Download Ratio of DNA Concentrations

10th Grader at Lincoln Park Academy
My name is Robert Bacchus, and I am a sophomore participating in the
International Baccalaureate (IB) Program at Lincoln Park Academy (LPA). I
am dual-enrolled at Indian River State College, and pursue other courses of
interest, such as Spanish and Journalism, through the Florida Virtual School
(FLVS).
I have taken innumerable science and mathematics courses, including
Earth & Space Science, Physical Science, Biology, Chemistry, Algebra 1,
Geometry, Algebra 2, Liberal Arts Mathematics, Intermediate Algebra,
College Algebra, Precalculus, and Trigonometry. My wide-spread interests
have led me to be involved in a number of clubs, and assume various
leadership positions. I am Class President at LPA, President of my local Red
Cross Youth Council, a competitor in Precalculus for my school’s math team,
a Cross Country runner, a French hornist in both the LPA Wind Ensemble
and the Treasure Coast Youth Symphony, as well as a member of the LPA
Odyssey of the Mind team.
My demanding schedule has greatly contributed to the development and
execution of my scientific research!
Growing up on the Caribbean island of Jamaica exposed me to the island’s
enclave of poverty, where I saw many people struggle for the basic necessities of life
– children begging in the streets, individuals stricken with diseases, others praying
each day would be their last. The sense of fear and helplessness this exposure
created within me resulted in my affinity for medical and biological sciences. I aspire
to tackle some of the many enigmas in science, especially those relating to the
human body.
In 2010 I began my studies to improve biotechnological approaches to DNA.
These studies, conducted in the Medical Lab at Indian River State College, have
qualified me for the Florida State Science & Engineering Fair (SSEF) for three
consecutive years. The state fair was an entirely new atmosphere in which my desire
to develop novel scientific research thrived! I am a founding member of the Research
Coast Florida Junior Academy of Science, and have been given the opportunity to
present my research at the University of Florida’s Junior Science, Engineering, and
Humanities Symposium (JSEHS), as well as at the Florida Junior Academy of
Sciences 2012 competition.
I chose to submit my research for the Virtual Science Fair, because the
independence that Florida Virtual School promotes was a key factor that helped me
balance both my academic interests, as well as time in the lab to research, conduct
and replicate my experiments.
Third Year Study
Manipulating the nucleic acid
thermodynamics of Musa acuminata to
evaluate DNA purity and improve
biotechnological approaches to DNA.
Introduction:
DNA isolation is a common
and almost vital procedure
for most medical and
biological research labs, with
a number of applications…
It can be used for the
functional analysis of genes,
to detect bacteria or viruses in
the environment, or to
diagnose infectious and
hereditary diseases such as
cancer and diabetes.
Figure 1. Clip art of DNA within banana.
The purity of the DNA
isolated is crucial.
Image taken from Scientific American.
Problem:
A current problem faced by
scientists isolating DNA is
contamination. Proteins not
properly deproteinized, such as
the enzyme deoxyribonuclease
(DNase), a nuclease that causes
cleaving of DNA and shearing in
isolations, are responsible for
these contaminations.
The present study hoped to
determine the role that the
temperatures relating to a cell’s
nucleic acid thermodynamics
could play in preventing these
contaminations and
deproteinizing DNase enzymes.
Figure 2. Me in the Medical Lab at Indian River State College
conducting my studies.
Photo taken by Rob Tack, Biotechnician at IRSC.
Hypothesis
Variables
1. Renaturation of DNA will
result in the highest
concentration of DNA.

Independent Variables:
 Thermodynamic levels used to treat
banana tissue.
2. Native DNA will provide
the lowest concentration
of DNA isolation.

Dependent Variables:
 Concentration of DNA isolated
 Purity of isolation
 Readings from the spectrophotometer

Constants:
 15mL of homogenized tissue used for
isolation
 Buffer solution
 Standards used to compare
spectrophotometric analysis.

Control:
 Banana tissue not exposed to
temperature change (native DNA)
3. Thermal denaturation will
lead to fragmentation of
DNA.
4. Renaturation of DNA will
result in the purest DNA.
5. Native DNA will have the
highest levels of
contamination.
Use of the
Banana
7
6
(Musa acuminata)
The banana serves as an effective model
5
for my research because of how it
ripens.
4
As one of the world’s most common
exports, the fruit is readily available.
It is a eukaryotic plant, with membrane-
3
bound cells containing DNA in their
nucleus.
2
The fruit is categorized into seven
distinct colors of maturation.
1
As it ripens, the tissue softens, making
the cells and DNA easily accessible.
Figure 3. Seven ripening stages of the banana.
Image by Dr. Scott Nelson, University of Hawaii
Continuation
The present study is the third in a series
of studies aimed at improving
biotechnological approaches to DNA.
Results from the two previous studies
contributed greatly to this year’s
experiment.
Year 1 Study: Comparative study of
DNA concentrations in Musa acuminata
ripening stages.
Year 2 Study: Targeting homogenization of
banana tissue, and precipitation of DNA.
Based on the first year study, the banana
in its seventh stage yielded the highest
concentration of DNA, and was therefore
used in this experiment.
Results from the second year study
proved that 99% isopropyl alcohol was
the most effective precipitant in DNA
isolation, therefore that was the
precipitant used in isolations performed
in this experiment.
Background
Research & Discussion:
DNA: Deoxyribonucleic acid is the hereditary material in
humans and almost all other organisms. Most DNA is located
in the nucleus of membrane-bound cells, which constitutes
for nearly ever cell in a human’s body. To release DNA, the
cell membranes must be lysed. The sugar and phosphate
components located on the backbone of DNA are soluble in
water. The phosphate groups on the outside of DNA carry a
negative charge, which are attracted to and neutralized by
cations such as sodium. With the presence of salt, protein
molecules precipitate from the solution. DNA is insoluble in
ethanol. When added to a solution containing DNA, ethanol
will come out of solution and stick to whatever it is around.
Precipitation via isopropyl alcohol is a common method
used for simple isolation of DNA. These methods involve
three main steps: homogenization, deproteinization, and
precipitation of DNA. Homogenization involves heating and
blending the tissue to expose the cells. Under common
protocols, the tissue is vortexed and then heated in a water
bath at 60ºC for 10 minutes. These exposed cells are then
deproteinized by implementing a buffer solution of sodium
hydroxide, table salt, SDS, and papain enzymes. The purpose
of this buffer solution is to emulsify any proteins clinging to
the nucleus, lyse the cell, and expose the DNA. By adding a
precipitant of 70% isopropyl alcohol, the DNA clumps
together (precipitates) and is able to be measured, extracted,
and analyzed.
Analysis by spectrophotometer: A spectrophotometer can be
used to evaluate DNA purity. The absorbance of DNA at 260
nm can be taking and compared to biochemical standards for
denatured, renatured, and native DNA at that frequency to
identify and deviance in the data – caused by contamination
or fragmentation. The standards used were obtained from
Pearson Edu.
Nucleic Acid
Thermodynamics
Nucleic acid thermodynamics: The temperatures that
affect the structure of DNA. Native DNA is DNA in its
native state as a double helical structure, with
complementary chemical structures. Denatured DNA
forms when the hydrogen bonds at the bases of DNA are
broken, causing DNA to unwind into single strands.
Renatured DNA occurs when the single strands of
denatured DNA reform to form a polynucleotide, a DNA
structure reformed after denaturation.
DNA denaturation is the process of breaking a
double-stranded DNA to generate two single strands.
There are a number of ways to denature the DNA within
a cell. The most common is by using the DNA’s melting
temperature (or Tm). When a DNA solution is heated
enough, the non-covalent forces that hold the two strands
together weaken and finally break. High pH also
promotes DNA denaturation. Solvents such as dimethyl
sulfoxide and formamide disrupt the hydrogen bonding
between DNA strands. Lowering the salt concentration of
the DNA solution also aids denaturation by removing the
ions that shield the negative charges on the two strands
from one another. At low ionic strength, the mutually
repulsive forces of the negative charges are strong enough
to denature the DNA at a relatively low temperature.
Renaturation of DNA, also known as annealing, is the
reformation of complementary strands in DNA separated
during denaturation. Usually the term refers to the
binding of a DNA probe, or the biding of a primer to a
DNA strand during a polymerase chain reaction (PCR).
Image taken from Pearson Edu.
Further Applications
The role of temperatures is especially
relevant today, because of their use in
such processes as gel electrophoresis and
polymerase-chain-reactions. DNA
denaturation is used in gel
electrophoresis through such processes as
denaturing gradient gels and
temperature gradient gels that can be
used to detect the presence of small
mismatches between two sequences
(temperature gradient gel
electrophoresis). DNA annealing is often
used in PCR. Which often involves the
binding of a DNA probe (fragment of
DNA used in DNA samples to detect the
presence of nucleotide sequences
complementary to the sequence in the
probe), or the binding of a primer to a
DNA strand.
Understanding the effect of both
renaturation and denaturation (as my
experiments aim to do), can improve
biotechnological approaches to DNA.
Works Cited
“Banana Ripening Stages” 24 November 2010
<http://www.bananaland.com.au/info/facts/banana_details_ripe
ning_stages.php>
Caprette, David R. “Absorbance Assay (280nm)” 24 May 1995. 7
December 2011.
<http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.ht
ml>
Mapson, L.W. & Robinson, J.E. (2007, June 28) Relation between
oxygen tension, biosynthesisf ethylene, respiration and ripening changes
in banana fruit. Retrieved January 31, 2012, from International
Journal of Food Science & Technology.
Rychlik, W., Spencer, W.J., Rhoads, R.E. (1990) Optimization of the
annealing temperature for DNA amplification in vitro. Retrieved
January 31, 2012 from Nucleic Acids Research, Volume 18, page 21.
Santos, E., Remy, S., Thiry, E., Windelinckx, S., Swennen, R., Sági, L.
(2009, June 24) Characterization and isolation of a T-DNA tagged banana
promoter active during in vitro culture and low temperature stress.
Retrieve January 31, 2012 from BMC Plant Biology.
Ussery, D.W. “DNA Denaturation” 2001. The Academic Presss
http://www.cbs.dtu.dk/staff/dave/genomics_course/2001_DNA
denature.pdf>
Watson, J.D. (2004) DNA: The Secret of Life. Arrow Books: New York.
Methodology
Materials
Musa acuminata in each of the sevens stages of maturation
99% Isopropyl Alcohol
Powdered Solution consisting of:
21.5% consisting of NaCl Sodium Chloride
77.3% consisting of NaHCO3
1.2% consisting of Papain Enzyme
Timer/Stopwatch with second accuracy
10mL Graduated Test Tubes
Graduated Eye Dropper
1% Methylene blue dye
Nylon Filter
Glass Extraction Rod
250mL Pyrex® Measuring Cup
Procedure:
250-500mL Beakers
1. Preparation of the
cell lysing buffer
Plastic 120mL Squeeze Bottle
10% Sodium Dodecyl Sulfate (SDS)
Cutting Board
Kitchen Knife
Vortex
Mortar and Pestle
4L of Distilled Water
3 pipets (1mL, 5mL, 10mL)
Pipet bulb
Spectrophotometer with cuvettes
Compound Microscope
Figure 4. Me in the Medical Lab at Indian River State
College conducting my studies.
2. Homogenization of
banana tissue
3. Precipitation of DNA
4. Determining DNA
concentrations
5. Analysis by
spectrophotometry
Figure 5, 6. Materials used in experiment.
Figure 4 taken by Rob Tack, Biotechnician at IRSC.
Figure 5 and 6 taken by me.
Preparation of the
Cell-Lysing Buffer
To isolate DNA, the banana’s
cells needed to be lysed. The
cell-lysing buffer used in this
experiment consisted of
sodium hydroxide, table salt,
sodium dodecyl sulfate, and
papain enzymes.
These solutions emulsify
proteins clinging to the
nucleus and prepare the DNA
for isolation.
The buffer was kept chilled at
10ºC in an ice water bath.
Figure 7. Cell-lysing buffer
Figure 8. Ice water bath for celllysing buffer.
Photos taken by me.
Homogenization of
Banana tissue
Homogenization involves heating
Figure 9. Banana chunks being
mashed in mortar and pestle.
and blending the banana tissue to
expose the cells.
1. Banana chunks placed in mortar
with distilled water, mashed
with a pestle.
Figure 10. Banana tissue
being treated to heat in water
bath.
2. Exposed tissue treated to varying
thermodynamic levels in water
bath:
1.
From 55-65º C to denature DNA.
2.
Heated, then cooled to 4-8º C
for annealing
Figure 11, 12. Labeled beakers
with treated banana tissue.
Photos taken by me.
Precipitation
of DNA
After the heat treatment, the
exposed banana cells were
deproteinized by
implementing the cell-lysing
buffer.
By adding a precipitant of
99% isopropyl alcohol, DNA
clumped together at the
interface of the solution, and
was able to be isolated and
measured.
Figure 14 taken by Rob Tack, Biotechnician at IRSC.
Figure 13 and 15 taken by me.
Figure 13. Banana tissue mixed with buffer solution.
Figure 14. Me adding 99%
isopropyl alcohol to solution.
Figure 15. Three layers form in
the test tube – the top, alcohol,
center, precipitated DNA,
bottom, banana filtrate.
DNA Concentrations:
Calculations & Results
Results showed that
denatured DNA had the
highest yield, that renatured
DNA had low concentrations,
and that little to no native
DNA was isolated.
Ratio of DNA Concentrations (±1)
Volume of DNA in mL/mL
DNA concentrations were
determined by applying a
biochemical formula, which
involved placing the amount
of DNA isolated over the
amount of banana tissue it
was isolated from.
1.4
1.2
1
0.8
Trial 1
0.6
Trial 2
0.4
Trial 3
0.2
Trial 4
0
Denatured
Renatured
Native
Manipulations
Figure 16. Bar graph representing ratio of DNA
concentrations.
Analysis by
Spectrophotometry
DNA isolated was then
placed in a cuvette of distilled
water, and analyzed with a
spectrophotometer at 260 nm.
These readings were then
compared to standards for
denatured, renatured, and
native DNA at a wavelength
of 260 nm.
Figure 17. DNA standards used, obtained from Pearson Edu.
Results from Spectrophotometric Analysis
Spectrophotometric Analysis of DNA Purity
Relative Absorbance at 260 nm
1.4
1.2
1
Denatured
DNA
Trial 1
Trial 2
0.8
0.6
Renatured
DNA
Trial 3
Native DNA
Trial 4
0.4
0.2
0
Figure 18. Line graph presenting the relative absorbance of
denatured, renatured, and native DNA at 260nm.
Conclusion
Results from four trials
proved that my hypothesis
was wrong, and that
denaturation of DNA was an
effective method for protocols
isolating DNA.
DNA Concentrations:
• Most denatured DNA
isolated
• Least native DNA isolated
Spectrophotometer:
• Denatured DNA deviated
least from standards
• Native DNA deviated
entirely
Discussion
Denaturing DNA was
effective in deproteinizing
any proteins and removing
deoxyribonuclease (DNase)
enzymes. The same results
were shown for renatured
DNA, however the process to
renature it seemed to cause
fragmentation of the isolation.
DNase enzymes were still
present in native DNA, which
shows that heat is necessary
for isolation of DNA, and that
denaturation will provide the
highest success of isolation.
Acknowledgments:

God, who has played an important role in my life, as well as my achievements in
science.

Rob Tack, a Biotechnician at the Medical Lab at Indian River State College who
photographed some of the images of me conducting my studies.

Marilyn Barbour, Director at the IRSC Medical Lab, who granted me lab access.

Francine, Robert & Sophia Bacchus, my sister, father, and mother who have
supported me greatly throughout my three years of research

Florida Virtual School, for providing me with this exceptional opportunity to
present my research.
Bibliography
“Banana Ripening Stages” 24 November 2010
<http://www.bananaland.com.au/info/facts/banana_details_ripening_stag
es.php>
Caprette, David R. “Absorbance Assay (280nm)” 24 May 1995. 7 December
2011. <http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html>
Mapson, L.W. & Robinson, J.E. (2007, June 28) Relation between oxygen tension,
biosynthesisf ethylene, respiration and ripening changes in banana fruit. Retrieved
January 31, 2012, from International Journal of Food Science & Technology.
Rychlik, W., Spencer, W.J., Rhoads, R.E. (1990) Optimization of the annealing
temperature for DNA amplification in vitro. Retrieved January 31, 2012 from
Nucleic Acids Research, Volume 18, page 21.
Santos, E., Remy, S., Thiry, E., Windelinckx, S., Swennen, R., Sági, L. (2009,
June 24) Characterization and isolation of a T-DNA tagged banana promoter active
during in vitro culture and low temperature stress. Retrieve January 31, 2012
from BMC Plant Biology.
Ussery, D.W. “DNA Denaturation” 2001. The Academic Presss
http://www.cbs.dtu.dk/staff/dave/genomics_course/2001_DNAdenature.
pdf>
Watson, J.D. (2004) DNA: The Secret of Life. Arrow Books: New York.
“Banana Ripening Stages” 24 November 2010
<http://www.bananaland.com.au/info/facts/banana_details_ripening_stag
es.php>
Caprette, David R. “Absorbance Assay (280nm)” 24 May 1995. 7 December
2011. <http://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html>
Dollard, Kate. “DNA Isolation from Onion” 07 December 2011.
<http://www.accessexcellence.org/AE/AEC/AEF/1994/dollard_onionDN
A.php?>
“Extraction of DNA from White Onion” 24 November 2010 <http://
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Hays, Lana “Introduction to DNA Extractions” 24 November 2010. <http://
<www.accessexcellence.org/AE/AEC/CC/DNA_extractions.php>
Klibaner, Edwin “Extraction of DNA from White Onion” 25 November 2010
<http://www.scienceteacherprogram.org/biology/oniondna.html>
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November 2010. <http://www.wisegeek.com/what-is-cell-lysis.htm>