Ee Draft Final1

SoYon Kim Biology Extended Essay
An investigation into five different extraction methods to best detect peanut DNA using PCR and visualise the amplified DNA through gel electrophoresis.
Candidate Number: School: North London Collegiate School Jeju School Number: Student: So Yon Kim Subject: Biology Supervisor: P. Brannac Word count: 3990 words Date: November 2013
Biology Extended Essay
Abstract
This investigation aims to find the most sensitive method for extracting peanut DNA that can be amplified through PCR and visualised by gel electrophoresis. In order for PCR to successfully amplify specific DNA, the template DNA solution should contain minimal impurities like proteins, DNases, lipids and chemicals. Impurities are present because different peanut parts have different functions. For example, since the embryo does not have storage or structural functions, my hypothesis is that its DNA is least likely to contain contaminants. These impurities are removed in the extraction and purification process. In Experiment 1, different parts of peanut (seed coat, cotyledon and embryo) of different mass is extracted and amplified through PCR. Then, in Experiment 2, to find the effect of extending or reducing incubation time, unsuccessful samples had their incubation time extended from 1 hour to 24 hours. Successful samples had their incubation time reduced to 20 minutes. In Experiment 3, the effect of Proteinase K is investigated by repeating the experiment without using Proteinase K for samples that showed positive results in Experiment 1. The experiment proved that long incubation time is more important to the extraction than the mass of the sample used for DNA extraction. Also, although the use of Proteinase K helps eliminating impurities, it is not necessary in all situations. The most sensitive detection was the detection of 0.0613g of embryo through a 24 hour incubation period and Proteinase K. For future studies, I could experiment by testing different foods to find out if they contain traces of peanut. (Word count : 255)
Table of Contents
Abstract 1.Introduction 2. Hypothesis 3. Investigation 4. Method 4.1 Modifying pH of Solutions 4.2 Extraction 4.3 Purification 4.4 Ethanol Precipitation 4.5 PCR (Polymerase Chain Reaction) 4.6 Gel electrophoresis 5. Results 6. Conclusion & Analysis 7. Evaluation 8. Appendix 2 4 8 8 10 12 12 14 15 16 17 19 22 26 29
1.Introduction
Allergies are among the most common diseases that many people suffer from. Allergic reactions are hypersensitive reactions towards foreign substances by the human auto immune system. Among many food products that have the potential to cause allergic reactions in humans, the peanut is known to be one of the most severe types. It can be fatal to its sufferers with a minimal dose ingested. Also the allergy lasts for lifetime so individuals always need to be cautious, avoiding even the smallest trace of peanut.1 According to research, a peanut seed storage protein Ara h 2 is the allergen; the main cause of allergic reaction2 and it is recognized by 90%-100% of peanut allergy patients3. Screening for traces of peanut in food is useful in order to avoid any contact with that allergen for sensitive people. In order to do that, peanut free food produced from peanut free environment should be guaranteed. First developed in 1984 by Kary Mullis (it granted him a Nobel Prize4), Polymerase Chain Reaction (PCR)5 is widely used to detect traces of a substance or for genetic fingerprinting due to its high
Figure 1 Polymerase Chain Reaction
1 Elena Scaravelli. (2008). Development of Three Real-Time PCR Assays to Detect Peanut Allergen
Residues in Processed Food Products.European Food Research and Technology. 227 (x), p857-869.
2 James Huntley, Soheila Maleki, Peggy Ozias-Akins. (2009). Identification and characterization of a
hypoallergenic ortholog of Ara h 2.01. Plant Molecular Biology. 69 (Issue 3), p325.
3
Dang TD, Tang M, Choo S, Licciardi PV, Koplin JJ, Martin PE, Tan T, Gurrin LC, Ponsonby AL, Tey D, Robinson M, Dharmage SC, Allen KJ; HealthNuts study. (2012). Increasing the accuracy of peanut allergy diagnosis by using Ara h 2.. Journal of Allergy and Clinical Immunology. 129 (Issue 4), p1056 1063.
4 The Nobel Prize in Chemistry 1993. 2013. The Nobel Prize in Chemistry 1993. [ONLINE] Available
at:http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/. [Accessed 01 November 2013].

5 Bruce Alberts, Alexander Johnson, Julian Lewis , Martin Raff , Keith Roberts, Peter Walter
(2008). Molecular Biology of the Cell. 5th ed. United States of America: Garland Science. P544-547.

specificity. For instance, in UKs horsemeat scandal, PCR was used to detect the presence of horsemeat in commercial food products.6 As shown in Figure 1, the template DNA extracted from peanut is denatured so that the double-stranded DNA separates into two single strands of DNA at 95C.
Figure 2 Complementary Base Pairing7
Then, at lower temperature, 56C Annealing step, primers bind to two single strands of DNA. (50C Annealing step did not work, thus altered to 56C using Tm calculator8. Annealing temperature depends on Guanine/Cytosine content since they have three hydrogen bonds linking them; higher GC content requires higher temperature for denaturation.) Temperature is raised to 72C9 in the next step extension, where thermo-stable Taq polymerase binds to the primers and extends the primer to make a complementary strand to the template. This forms one cycle. In about 30 cycles, the DNA samples would be amplified enough to be detected under gel electrophoresis that visualises DNA by separating DNA according to their mass (size) when charge is applied so that the negatively charged nucleic acids can move towards the positive side of the electric field.
Mun-Keat Looi. (2013). Flogging a dead horse: genetics and the UK horsemeat scandal. Available: http://blog.wellcome.ac.uk/2013/03/11/flogging-a-dead-horse-genetics-and-the-uk-horsemeat-scandal/. Last accessed 13th Oct 2013.
7
DNA Double Helix. 2013. DNA Double Helix. [ONLINE] Available at:http://www.elmhurst.edu/~chm/vchembook/582dnadoublehelix.html. [Accessed 01 November 2013].
8
Tm Calculator | New England Biolabs . 2013. Tm Calculator | New England Biolabs . [ONLINE] Available at: https://www.neb.com/tools-and-resources/interactive-tools/tm-calculator. [Accessed 01 November 2013].
9
Mark V.Bloom, Greg A.Freyer, David A.Micklos (1996). Laboratory DNA Science. Menlo Park, CA: The Benjamin/Cummings Publishing Company, Inc. p281-301.
In order to achieve successful detection of DNA, the template DNA should be stable. To do that, DNases(Deoxyribonucleases), which breaks down DNA, should be removed so that it can not damage the DNA. Also, it is vital to extract DNA with minimum impurities like lipids, polysaccharides, proteins and some chemicals, since those impurities can stop PCR. In this investigation, the extraction method that has the best possibility of achieving the most sensitive detection of peanut DNA with the least impurities will be discussed. The interference of impurities will be shown through smeary bands or even no detection through gel electrophoresis. The detection method that will be discussed includes the amount of peanut, the time of incubation and the use of Proteinase K. Also, the position from which the peanut tissue is taken was tested. They are; the seed coat, the cotyledon and the embryo were tested separately in each trial. The goal for this investigation is to optimise DNA extraction method to detect peanut DNA in smallest amounts with least impurities.
Figure 3. The structure of a peanut.10
The development of PCR is significant that it allowed the detection of substances and organisms through their DNA. Also, one of the modern uses of PCR includes its application to forensic science, DNA-fingerprinting. Especially, it offered more cost effective method for detection of DNA and proteins with higher specificity compared to a more common method for allergen detection by Enzyme-linked immunosorbent
10
Peanut Plant - How does the groundnut grow?. 2013. Peanut Plant - How does the groundnut grow?. [ONLINE] Available at: http://www.boiled-peanut-world.com/peanut.html. [Accessed 30 September 2013].

assays (ELISAs) that use antibodies in detection.11 Also some ELISA cannot detect denatured proteins because as they are denatured, the epitope where the antibody recognizes can be altered. By investigating the extraction method that would result in the most sensitive detection of peanut DNA it will help to find traces of peanut in food products and potentially prevent peanut allergic people from consuming the product, by ensuring the production of peanut-free food.
11 Overview of ELISA. 2013. Overview of ELISA. [ONLINE] Available
at:http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403. [Accessed 29 September 2013].
2. Hypothesis
Purer and more DNA dense samples will need more intensive extraction techniques.
3. Investigation
To test this hypothesis, I would be investigating four factors. 1. Purity of peanut sample. Different tissues have different adapted functions and therefore different contents, which will lead to different impurities.
Figure 4. Mass, DNA content, DNA density in different parts of a soybean.12
2. Mass of extraction samples. The greater the amount of DNA sample, the greater DNA present in the peanut sample. With greater quantity of DNA, more nucleic acid will be extracted successfully.
12
Wen-Tao Xu, Kun-Lun Huang, Ai-Ke Deng, Zhi-hong Liang, Yun-Bo Luo. (2007). Variations of tissue DNA density and nuclear DNA content in soybean lines and their impacts on the GMO quantification. Food Control. 18, p1300-1306.
3. Use of an enzyme to remove macromolecules. Purer template DNA will be achieved through the use of Proteinase K since Proteinase Ks main role is to digest DNases and other proteins that potentially damage nucleic acids or prevents successful PCR. 4. Length of Lysis incubation. As there is more Lysis incubation time, Proteinase K will have more time to break open cell walls and membranes and remove more proteins and DNases.
4. Method13
Variables Independent Variables: Four DNA extraction variables were tested: 1. The part of the peanut. (seed coat, cotyledon, embryo) 2. The amount of peanut sample used in purification. (0.0613g, 0.125g, 0.25g, 0.5g) 3. Time of incubation in Lysis buffer. (24hours, 20 minutes) 4. The existence of Proteinase K. (with Proteinase K, without Proteinase K) Dependent Variable : The detection of amplifiable DNA through PCR visualised by gel electrophoresis. (A clear band of DNA will be shown) Conditions for independent Variables: * Positive and negative results refer to the success or failure to detect peanut DNA through electrophoresis. <Experiment 1> Code 1A1 1A2 1A3 1B1 1B2 1B3 1C1 1C2 1C3 1D1 1D2 1D3 The location of Peanut DNA. Seed coat Cotyledon Embryo Seed coat Cotyledon Embryo Seed coat Cotyledon Embryo Seed coat Cotyledon Embryo The amount of peanut DNA. (g) 0.0613 Time of incubation.(minutes) 60 minutes Use of Proteinase K. Yes
0.125
60 minutes
Yes
0.25
60 minutes
Yes
0.5
60 minutes
Yes
Table 1 Experiment Variables - Experiment 1
13 Mark V.Bloom, Greg A.Freyer, David A.Micklos (1996). Laboratory DNA Science. Menlo Park, CA:
The Benjamin/Cummings Publishing Company, Inc. p281-301.
10

<Experiment 2> *Based on results from Experiment1, the only trials that showed negative results (1D1, 1C2, 1A3) had their incubation time extended to 24hours to see if longer incubation time will show positive result. The only trials that succeeded in the detection of DNA(1D2, 1B3) had their incubation time reduced to 20 minutes to see if they still work with low incubation time. Seed coat is not tested because its DNA failed to be amplified with more incubation time. Code 2A1 2A2 2A3 2B1 2B2 2B3 The location of peanut DNA. Seed coat Cotyledon Embryo Cotyledon Embryo The amount of peanut DNA. (g) 0.5 0.25 0.0613 0.5 0.125 Time of incubation. (minutes) 60 X 24 = 1440 (24hr) 1440 1440 20 20 Use of Proteinase K. Yes Yes Yes Yes Yes
<Experiment 3> *Based on results from Experiment1, the only trials that showed positive results (1B3 1D2) were tested again without Proteinase K.
Code 3A 3B 3C
The location of peanut DNA. Cotyledon Embryo
The amount of peanut DNA. (g) 0.5 0.125
Time of incubation. (minutes) 1440 1440
Use of Proteinase K. No No
For all trials, the same PCR conditions14 are used. Denaturation - 95C, 75 seconds Annealing - 56C, 30 seconds Extension - 72C, 120 seconds
14 Standard PCR Conditions . 2013. Standard PCR Conditions . [ONLINE] Available at: https://www.k-
state.edu/hermanlab/protocols/StandardPCRConditions.html. [Accessed 03 October 2013].
11
Figure 5. PCR Condition
*Control variables will be discussed in depth in the Method. *List of reagents and equipment is stated in the Appendix1 and 2.
4.1 Modifying pH of Solutions

See Appendix3.
4.2 Extraction
1. Classify peanuts into 3 parts: seed coat, cotyledon and embryo. 2. Crush each part with mortar and pestle separately. To avoid contamination, use different mortars and pestles for each type of peanut sample. This mechanically destroys peanut cell walls.
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TE buffer
SDS from washing up liquid
Proteinase K
Figure 6 DNA Extraction equipment and reagents
3. Put crushed peanut tissue in a 200ul PCR tube and add Lysis buffer15 (See Appendix7 for composition.) SDS in washing up liquid (Although not ideal, washing up liquid16 containing dye is used because the school lab did not have SDS.) in Lysis buffer demolishes the cell membrane composed of fats and nuclear membrane.17 This exposes the DNA to the solution. Proteinase K digests cell membranes that are made up of protein and also digests DNases that damage the DNA. Also, EDTA in TE (Tris-EDTA) buffer removes metal ions required by active sites of DNases so that they cannot degrade the DNA.
15 DNA Isolation Protocol. 2013. DNA Isolation Protocol. [ONLINE] Available
at: http://web.mit.edu/jacks-lab/protocols/DNA_Isolation_tables.html. [Accessed 01 November 2013].

16 Lysis buffer - Wikipedia, the free encyclopedia. 2013. Lysis buffer - Wikipedia, the free encyclopedia.
[ONLINE] Available at:http://en.wikipedia.org/wiki/Lysis_buffer. [Accessed 01 November 2013].

17
SEP staff.Strawberry DNA Extraction. Available: http://seplessons.ucsf.edu/node/217. Last accessed 25th Aug 2013.
13
60C Water bath Weighing boat PCR tubes
Figure 7 DNA Extraction - Incubation of peanut sample in 60C water bath.
4. Place the PCR tube in 60C water bath for set incubation time. (Puncture weighing boat to hold PCR tubes.) This denatures the cytoplasmic enzymes that break the DNA apart.18
4.3 Purification1920
See Appendix 5. The method is identical to the method from the source.
Aqueous phase Organic phase

Figure 8.1 DNA Purification - Addition of Phenol Figure 9.2 DNA Purification Addition of Chloroform
18
Department of Biochemistry and Molecular Biophysics. (2002). DNA Extraction from Cheek Cells. Available: http://webcache.googleusercontent.com/search?q=cache:mosvrsbY7tgJ:biology.arizona.edu/sciconn/les sons2/vuturo/vuturo/dna.htm+&cd=1&hl=en&ct=clnk&gl=kr. Last accessed 25th Aug 2013.
19 Phenol/chloroform extraction - OpenWetWare. 2013. Phenol/chloroform extraction - OpenWetWare.
[ONLINE] Available at:http://openwetware.org/wiki/Phenol/chloroform_extraction. [Accessed 01 November 2013].

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4.4 Ethanol Precipitation2122

In the ethanol precipitation step, the DNA is precipitated by freezing it in ethanol. 1.Add 20ul of 3M Sodium Acetate to PCR tube from step 11. Addition of 3M Sodium Acetate makes the solution extremely polar, forcing DNA out of the solution. 2. Add 200ul of ethanol and put it in a -20C freezer and freeze them overnight. Since DNA, being polar, is insoluble in alcohol, while other cell parts are, DNA precipitates out of the emulsion and submerges to the bottom of the PCR tube. 3. Take the tube out of the freezer and spin it in the centrifuge for 30 minutes. 4. Take the tube out and carefully tilt the tube to pour out most of the liquid out. If you can see smudge-like, fog-like precipitates in the PCR tube, they are the precipitated DNA. All other impurities in the solution are poured away except for the precipitated DNA at the bottom of the PCR tube. 5.Dry the PCR tube. Leave the cap open in the air for 2-5 hours so that remaining ethanol evaporates. 6. To re-suspend DNA, add 50ul of TE buffer. TE buffer will ensure that nucleic acid is stored at pH8 and EDTA will chelate any remaining metal ions23 to disable remaining DNase to prevent degradation of DNA. 7. Spin it in the centrifuge to re-suspend nucleic acids.
21 Ethanol precipitation of nucleic acids - OpenWetWare. 2013. Ethanol precipitation of nucleic acids -
OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids. [Accessed 15 September 2013].


23
Ethanol precipitation of nucleic acids - OpenWetWare. 2013. Ethanol precipitation of nucleic acids OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids. [Accessed 15 September 2013].
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Precipitated DNA
Figure 9 Ethanol Precipitation - precipitated DNA before centrifugation
4.5 PCR (Polymerase Chain Reaction)

1.Take 8ul of re-suspended DNA solution and add it to the PCR tube containing the PCR bead. PCR bead (Edvotek) contains building blocks of nucleotides, which are Deoxyribonucleotides (dATP, dCTP, dGTP, dTTP), Taq Polymerase, reaction buffers24 2. In the same PCR tube, add 8ul of Primer1 and Primer2. 3. Place the tube in the thermo-cycler. 4. Set the thermo-cycler and start it. Denaturation - 95C, 75 seconds Annealing - 56C, 30 seconds Extension - 72C, 120 seconds
24
PuReTaq Ready-To-Go PCR Beads. 2013. . [ONLINE] Available at:https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/1314750913712/litd oc11002607AB_20110831023628.pdf. [Accessed 15 September 2013].
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4.6 Gel electrophoresis25

1. Pour 3% Agarose gel into the mold with comb. (3g of Agarose powder, 5ml of 20xTAE, 95ml of distilled water, 8ul Red Safe stain) 2. Put the gel into the solution of 1 litre TAE buffer so that the buffer solution covers the gel completely. (100ml 20xTAE, 900ml distilled water, 20ul Red Safe Stain) 3. Add 8ul of loading buffer to the PCR-processed DNA solution. 4.Transfer 20ul of this solution into the well on agarose gel. 5.The well should be on (-) side, facing (+) side, run it at 120V for 20 minutes.
Figure 10 Gel Electrophoresis - Experimental setup
Negatively charged DNA is attracted to the positively charged electrode, separating DNA by its size. 6. Observe whether a clear florescent band is present on the gel under a hand held UV light. For ones that did not work, a blue stain will be shown or nothing will be shown if the stain diffused away.
17
DNA detected
Figure 11 Gel Electrophoresis - Detection of DNA
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5. Results
Visual Results The amount of peanut 0.06125g
1A1 1A2
No bands detected in lane 1A1, 1A2 and 1A3.
1A3
0.125g
1B1 1B2 1B3

0.25g
Clear band detected for 1B3. No band detected in 1B1 and 1B2.
1C1 1C2 1C3
Clear band detected in 1C3. No band detected in 1C1 and 1C2.
0.5g
1D1 1D2 1D3
Clear band detected in 1D2 and 1D3. No band detected in 1D1.
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The time of incubation 24 hours Smeary band detected in both 2A1 and 2A3. A clear band detected in 2A2. No band detected for 2B2. A clear band detected for 2B3.
2A1 2A2 2A3
20 minutes
2B2 2B3
Existence of Proteinase K Without Proteinase K
3B 3C
No band detected for 3b. A clear band detected for 3C.
Table 4 Results Table - Detection of DNA, with fluorescent band
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Table of results
The amount of peanut (g) 0.0613 A. Seed coat 0.125 0.25 0.5
The time of incubation 24 hour 20 minutes
Without ProteinaseK
(1A1)
(1B1)
(1C1)
(1D1)
-smear
(2A1) B. Cotyledon
(1A2)
(1B2)
(1C2)
(1D2)
(2A2)
(2B2)
(3A)
C. Embryo
(1A3)
(1B3)
(1C3)
(1D3)
-smear
(2B3)
(3C)
(2A3)
Table 5 Results Table - Summary
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6. Conclusion & Analysis

* This is written under the assumption that there were no errors in Polymerase Chain Reaction and electrophoresis step because there were only one band of same size and there were many negative results. This suggests that the detected DNA is likely to be the same DNA. As anticipated in the hypothesis, the more extreme the method was, the better chance that it showed positive results. It is proven in Experiment 1 that DNA from the embryo is most likely to produce the template DNA that can be detected with least amount of sample. However, cotyledon was more prone to DNA detection than seed coat although the DNA density of cotyledon was less than seed coat. This shows that rather than DNA, the type of impurity in the sample matters more. The embryos main function is to produce daughter cells and to germinate, thus considered to contain purest DNA. The seed coat (testa) functions as a protective barrier against the external environment and pathogens, as a nutrient pathway for the cotyledon and the embryo and also controls the seeds dormancy and germination.26 The seed coat is mainly composed of structural polysaccharide complex, cellulose27, which is a known PCR inhibitor28, whereas, the cotyledon is mostly composed of lipids and proteins that serve as nutrients for the germination of the embryo. Protein-based impurities can be removed by Proteinase K and lipids are broken down by SDS. Since polysaccharides were not removed properly in this experimental condition, it could have inhibited PCR. However, impurities from seed coat was overcome by 24 hour incubation (2A1). In addition, the experiment showed that greater mass of sample leads to easier amplification of DNA. In Experiment 1, all 0.0613g of peanut samples failed to be amplified through PCR. This implies that Experiment 1s codition (1hour incubation,
26
Gurdev S. Khush, Darshan S. Brar, Bill Hardy (2003). Advances in Rice Genetics, Volume 1. Philippines: International Rice Research Institute(IRRI). p473-475.
27
N. S. Rangaswamy and Lakshmi Nandakumar. (1985). Correlative Studies on Seed Coat Structure, Chemical Composition, and Impermeability in the Legume Rhynchosia minima. Botanical Gazette. 146 (4), p501-509.
28
Monteiro, L. et al. (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J. Clin. Microbiol.35, 995 8.
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using Proteinase K) 0.0613g of peanut cannot be PCRed. However, as the mass of DNA sample increased, they were more likely to show positive results, although a more important factor in determining success of the extraction was the purity of the sample associated with tissues location. For example, 1C2 showed a negative result while 1D2 showed a positive result.
Figure 12.1 Structure of Proteinase K29
Figure 13.2 Structure of a Nucleosome
Proteinase K is an enzyme that breaks down proteins by slicing them into smaller polypeptide chains by hydrolysis.30 This is widely used for extraction of DNA since it is stable over wide range of pH from pH4 to pH12 (optimum: pH7-pH12). So it is not denatured with basic SDS. One of the most important roles of Proteinase K is to digest DNases and proteins. In plant cells, the main role of DNases is to combat viruses. Also, nucleases are shown to take part in DNA degradation as the cell dies31. Remaining DNA related proteins like histones could inhibit the action of Taq polymerase. Proteinase Ks eliminates those proteins after SDS breaks down nuclear membrane of the peanut cell and releases the DNA. Then, at the Phenol-Chloroform purification, the remaining lysate, which contains proteins that could contaminate the template DNA is easily removed. Most proteins can be eliminated to produce PCR-Quality lysate just
29Betzel
C, Singh TP, Visanji M, Peters K, Fittkau S, Saenger W, Wilson KS (July 1993). "Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution". J. Biol.Chem. 268 (21): 158548.
30
Hedstrom, L. (Dec 2002). "Serine protease mechanism and specificity.". Chem Rev 102 (12): 4501 24. Leniewicz K, Porba E, Smolarkiewicz M, Wolff N, Stanisawski S, Wojtaszek P. (2012). Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases.. BMC Plant Biology. 12. p195.
31
23

with Proteinase K and SDS32. However, due to high lipid (PCR inhibitor33) content of peanut and thick cell wall, purification step is essential for efficiency. Longer length of Lyis incubation increases the probability for successful amplification of DNA since the activity of Protienase K and SDS increase. This is because high temperature provides them of greater kinetic energy so that more successful collision between the enzyme and its substrate occurs, therefore, results in higher yield. The solubility of lipid bilayer, including the cellular membrane also increases because SDS helps break apart cell membrane to release DNA in to the solution. Thus, it will be more active at digesting unnecessary proteins for the purification of DNA. Furthermore, more incubation time means that more unnecessary proteins will be denatured and unfolded due to the higher temperature, which made conditions more favourable activity for Proteinase K. The most surprising result is the detection of DNA in 2A1, which Experiment1 proved to contain the most impurities, although the band is smeary. Smeary bands indicate nonspecific amplification, which will be further discussed in the evaluation. Use of Proteinase K suggests the kind of impurities sites of sample extraction has. In Experiment3, without Proteinase K, 3B showed negative results whilst 3C showed positive results. This shows that what inhibits PCR is removed by Proteinase K suggesting that impurities are protein-based and the type of impurities vary according to their sample extraction site. Assuming that impurities are considered as materials that inhibit PCR, the experiment proves that cotyledon has protein-based impurities while embryo does not. Embryos impurities are not significantly protein-based. In conclusion, Proteinase K was not essential in all cases, the more important factor that affected the DNAs ability to be PCRed was the length of incubation they undergo, so that possible PCR inhibitors are broken down. Even the microscopic amount of DNA has a possibility to be detected through PCR if they are incubated sufficiently like the most sensitive detection 2A1 shows. Also, different foods may need different
32
Gary E, Truett. (2006). Preparation of Genomic DNA from Animal Tissues. In: Jan Kieleczawa DNA Sequencing II: Optimizing Preparation and Cleanup, Volume 2 . Canada: Jones & Bartlett Learning. p3941.
33
H. Hird, J. Loyd, R. Goodier, J. Brown, P. Reece. (2003). Detection of peanut using real-time polymerase chain reaction. European Food Research and Technology. 217 (Issue 3), p265-268.
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methods of extraction to trace the existence of a substance depending on the amount and the kind of impurities they contain.
25
7. Evaluation
The smeary band appeared in samples 2A1 and 2A3 indicates that the DNA detected is non-specific since a single band represents an incorrectly amplified DNA sequence. Assuming the errors originated from extraction, it could be because there were contaminants in the template DNA. These remaining impurities could interfere with annealing step by stabilising nonspecific primers or interfere with the ability of Taq polymerase to recognise and add on complimentary bases. This results in amplifying nucleic acids of many different lengths. Then, in the electrophoresis step when the DNA is separated according to their mass, nucleic acids of different lengths will be shown as a smeary line.
Gel Loading Buffer
Figure 13 No DNA detected due to errors in PCR.
There were failed trials as shown in Figure 14, no DNA (fluorescent band) was detected, while the movement of the loading buffer (dark blue band) was shown. This is because there was no DNA in the sample. This could be caused by impurities in the template DNA that inhibits PCR. Impurities could not have been removed from the extraction and purification step. PCR conditions were originally from H.Hirds article Detection of peanut using real-time polymerase chain reaction but it did not work as shown in Figure 14. Dr. Hirds method did not contain 72C Extenstion step. As I included that step from Blooms Laboratory DNA Science, it worked. In Dr. Hirds reply to my email (see Appendix8), the reason why he did not include 72C step is because it is not essential in many cases because the rate of addition of bases for the enzyme is so high that it would have completed base addition before 72C step. Also, although 72C is the optimum temperature for Taq polymerase, it works at other temperatures with less
26

efficiency. Therefore, the problem could have been our Taq polymerase or the thermocycler.34
Figure 14 Temporary circuit with two power packs.
Figure 15 Difference in size between 1.5ml, 500ul, 200ul tube.
In addition, there are some human errors that could have harmed the accuracy and reliability of this experiment. First, during the experiment, two fuses of the thermocycler blew up. Thus, a temporary circuit connecting two power packs in series (Figure15) had to be constructed, so the voltage became 24V instead of 120V, which resulted in more diffused bands. Originally, all the extraction, purification and resuspension step should have been done with 1.5ml PCR tube. However, due to limited supply of them, most steps were done in 500ul and tubes. Moreover, the lab did not have a microfuge that could fit a 200ul PCR tube, so a centrifuge that neither 200ul nor 500ul PCR tube would fit, had to be used. This could have affected the re-suspension the precipitated DNA. Also, the centrifuge did not have a cooling system, so during resuspension, the temperature inside the centrifuge increased. This could yield less DNA precipitation. Thus in the future, experiment should be done with a microfuge with refrigeration system. Furthermore, there is a systematic error in using a micropipette, which was used throughout this experiment to transfer solutions. The lab only had a micropipette with accuracy range of 20ul to 200ul, but it could go down to 8ul. The accuracy of pipette is
34
H. Hird. Hez.Hird@fera.gsi.gov.uk. Hi there, Thank you for the interest in our work.. 31 Oct 2013.
27

checked through weighing 8ul pipetted with a balance that is accurate to 0.001g and it was proven accurate. However, it was not ideal for the accuracy of the results, so micropipettes with higher precision should be used in the future. Also, a UV lamp is required when observing whether DNA was detected and small hand-held battery-powered UV lamp with weaker light intensity was used. A clearer results could have been obtained with a brighter UV lamp. Due to limitations in resources, including PCR beads (only 1 pack of 25 beads), no repeats were done. Moreover, positive controls have been avoided to reduce the use of materials including PCR beads. To increase reliability of the result, this experiment should be repeated at least three times with positive controls. This could also allow more accurate data collection to see the correlation between the amount of template DNA (concentration) and how it affects its ability to be amplified. To make better use of this for food safety finding traces of allergy-provoking substances, this should be done with commercial food samples. Additionally, future investigation could change the Proteinase K concentration or even use different enzymes. For instance, Cellulase could be adopted instead of Proteinase K to observe if it makes structural polysaccharide based seed coat DNA to be extracted with fewer impurities. Furthermore, this investigation focused on applying slight changes to extraction methods, but for future work, investigation could be done to optimise PhenolChloroform extraction. Additionally, it could look at quantifying extracted DNA through its UV absorption rate35 or introduce different impurities like lipids to the tested samples. Also, extracting peanut DNA from possible peanut-containing commercial products like Nutella, will be a step toward to using PCR as a food safety checking method. Screening for traces of peanut in commercial food products are already helping peanut allergic people to avoid the consuming them. In the future, this experiment could be taken further to detect traces of peanut in food sources since this would be applicable to many real-life situations to provide guaranteed peanut free, safe food for people. (Word count: 3990 words)
35
Quantitation of DNA and RNA . 2013. Quantitation of DNA and RNA . [ONLINE] Available at:http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full. [Accessed 02 November 2013].
28
8. Appendix
Appendix1. List of Reagents36 Lysis Buffer Phenol (Tris buffered) pH.815 Chloroform 95% Ethanol 3M Sodium Acetate Peanut primers (pre-ordered) TE, 16ul washing up liquid (SDS), 8ul Proteinase K
PCR bead (Edvotek) Gel Loading buffer 10Xconcentration (Edvotek) Indicator solution RedSafe (iNtRON Biotechnologies) TAE buffer15 Tris-Cl buffer pH.815 Agarose Gel15 Distilled water
Primer1 (51-GCTCGAGAGGCCGAACCT-31)37 Primer2 (51-TCCTCGTCACGTTGGATATTC-31)38 Taq polymerase, MgCl2, dNTPs
Agarose, TAE, indicator solution Red Safe
Figure 16 Reagents used in the experiment.

36
Mark V.Bloom, Greg A.Freyer, David A.Micklos (1996). Laboratory DNA Science. Menlo Park, CA: The Benjamin/Cummings Publishing Company, Inc. p292-298.
37
38
29
Appendix2. List of Equipment and Supplies Mortar and pestle 200ul PCR tube Permanent marker 60C Water bath 8ul-100ul Micro pipettes Micro pipette tips (disposable) Weighing boat Hand held UV lamp Electrophoresis kit39 (Edvotek) Freezer (-20C) Centrifuge Thermocycler (Edvotek) Electronic balance accurate to 0.0001g
Gel tank, 4-toothed comb, wires with crocodile clip
Appndix3. Adjusting pH of solutions40 Modifying pH of solutions such as phenol is vital since pH8 inhibits DNase action (nonoptimal) and charges DNA so that it moves during electrophoresis. To adjust pH of Tris buffer, use pH metre to keep track of pH change. Keep adding HCl until there is no further change in pH although more acid is added. Then, Tris buffer becomes Tris-Cl with pH 8. To adjust pH of Phenol to pH8, Tris Base is added and mixed with magnetic stirrer. This is done in the fume hood and pH Strips were used to prevent pH probe being damaged by Phenol. Then, add drops of HCL to get desired pH8.41
Appendix5. Purification4243
39
Equipment and materials | DNA | Base unit. 2013. Equipment and materials | DNA | Base unit. [ONLINE] Available at:http://www.ncbe.reading.ac.uk/ncbe/materials/dna/baseunit.html. [Accessed 05 October 2013].
40
Phenol/chloroform extraction - OpenWetWare. 2013. Phenol/chloroform extraction - OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Phenol/chloroform_extraction. [Accessed 01 November 2013].
41
Preparation of phenol/phenol:chloroform. 2013. Preparation of phenol/phenol:chloroform. [ONLINE] Available at: http://www.mrc-lmb.cam.ac.uk/ms/methods/phenol.html. [Accessed 02 November 2013].
42 Phenol/chloroform extraction - OpenWetWare. 2013. Phenol/chloroform extraction - OpenWetWare.
[ONLINE] Available at:http://openwetware.org/wiki/Phenol/chloroform_extraction. [Accessed 01 November 2013].
30
1. In the PCR tube, add 120ul of Phenol (pH8) and shake it to mix it or use a micropipette to mix it. (1st Phenol) This step is very important since Phenol eliminates lipids and proteins, which are known PCR inhibitors. Also, it is important to use Tris-buffered pH8 phenol since only at slightly alkaline pH (pH7-8), 44DNA moves into the aqueous phase. This is because only at alkaline solutions, negatively charged phosphate diesters become more charged to make the DNA soluble in the aqueous phase. However in acidic conditions, where H+ ions are available in solution for negatively charged DNA to gain to become neutral, DNA becomes less soluble.

Figure 17.1 DNA Purification - Addition of Phenol Figure 17.2 DNA Purification Addition of Chloroform
2. Centrifuge the PCR tube so that the organic and aqueous phase separate. 3.Transfer the aqueous phase into a new PCR tube and add another 120ul of phenol. (2nd Phenol) 4. Repeat step 2,3 of Purification twice until 3rd Phenol. 5. Only transfer the aqueous phase into a new PCR tube and add 120ul of Chloroform. (1st Chloroform) 6. Mix and centrifuge it so that it divides into two layers of Chloroform and aqueous layer.

44
P. Zumbo. Phenol-chloroform Extraction . Available: http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOL-CHLOROFORM.pdf. Last accessed 11th Oct 2013.
31

7. Transfer the aqueous layer into a fresh PCR tube and add another 120ul of Chloroform. (2nd Chloroform) 8. Mix and centrifuge, let it divide into two layers. 9. Transfer the aqueous phase into a fresh PCR tube.
Appnedix6. Safety Lab coats should always be worn for protection. Goggles should always be worn for protection. Rubber gloves should always be worn for protection. Phenol According to CLEAPSS Student Safety sheet, Phenol is toxic and corrosive. Toxic by inhalation, in contact with skin and if swallowed. R48/20/21/22: Harmful: danger of serious damage to health by prolonged exposure through inhalation, in contact with skin and if swallowed. R34: Causes burns.45 Chloroform According to CLEAPSS Student Safety Sheet, Chloroform is harmful. R40: Limited evidence of a carcinogenic effect. Category 3 carcinogen. R22: Harmful if swallowed. R48/20/22: Harmful: danger of serious damage to health by prolonged exposure through inhalation and if swallowed. R38: Irritating to skin. 46
45
(2007). CLEAPSS Student Safety Sheet - Phenol. Available: http://www.cleapss.org.uk/attachments/article/0/SSS64.pdf?Secondary/Science/Student%20S afety%20Sheets/. Last accessed 12th Sept 2013. 46 (2007). CLEAPSS Student Safety Sheet - Chloroform. Available: http://www.cleapss.org.uk/attachments/article/0/SSS64.pdf?Secondary/Science/Student%20S afety%20Sheets/. Last accessed 12th Sept 2013.
32

Appendix7. Formulating Solutions Lysis Buffer 8ul Proteinase K 16ul washing up liquid 16ul TE buffer 0.5M EDTA (pH8)47 186.1g disodium ethylenedamine tetraacetate 800g of distilled water stir vigorously on a magnetic stirrer Adjust pH by adding NaOH until it reaches pH8 50X TAE 242g Tris base 37.2g EDTA 900ml distilled water 57.1ml glacial acetic acid Fill the remaining volume with distilled water to 1 litre. TE buffer 10mM Tris-HCl (pH8) 1mM EDTA
47 Recipes for Common Laboratory Solutions. 2013. [ONLINE] Available
at:http://www.promega.com/~/media/Files/Resources/Technical%20References/Recipes%20For%20Co mmon%20Lab%20Solutions.pdf. [Accessed 06 October 2013].
33

Appendix8. Email reply from Dr. Hird
34
Appendix9.Photos of the process of experiments.
Appendix 9.1 Thermocycler used in the experiment.
Appendix 9.2 Phenol-Chloroform Purification, 2nd Phenol
35
Appendix 9.3 Reagents used in the Ethanol Precipitation step.
Appendix 9.4 Wells filled with DNA solution in electrophoresis plate.
36
9. Bibliography
1. Betzel C, Singh TP, Visanji M, Peters K, Fittkau S, Saenger W, Wilson KS (July 1993). "Structure of the complex of proteinase K with a substrate analogue hexapeptide inhibitor at 2.2-A resolution". J. Biol.Chem. 268 (21): 158548. 2. Bruce Alberts, Alexander Johnson, Julian Lewis , Martin Raff , Keith Roberts, Peter Walter (2008). Molecular Biology of the Cell. 5th ed. United States of America: Garland Science. P544-547 3. CLEAPSS (2007). CLEAPSS Student Safety Sheet - Phenol. Available: http://www.cleapss.org.uk/attachments/article/0/SSS64.pdf?Secondary/Science/Stud ent%20Safety%20Sheets/. Last accessed 12th Sept 2013. 4. CLEAPSS (2007). CLEAPSS Student Safety Sheet - Chloroform. Available: http://www.cleapss.org.uk/attachments/article/0/SSS64.pdf?Secondary/Science/Stud ent%20Safety%20Sheets/. Last accessed 12th Sept 2013. 5. Dang TD, Tang M, Choo S, Licciardi PV, Koplin JJ, Martin PE, Tan T, Gurrin LC, Ponsonby AL, Tey D, Robinson M, Dharmage SC, Allen KJ; HealthNuts study. (2012). Increasing the accuracy of peanut allergy diagnosis by using Ara h 2.. Journal of Allergy and Clinical Immunology. 129 (Issue 4), p10561063. 6. Department of Biochemistry and Molecular Biophysics. (2002). DNA Extraction from Cheek Cells. Available: http://webcache.googleusercontent.com/search?q=cache:mosvrsbY7tgJ:biology.arizona .edu/sciconn/lessons2/vuturo/vuturo/dna.htm+&cd=1&hl=en&ct=clnk&gl=kr. Last accessed 25th Aug 2013. 7. DNA Double Helix. 2013. DNA Double Helix. [ONLINE] Available at:http://www.elmhurst.edu/~chm/vchembook/582dnadoublehelix.html. [Accessed 01 November 2013]. 8. DNA Isolation Protocol. 2013. DNA Isolation Protocol. [ONLINE] Available at: http://web.mit.edu/jacks-lab/protocols/DNA_Isolation_tables.html. [Accessed 01 November 2013]. Elena Scaravelli. (2008). Development of Three Real-Time PCR Assays to Detect Peanut Allergen Residues in Processed Food Products.European Food Research and Technology. 227 (x), p857-869. 9. Equipment and materials | DNA | Base unit. 2013. Equipment and materials | DNA | Base unit. [ONLINE] Available at:http://www.ncbe.reading.ac.uk/ncbe/materials/dna/baseunit.html. [Accessed 05 October 2013]. 10. Ethanol precipitation of nucleic acids - OpenWetWare. 2013. Ethanol precipitation of nucleic acids - OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids. [Accessed 15 September 2013].
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11. Gary E, Truett. (2006). Preparation of Genomic DNA from Animal Tissues. In: Jan Kieleczawa DNA Sequencing II: Optimizing Preparation and Cleanup, Volume 2. Canada: Jones & Bartlett Learning. p39-41. 12. Gurdev S. Khush, Darshan S. Brar, Bill Hardy (2003). Advances in Rice Genetics, Volume 1. Philippines: International Rice Research Institute(IRRI). p473-475. 13. Hedstrom, L. (Dec 2002). "Serine protease mechanism and specificity.". Chem Rev 102 (12): 450124. 14. H. Hird. Hez.Hird@fera.gsi.gov.uk. Hi there, Thank you for the interest in our work. 31 Oct 2013. 15. H. Hird, J. Loyd, R. Goodier, J. Brown, P. Reece. (2003). Detection of peanut using realtime polymerase chain reaction. European Food Research and Technology. 217 (Issue 3), p265-268. 16. Leniewicz K, Porba E, Smolarkiewicz M, Wolff N, Stanisawski S, Wojtaszek P. (2012). Plant plasma membrane-bound staphylococcal-like DNases as a novel class of eukaryotic nucleases.. BMC Plant Biology. 12. p195. 17. Lysis buffer - Wikipedia, the free encyclopedia. 2013. Lysis buffer - Wikipedia, the free encyclopedia. [ONLINE] Available at:http://en.wikipedia.org/wiki/Lysis_buffer. [Accessed 01 November 2013]. 18. Mark V.Bloom, Greg A.Freyer, David A.Micklos (1996). Laboratory DNA Science. Menlo Park, CA: The Benjamin/Cummings Publishing Company, Inc. p281-301. 19. Monteiro, L. et al. (1997) Complex polysaccharides as PCR inhibitors in feces: Helicobacter pylori model. J. Clin. Microbiol.35, 9958. 10. Mun-Keat Looi. (2013). Flogging a dead horse: genetics and the UK horsemeat scandal. Available: http://blog.wellcome.ac.uk/2013/03/11/flogging-a-dead-horsegenetics-and-the-uk-horsemeat-scandal/. Last accessed 13th Oct 2013. 21. N. S. Rangaswamy and Lakshmi Nandakumar. (1985). Correlative Studies on Seed Coat Structure, Chemical Composition, and Impermeability in the Legume Rhynchosia minima. Botanical Gazette. 146 (4), p501-509. 22. Overview of ELISA. 2013. Overview of ELISA. [ONLINE] Available at:http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E836FE09D8403. [Accessed 29 September 2013]. 23. Peanut Plant - How does the groundnut grow?. 2013. Peanut Plant - How does the groundnut grow?. [ONLINE] Available at: http://www.boiled-peanutworld.com/peanut.html. [Accessed 30 September 2013]. 24. Phenol/chloroform extraction - OpenWetWare. 2013. Phenol/chloroform extraction OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Phenol/chloroform_extraction. [Accessed 01 November 2013].
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25. Preparation of phenol/phenol:chloroform. 2013. Preparation of phenol/phenol:chloroform. [ONLINE] Available at: http://www.mrclmb.cam.ac.uk/ms/methods/phenol.html. [Accessed 02 November 2013]. 26. PuReTaq Ready-To-Go PCR Beads. 2013.[ONLINE] Available at:https://www.gelifesciences.com/gehcls_images/GELS/Related%20Content/Files/13 14750913712/litdoc11002607AB_20110831023628.pdf. [Accessed 15 September 2013]. 27. P. Zumbo. Phenol-chloroform Extraction . Available: http://physiology.med.cornell.edu/faculty/mason/lab/zumbo/files/PHENOLCHLOROFORM.pdf. Last accessed 11th Oct 2013. 28. Quantitation of DNA and RNA . 2013. Quantitation of DNA and RNA . [ONLINE] Available at:http://cshprotocols.cshlp.org/content/2007/11/pdb.ip47.full. [Accessed 02 November 2013]. 29. Recipes for Common Laboratory Solutions. 2013. [ONLINE] Available at:http://www.promega.com/~/media/Files/Resources/Technical%20References/Rec ipes%20For%20Common%20Lab%20Solutions.pdf. [Accessed 06 October 2013]. 30. SEP staff.Strawberry DNA Extraction. Available: http://seplessons.ucsf.edu/node/217. Last accessed 25th Aug 2013. 31. Standard PCR Conditions . 2013. Standard PCR Conditions . [ONLINE] Available at: https://www.k-state.edu/hermanlab/protocols/StandardPCRConditions.html. [Accessed 03 October 2013]. 32. The Nobel Prize in Chemistry 1993. 2013. The Nobel Prize in Chemistry 1993. [ONLINE] Available at:http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/. [Accessed 01 November 2013]. 33. Tm Calculator | New England Biolabs . 2013. Tm Calculator | New England Biolabs . [ONLINE] Available at: https://www.neb.com/tools-and-resources/interactivetools/tm-calculator. [Accessed 01 November 2013]. 34. James Huntley, Soheila Maleki, Peggy Ozias-Akins. (2009). Identification and characterization of a hypoallergenic ortholog of Ara h 2.01. Plant Molecular Biology. 69 (Issue 3), p325. 35. Wen-Tao Xu, Kun-Lun Huang, Ai-Ke Deng, Zhi-hong Liang, Yun-Bo Luo. (2007). Variations of tissue DNA density and nuclear DNA content in soybean lines and their impacts on the GMO quantification. Food Control. 18, p1300-1306.
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at:http://www.nobelprize.org/nobel_prizes/chemistry/laureates/1993/. [Accessed 01 November 2013].

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Figure 2 Complementary Base Pairing7

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Figure 3. The structure of a peanut.10

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11 Overview of ELISA. 2013. Overview of ELISA. [ONLINE] Available

at:http://www.piercenet.com/browse.cfm?fldID=F88ADEC9-1B43-4585-922E-836FE09D8403. [Accessed 29 September 2013].

Biology Extended Essay

Figure 4. Mass, DNA content, DNA density in different parts of a soybean.12

Biology Extended Essay

Biology Extended Essay

Table 1 Experiment Variables - Experiment 1

The Benjamin/Cummings Publishing Company, Inc. p281-301.

Biology Extended Essay

Table 2 Experiment Variables - Experiment 2

The location of peanut DNA. Cotyledon Embryo

The amount of peanut DNA. (g) 0.5 0.125

Time of incubation. (minutes) 1440 1440

Table 3 Experiment Variables - Experiment 3

state.edu/hermanlab/protocols/StandardPCRConditions.html. [Accessed 03 October 2013].

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Figure 5. PCR Condition

4.1 Modifying pH of Solutions

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SDS from washing up liquid

15 DNA Isolation Protocol. 2013. DNA Isolation Protocol. [ONLINE] Available

at: http://web.mit.edu/jacks-lab/protocols/DNA_Isolation_tables.html. [Accessed 01 November 2013].

[ONLINE] Available at:http://en.wikipedia.org/wiki/Lysis_buffer. [Accessed 01 November 2013].

Biology Extended Essay

60C Water bath Weighing boat PCR tubes

Figure 7 DNA Extraction - Incubation of peanut sample in 60C water bath.

Aqueous phase Organic phase

Aqueous phase Organic phase

[ONLINE] Available at:http://openwetware.org/wiki/Phenol/chloroform_extraction. [Accessed 01 November 2013].

The Benjamin/Cummings Publishing Company, Inc. p292-298.

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4.4 Ethanol Precipitation2122

OpenWetWare. [ONLINE] Available at:http://openwetware.org/wiki/Ethanol_precipitation_of_nucleic_acids. [Accessed 15 September 2013].

The Benjamin/Cummings Publishing Company, Inc. p292-298.

Biology Extended Essay

Figure 9 Ethanol Precipitation - precipitated DNA before centrifugation

4.5 PCR (Polymerase Chain Reaction)

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4.6 Gel electrophoresis25

Figure 10 Gel Electrophoresis - Experimental setup

The Benjamin/Cummings Publishing Company, Inc. p292-298.

Biology Extended Essay

Biology Extended Essay

No bands detected in lane 1A1, 1A2 and 1A3.

1B1 1B2 1B3

1C1 1C2 1C3

Clear band detected in 1C3. No band detected in 1C1 and 1C2.

1D1 1D2 1D3

Clear band detected in 1D2 and 1D3. No band detected in 1D1.

Biology Extended Essay

2A1 2A2 2A3

No band detected for 3b. A clear band detected for 3C.

Table 4 Results Table - Detection of DNA, with fluorescent band