in
the buffer providing high ionic strength for DNA precipitation (Isleib, 2012).
The addition of chloroform was to separate soluble materials such as proteins on other material
followed by centrifugation while insoluble particles were removed through centrifugation as a way
of purifying the DNA (Aimar et al., 2015). The isopropanol and ethanol also added assisted in
effective precipitation of DNA and making the DNA more stable through formation hydrogen
bond formation. Ammonium acetate neutralized the charges on the phosphate backbone of the
DNA thereby reducing its solubility and caused it to precipitate out of solution facilitating
precipitation of DNA (Delvin, 2011).
The DNA was washed with 70 % ethanol to remove any salt that may remain bound to the DNA
pellet before resuspending it in 100 % ethanol to completely evaporate any water molecules on the
DNA pellet thereby keeping it dried. Tris EDTA (TE) buffer was used to maintain the integrity of
the pellet DNA. Agarose gel separated molecules by size and charge but because nucleic acids had
similar negative charges due the presence of negative charge on the phosphate group, hence their
movement was based purely on size (Isleib, 2012).
The subjection of the DNA to agarose gel electrophoresis was to obtain highly purified DNA.
When DNA was loaded on to agarose gel, they moved from the cathode to the anode as shown in
figures 2.0 to 2.2. RNA moved faster than DNA to the anode because it was smaller as shown in
figure 2.0 (Wilson and Walker, 2010) but there was low yield after fungal DNA extraction
19
probably due to some human errors such as not adding TE buffer to the DNA just when it was. For
this reason, DNA from E. coli was extracted and had higher yield as shown in figure 2.1.
The loading dye gave weight to the DNA to sink into the well for separation and also to monitor
the movement of DNA through the gel. The ethidium bromide a fluorophore, used to prepare the
gel enabled the DNA to be visualized by intercalating into the DNA as shown in figure 2.2 (Wilson
and Walker, 2010). At high concentration of acid there was depurination of RNA, cleaving the Nglycosidic bond and distorted the hydrogen bonds holding the double strands in DNA, thereby
causing denaturation accounting for the reason why no bands were observed for well 5 as shown
in figure 2.2 according to Ageno et al. (1969).
DNA double strands were held together by hydrogen bonds which are considerably weaker than
covalent phosphodiester bonds that join the nucleotides of a strand according to Galewska et al,
(2013). Unlike RNA, DNA lacked a hydroxyl group on the 2' position in each sugar group and
was much more stable in alkaline solution. RNA underwent cleaving in alkaline solution due to
deprotonation of the 2-OH group which facilitated its nucleophilic attack on the adjacent
phosphorus atom, thereby cleaving the RNA backbone (Wilson and Walker, 2010).
In high concentration an alkaline solution (NaOH), DNA was denatured and not cleaved. The
hydrogen bond holding the double strands was distorted (Isleib, 2012), thereby separating the
strands which accounted for the reason why no bands were observed for well 4.
Vortexing DNA and RNA for a long period of time breaks both DNA and RNA backbone (Delvin,
2011), giving smaller fragments and hence there were expectation of smaller bands with higher
movement as opposed to the sharp band observed for well 3 suggesting that there were little effects
on the DNA right after vortexing as it band corresponded to that of the control.
20
With regards to the results observed in the wells containing DNA that had been placed in
environments with high temperature, it was expected that there would have been denaturation and
hence no visible DNA bands on the electrophoregram according to Galewska et al, (2013). Heating
at high temperature denatures both DNA and RNA (Campbell, 1999) hence accounted for the
reason why band seen for well 2 had a long migration compared to that of the control. However,
since the control in well 1 was not subjected to any of the above conditions, normal separations
were obtained (González-Mendoza et al., 2010).
6.0 CONCLUSION
The Genomic DNA of the fungal sample was not successfully but that of E. coli was successfully
extracted and can be used for further characterization. The effect of heating, vortexing, alkaline
and acidic conditions on the extracted DNA were noticeably observed.
21
7.0 REFERENCES
Aamir S., Sutar S., Singh S. K. and Baghela A. (2015). A rapid and efficient method of fungal
genomic DNA extraction, suitable for PCR based molecular methods. Plant Pathology &
Quarantine, 5(2), 74–81.
Ageno M., Dore E. and Frontali C. (1969). Alkaline Denaturation of DNA. Biophysical Journal,
9, 1281-1312.
Campbell, M. (1999). Biochemistry. Sea Harbors Drive-Florida: Harcourt Brace College
Publishers.
Delvin, M. T. (2011). Biochemistry With Clinical correlations (7th ed.). Hoboken: John Willey
& Sons, Inc.
Galewska Z., Gogiel T., Malkowski A., Romanowicz L., Sobolewski K. & Wolanska M. (2013).
Biochemistry Workbook. Bialystok: Medical University of Bialystok.
González-Mendoza D., Argumedo-Delira R., Morales-Trejo A. and Pulido-Herrera L. A. (2010).
A rapid method for isolation of total DNA from pathogenic filamentous plant fungi (2nd
ed.). Funpec-Mexico: Medellinks.
Isleib, J. (2012). Signs and symptoms of plant disease: Is it fungal, viral or bacterial? Michigan
State University: Michigan Extension Press.
Voet D. and Voet J. G. (2011). Biochemistry. (4th, Ed.) New Jersey: John Wiley & Sons, Inc.
Wilson, K. and Walker, J. (2010). Principles and Techniques of Biochemistry and Molecular
Biology (7th ed.). New York: Cambridge University Press.
Wu H., Yang H., You X. and Li Y. (2012). Isolation and Characterization of Saponin-Producing
Fungal Endophytes from Aralia elata in Northeast China. International Journal of
Molecular Sciences, 13, 16255-16266.
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