Robert Bacchus, Jr. 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!

Affinity for Science 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.

Manipulating the nucleic acid thermodynamics of Musa

acuminata to evaluate DNA purity and improve

biotechnological approaches to DNA.

Banana Split! Third Year Study

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.The purity of the DNA

isolated is crucial.

Figure 1. Clip art of DNA within banana.

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 Independent Variables:

Thermodynamic levels used to treat banana tissue.

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)

1. Renaturation of DNA will result in the highest concentration of DNA.

2. Native DNA will provide the lowest concentration of DNA isolation.

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.

Variables

Use of the Banana

(Musa acuminata)The banana serves as an effective model for my research because of

how it ripens.As one of the world’s most

common exports, the fruit is readily available.

It is a eukaryotic plant, with membrane-bound cells containing

DNA in their nucleus.The fruit is categorized into seven

distinct colors of maturation. As it ripens, the tissue softens,

making the cells and DNA easily accessible.

1

2

3

4

5

6

7

Figure 3. Seven ripening stages of the banana.

Image by Dr. Scott Nelson, University of Hawaii

BackgroundResearch & 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.

ContinuationThe 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.

Nucleic Acid Thermodynami

cs 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 ApplicationsThe 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.

“Banana Ripening Stages” 24 November 2010 <http://www.bananaland.com.au/info/facts/banana_details_ripening_stages.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.

Works Cited

MethodologyMaterialsMusa 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

250-500mL Beakers

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

Procedure:1. Preparation of the cell lysing buffer2. Homogenization of banana tissue3. Precipitation of DNA4. Determining DNA concentrations5. Analysis by spectrophotometry

Figure 4. Me in the Medical Lab at Indian River State College conducting my studies.

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 cell-lysing buffer.

Photos taken by me.

Homogenization of

Banana tissueHomogenization involves heating and blending the

banana tissue to expose the cells.

1. Banana chunks placed in mortar with distilled water, mashed with a pestle.

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 9. Banana chunks being mashed in mortar

and pestle.

Figure 10. Banana tissue being treated to heat in

water bath.

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 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.Figure 14 taken by Rob Tack, Biotechnician at IRSC.

Figure 13 and 15 taken by me.

DNA Concentrations:

Calculations & Results

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.Results showed that

denatured DNA had the highest yield, that

renatured DNA had low concentrations, and that

little to no native DNA was isolated.

Denatured Renatured Native00.20.40.60.8

11.2Ratio of DNA Concentrations (±1)

Trial 1Trial 2Trial 3Trial 4

Manipulations

Volu

me

of D

NA

in m

L/m

L Figure 16. Bar graph representing ratio of DNA

concentrations.

Analysis by Spectrophotome

try

Figure 17. DNA standards used, obtained from Pearson Edu.

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.

Results from Spectrophotometric Analysis

50ºC

55ºC

60ºC

65ºC 8º

C6º

C4º

C

No tem

perau

re0

0.2

0.4

0.6

0.8

1

1.2Spectrophotometric Analysis of DNA Purity

Trial 1Trial 2Trial 3Trial 4

Rela

tive

Abs

orba

nce

at 2

60 n

m

Denatured DNA

Renatured DNA

Native DNA

Figure 18. Line graph presenting the relative absorbance of denatured, renatured, and native DNA at

260nm.

ConclusionResults 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

isolatedSpectrophotometer:• Denatured DNA

deviated least from standards

• Native DNA deviated entirely

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.

Discussion

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_stages.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_stages.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_onionDNA.php?> “Extraction of DNA from White Onion” 24 November 2010 <http:// dwb4.unl.edu/Chem/CHEM869N/CHEM869NLinks/cpmcnet.columbia.edu/dept/physio/tchrplan/oniondna.html>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>Liden, Daniel and Jenn Webb “What is Cell Lysis?” 09 September 2010. 24 November 2010. <http://www.wisegeek.com/what-is-cell-lysis.htm>“What is DNA?” 5 December 2010. 24 November 2010. <http://ghr.nlm.nih.gov/handbook/basics/dna>