Leo Forster
2213-018
International Baccalaureate Extended Essay: Biology
Comparison of Phenol Chloroform, Proteinase K, and a combination of the two in respect to the purity attained in DNA extracted from onions.
3,995 Words
Leo Forster 2213-018
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Leo Forster
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Abstract It is in the interests of secondary schools and universities to have purified DNA for lab use and experimentation. As pure DNA is expensive and unavailable or impractical, a cost-effective and efficient alternative for purifying DNA was investigated.
Extraction of DNA from onions was done using a SDS/NaCl-based Lysis solution. The extracts were filtered and DNA precipitated out using ice-cold 95% ethanol. They were stored at 4째C and then purified using different purification protocols. In one case, Phenol Chloroform was added to the sample and DNA precipitated out of the resulting supernatant. Two variations of Phenol Chloroform purification were carried out: in one, the process was repeated once (1x PC); in the other it was repeated thrice (3x PC). Alternatively, the enzyme Proteinase K was added and incubated at 55째C for an hour. Two variations of this also were carried out: one included only the enzyme (PK), while the other added a treatment with Phenol Chloroform (PC+PK). Samples were dissolved in TE Buffer, and 260/280nm absorbance ratio was determined using a UV Spectrophotometer. Gel electrophoresis was also carried out to verify the quantitative data.
Results indicate that with a 260/280nm absorbance ratio of 1.774, 3x PC was able to produce the purest DNA; while 1x PC and PC+PK gave ratios of 1.585 and 1.621 respectively, and PK alone gave a ratio of 1.216. Purity was interpreted based on the assumption that a ratio of 1.8 indicated pure DNA. A greater deviation from 1.8 indicates less-pure DNA. These results were reaffirmed in gel electrophoresis, whereby single bands of DNA were observed for 3x and 1x PC while other samples contained extensive smearing with very faint or no bands.
In conclusion, it was found that 3x PC is a feasible method of producing pure DNA and a viable substitute for expensive commercially purchased DNA. (299 Words)
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Contents Abstract............................................................................................................................... 1 Contents.............................................................................................................................. 2 Chapter 1: Introduction...................................................................................................... 4 1.1
Rationale of Study:.............................................................................................. 4
1.2
Background: ........................................................................................................ 5
1.3
Purification:......................................................................................................... 7
1.3.1
Proteinase K Incubation (PK) ...................................................................... 8
1.3.2
Phenol Chloroform Suspension (PC)........................................................... 9
1.4
Aim: ................................................................................................................... 10
1.4.1 1.5
Objectives of Study .................................................................................. 10
Theoretical Basis: .............................................................................................. 11
1.5.1
DNA Extraction.......................................................................................... 11
1.5.2
Precipitation of DNA ................................................................................. 12
1.5.3
Absorbance Ratio (UV Spectrophotometer)............................................. 13
1.5.4
Gel Electrophoresis ................................................................................... 15
Chapter 2: Methodology .................................................................................................. 16 2.1
Hypothesis: ....................................................................................................... 16
2.2
Procedures: ....................................................................................................... 17
2.2.1
DNA Extraction and Precipitation ............................................................. 18
2.2.2
Purification by means of Phenol Chloroform (1x and 3x)......................... 20
2.2.3
Purification by means of Proteinase K (1x and Combination).................. 21
2.2.4
Preparation for measurement of Absorption Ratio ................................. 21
2.3.5
Measurement of Absorption Ratio ........................................................... 22
2.2.6
Gel Electrophoresis ................................................................................... 23
Chapter 3: Data Collection and Presentation.................................................................. 24 3.1
Quantitative Raw Data..................................................................................... 24
3.1.1
Establishment of Absorbance-Concentration Relationship...................... 24
3.1.2
Establishment of DNA Degradation over Time......................................... 25
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Data Presentation ............................................................................................. 26
3.2.1
Standard Calibration Curve for Pure DNA ................................................ 26
3.2.2
Absorbance Ratio vs Time & Absorbance Ratio vs DNA Concentration... 27
3.3
260/280nm Absorbance Ratio Data ................................................................. 28
3.4
Qualitative Raw Data ........................................................................................ 30
3.4.1
Precipitation:............................................................................................. 30
3.4.2
Gel Electrophoresis Results...................................................................... 31
Chapter 4: Discussion of Data.......................................................................................... 33 4.1
Interpretation of Results................................................................................... 33
4.1.1
260/280nm Absorbance Ratio (Quantitative) ......................................... 33
4.1.2
Gel Electrophoresis (Qualitative)............................................................. 35
Chapter 5: Evaluation....................................................................................................... 36 5.1
Limitations and Improvements......................................................................... 36
5.1.1
Phenol Chloroform Contamination........................................................... 36
5.1.2
Time Frame .............................................................................................. 36
5.1.3
UV Spectrophotometer............................................................................ 36
5.2
Further Investigation ........................................................................................ 37
5.2.1
Solvent...................................................................................................... 37
5.2.1
230/260 & Other Ratios........................................................................... 37
5.2.2
DNA Source ............................................................................................... 37
Chapter 6: Conclusion ...................................................................................................... 38 Chapter 7: Bibliography ................................................................................................... 39 8.1
Citations ............................................................................................................ 39
8.2
References........................................................................................................ 40
Chapter 8: Appendices ..................................................................................................... 42
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Chapter 1: Introduction 1.1
Rationale of Study:
Finding a purification protocol which will produce highly pure DNA at minimal expenditure allows secondary schools and universities to give a hands-on experience with DNA within the lab. An institute wishing to use pure DNA must purchase purified Lambda DNA1 from commercial retailers. This is inefficient and impractical because the DNA is expensive and highly concentrated. Secondary institutes which allow their students to experience DNA are rare. The result of this study can be used to replace Lambda DNA in the classroom, opting instead to use purified onion DNA.
This lack of affordable and easily produced pure DNA became evident during an in-class investigation into the effects several restriction enzymes on DNA, during which the class had to share the small amount of available Lambda DNA and several students sat out altogether. As this is not optimal and does not enhance the learning of each individual (and as the class can consider itself lucky to have been able to conduct such an experiment at all), the goal of this study is to compare several DNA purification protocols, hoping to find one which optimally balances cost and effectiveness.
1
Double-stranded linear DNA extracted from Lambda phage, a virus which infects E. Coli
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Background:
This investigation is designed to provide a cheaper alternative for pure DNA used in research, development, and schools. It would allow more students to interact with and learn about DNA while also saving the institutes a significant amount of money: commercially available Lamda DNA costs $100 per 0.1mg. In this study, DNA extracted from Allium cepa, the common onion, will be purified using different purification protocols. Onions were chosen because of their availability and cheapness, and because they are a great source of DNA due to the ease of extraction and the lack of safety risks involved as compared to alternatives2. The emphasis of this investigation lies with the further purification of DNA. This purification involves removing proteins and other contaminants from the DNA in the onion cells.
Figure 1: Allium cepa, the common onion
2
DNA extractions from wheat germ and lambda phage are also commonplace
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Of the contaminants which are removed through DNA purification, the most common are the histone proteins (See Figure 2). The histones serve to provide structure to the DNA and allow it to supercoil3.
Figure 2: Histones within a strand of DNA
Other impurities include RNA and cell matter as well as enzymes and proteins such as DNase4, an enzyme which would otherwise catalyze the hydrolytic cleavage5 of phosphodiester bonds6 in the DNA backbone.
3
When helical strands of DNA coil around themselves to conserve space Deoxyribonuclease, and enzyme which will digest and break down DNA strands 5 A chemical reaction resulting in the opening of the DNA double helix 6 The link between the 3’ and 5’ ends of phosphate groups in DNA molecules 4
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Purification:
The substance of this study is to compare several different purification protocols and find one which best balances cost and effectiveness.
Effectiveness will be determined with a UV Spectrophotometer (See 1.5.3, Absorbance Ratio). DNA absorbs light at 260 nm, while most contaminants absorb light at 280nm [1]. Hence, a ratio of the two absorbances can be used to estimate DNA purity. This ratio is known as the 260/280 Absorbance Ratio.
The protocols being evaluated are:
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1.3.1 Proteinase K Incubation (PK) The enzyme, Proteinase K, is used to digest nucleic acid proteins and remove contaminants from DNA. It was discovered in 1974 in Engyodontium album7. Though it is very effective in its applications, it is expensive. In this investigation, it is used as a comparison to judge the effectiveness of Phenol Chloroform rather than as a primary purification protocol.
The enzyme is extracted and is stored in powder form until activated in the presence of a buffer. Upon being activated, it can simply be added to the nucleic acid extract and incubated with the sample for it to function. Proteinase K is functional in temperatures ranging from 0-65°C, and pH ranging from 4-12 [3].
The enzyme becomes activated in the presence of Ca2+ ions8. This is an issue in nucleic acids prepared using EDTA9, as EDTA will attack Ca2+ ions. The enzyme is not significantly inhibited in the presence of EDTA as EDTA will also weakening protein structures [4].
Pros
Cons
Versatile
Degrades / can be inhibited Expensive ($81.5 per 50Âľl)
Harmless Contaminates samples, lowers purity Table 1: Pros and cons of using PK in purification
7
Formerly Tritirachium album, a microscopic fungus. [2] The Proteinase K buffer will activate the enzymes. Hence they are stored in powder form until used. 9 Ethylenediaminetetraacetic acid, used because it attacks ions which would otherwise degrade DNA 8
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1.3.2 Phenol Chloroform Suspension (PC) Phenol Chloroform is cheap to obtain and effective in its application. It provides an alternative to conventional purification methods. It is added to DNA and after a short incubation, is centrifuged, and the DNA precipitated out of solution.
The compound removes proteins from the nucleic acid through interactions between the Phenol and Water which cause proteins to undergo a conformational change and exit the aqueous- and enter the organic10 solution [5]. The two layers are partitioned and the aqueous solution can be removed.
A 25:24:1 solution of Phenol-Chloroform-Isoamyl Alcohol solution was used in this study, though the Isoamyl Alcohol is not required. It is important it be kept at pH 7 during purification, as a more acidic solution would result in a separation of RNA molecules into phases instead of DNA [6].
Here, the effects of doing a single Phenol Chloroform purification compared to doing three were assessed. It was thought that subsequent purification would remove a larger percentage of the total proteins within the solution.
Pros Cheap ($35 for 200ml)
Cons Toxic
Simple to use Fast Table 2: Pros and Cons of PC purification
10
Phenol, so called because of it’s Carbon-based structure.
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Aim:
The aim of this study is to explore which DNA purification protocols are able to remove contaminants from DNA; hence finding the most practical and effective.
The purification protocols used in this study are incubation with the enzyme Proteinase K, suspension in Phenol Chloroform solution, as well as a mixture of the two (incubation with Proteinase K, followed Phenol Chloroform purification).
Hence, the precise research topic involved with this study is: Comparison of Phenol Chloroform (1x and 3x), Proteinase K, and a combination of the two in respect to the purity attained in DNA extracted from onions.
A UV Spectrophotometer will be used to quantify purified DNA and provide data as to the absorption (concentration) of DNA, and will also be used to produce an absorbance ratio to compare the purity of individual samples. Gel electrophoresis of produced samples will be used to give qualitative data of sample purity.
1.4.1 Objectives of Study Hence, the objectives of this study are as follows: •
Investigating Phenol Chloroform and Proteinase K’s ability to purify DNA extracts.
•
Assessing purity of sample via data from UV Spectrophotometer
•
Separation of DNA extract bands using gel electrophoresis
•
Comparing the above on the criteria of cost-effectiveness and efficiency.
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Theoretical Basis: 1.5.1 DNA Extraction
Extraction of the DNA is the most important step within this study. If the extract does not contain DNA, any attempts at further purification will be invalid.
During extraction, onions cells are ruptured using a lysis11 solution. Being composed of SDS12, NaCl, and EDTA13, the lysis solution functions in that SDS disrupts the hydrophilic14 nature of the cell membrane, effectively breaking it open so that DNA can enter the aqueous solution [7].
Hence, DNA strands and onion cell remnants are dissolved in the aqueous solution and can be precipitated using ethanol or isopropanol.
11
Solution used to destroy cell membranes, allowing DNA to exit the cell. For preparation, see Appendix 1. Sodium Dodecyl Sulphate, used to denature the proteins in the cell membrane; damaging the membrane and breaking the cell open. 13 Ethylenediaminetetraacetic acid, used because it attacks ions which would otherwise degrade DNA 14 Water-loving; an important aspect of what holds together the cell membrane. 12
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1.5.2 Precipitation of DNA Remnants of the onion cell organelles and cytoplasm are present within the aqueous solution, but will be separated from the DNA through precipitation.
Precipitation functions in that the added ethanol makes it much easier for the Na+ (from NaCl) to interact with the PO4(3-) 15 on the DNA, causing the nucleic acid to become less positively charged and hence less hydrophilic - leading it to leave the aqueous solution and enter the ethanol [8].
It is important that the ethanol used in precipitation be as cold as possible. In this study, the ethanol used was about -5째C.
Also, during precipitation proceeding Phenol Chloroform purification, 1/10th volume of 3M Sodium Acetate16 is added to the aqueous solution.
15 16
Phosphate groups found on DNA strands. Sodium Acetate, NaAC added because it facilitates the pelleting of DNA after precipitation.
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1.5.3 Absorbance Ratio (UV Spectrophotometer) Knowing that DNA absorbs light at 260nm while contaminants absorb light at 280nm, it is possible to, by comparison of the respective absorbance bell curves, assess the ratio of DNA to contaminants within a solution. This is because the more light is absorbed by the sample, the higher the concentration of nucleic acid or contaminants within the sample. A sample of pure DNA will have a high 260nm absorbance and a low 280nm one. Hence the ratio of absorbances will be proportionally higher.
This ratio of absorbances is indicative of DNA concentration, as per the Beer Lambert Law17 where it is possible to relate the light absorbed to the concentration of the absorbing molecule. The following table describes what compounds absorb light at which wavelength.
Wavelength / nm
Chief Absorbing Compound
230
Organic or carbohydrate contaminants
260
DNA and RNA
270
Phenol
280
Proteins
Table 3: The chief absorbing compound at varying wavelengths [9]
This justifies use of the 260/280nm Absorbance Ratio due to the fact that proteins are the major contaminating factor in the extract. The 260/280nm incorporates this into its estimation, so it is optimal when considering DNA purity.
17
http://elchem.kaist.ac.kr/vt/chem-ed/spec/beerslaw.htm
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The following table describes what can be expected of the components of a sample based on the calculated Absorbance Ratio.
260/280 Ratio
Sample Consistency
1.3
<50% Contaminants
1.5
50% nucleic acid, 50% contaminants
1.8
100% DNA
2.0
100% RNA / Phenol contamination18
Table 4: Components of a sample at varying Absorbance Ratio [10]
It should be noted that the DNA sample may not be pure though its ratio is 1.8. This is because other contaminants may not be absorbing at 280nm.
This information was applied in the case of this study, and a UV Spectrometer with the capability to produce a 260/280nm absorbance ratio was used for quantification.
18
Phenol absorbs at 270nm, so large amounts of residual phenol can create an artificially high ratio.
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1.5.4 Gel Electrophoresis Gel electrophoresis is used to provide qualitative data to reinforce the conclusions drawn from the quantitative data. It consists of allowing samples to travel through a gel because of an electrical current. Due to varying densities and sizes of DNA and contaminants, they will separate visibly within the gel.
When placed under a current the DNA in the gel travels through it. The end-position of the DNA will vary depending on its size and the concentration of gel used.
After this, the gel is dyed so that DNA bands can be seen. Methylene Blue was used as a dye because it is cheap and effective. The gel is submersed in water and destained so a picture can be taken (See Methodology: 2.2.6, Gel Electrophoresis).
(See Appendix 2, for more info).
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Chapter 2: Methodology 2.1
Hypothesis:
It is hypothesized that the 3x Phenol Chloroform purification will produce the DNA of highest purity because through successive Phenol Chlorform purifications proteins which had escaped previously will be removed.
It is hypothesized that the Proteinase K purification will produce DNA of comparably lower purity because Proteinase K itself is an enzyme (that is, a protein) and will remain in the sample during quantification. Hence, the enzyme itself will be measured as an impurity and will lower the absorbance ratio.
It is hypothesized that the mixture of Proteinase K and Phenol Chloroform will be able to produce DNA of similar purity as the Phenol Chloroform protocol because Proteinase K will digest all proteins existing within the extracted DNA, while subsequent Phenol Chloroform purifications will remove all remnants of Proteinase K enzymes from the sample.
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Procedures:
The overall methodology of this experiment was as follows:
Diagram 1: Overview of extraction & purification process.
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2.2.1 DNA Extraction and Precipitation For DNA extraction from onions: •
Onion was sliced into cubes with maximum dimension of 1 x 1 x 1 cm and put in a beaker.
•
Sufficient lysis solution to cover the cubes was added.
•
Beaker was placed on a hot plate kept at 60-65°C 19 for 15 minutes, while stirring.
•
Solution was placed in an ice bath for several minutes, while still stirring.
•
Solution was filtered into a new beaker using filter paper, and filtered once more after that.
For precipitation of extracted DNA: •
Filtered solution was poured into test tubes, with approximately 20ml of solution in each 50ml test tube.
•
Test tubes were tilted to increase the surface area for reaction, and ice cold 95% ethanol 20 was slowly added.
•
19 20
Test tubes were capped and refrigerated overnight.
Solution’s temperature must not exceed 65°C 95% denotes a solution of 95 parts Ethanol and 5 parts distilled water.
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For preparation of DNA for purification: •
Using a 1,000μl micropipette with the end cut off, the cloudy/stringy interphase (See Figure 3) which had appeared was extracted and placed into microcentrifuge tubes.
Figure 3: DNA present within added ethanol
•
Microcentrifuge tubes were centrifuged for several minutes at highest speed (10,000+ RPM21).
•
The supernatant was removed, leaving pellet untouched.
•
Ice-cold 70% ethanol22 was added to the microcentrifuge tubes. The pellet was dislodged and agitated and then centrifuged at maximum speed for 2-3 minutes.
•
Previous two steps were repeated two more times (Washing with 70% ethanol).
•
Supernatant was removed and replaced with approximately 500μl of TE Buffer23.
21
Revolutions Per Minute 70% denotes a solution of 70 parts Ethanol and 30 parts distilled water. 23 Buffer consisting of Tris base and EDTA. Protects DNA from degradation while rendering it soluble. 22
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2.2.2 Purification by means of Phenol Chloroform (1x and 3x) •
200μl of TE Buffer + DNA was split into two microcentrifuge tubes containing 100μl of solution each. 100μl of Phenol Chloroform was added to each.
•
The microcentrifuge tubes were centrifuged for 5 minutes at highest speed.
•
The aqueous phases (See Figure 4) of the resulting partitioned solutions were removed and placed into new tubes. Care was taken not to disturb the existing inter- or organic phases.
Figure 4: Phase Separation after addition of Phenol Chloroform
•
One tube was placed in the refrigerator. (this is 1x PC)
•
100μl of Phenol Chloroform was added to the other tube and spun in a centrifuge for five minutes at maximum speed (10,000+ RPM).
•
Aqueous phase was removed and transferred into a new microcentrifuge tube.
•
The previous two steps were repeated once more, and the resulting tube was placed in the refrigerator. (this is 3x PC)
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2.2.3 Purification by means of Proteinase K (1x and Combination) •
200μl of TE Buffer + DNA was split into two microcentrifuge tubes containing 100μl of solution each. 50μl of activated Proteinase K enzyme was added to each tube.
•
Tubes were left in a waterbath at 55°C for one hour.
•
One of the tubes was refrigerated. (this is 1x PK)
•
200μl Phenol Chloroform was added to the tube and spun in a centrifuge for 5 minutes at maximum speed.
•
Aqueous phase was removed and transferred into a new microcentrifuge tube.
•
Tube was refrigerated. (this is 1x PC+PK) 2.2.4 Preparation for measurement of Absorption Ratio
•
20μl of Sodium Acetate was added to each tube of 1x PC, 3x PC, and 1x PC+PK.
•
Cooled 95% ethanol was added to each tube in a 2:1 ratio of ethanol to solution. Tubes were mixed by inversion and agitation, and were refrigerated for at least 12 hours.
•
The precipitated DNA (see Figure 5) was extracted and placed into a new microcentrifuge tube.
Figure 5: Precipitated DNA after Phenol Chloroform purification
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The contents of each tube were centrifuged at maximum speed for several minutes, and were washed with 70% ethanol (see above, 2.2.1 page 19).
•
100μl of TE buffer was added to each tube, and tube was refrigerated. 2.3.5 Measurement of Absorption Ratio
Figure 6: UV Spectrometer used, model Optizen 2120 UV •
A Cell was filled with 100μl TE buffer and was used to autozero the UV Spectrometer (See Figure 6).
•
A cell was filled with 100μl of 3x PC and the 260/280nm absorbance ratio was measured using the UV Spectrometer.
•
The cell was emptied of 3x PC and rinsed with TE buffer repeatedly.
•
Previous two steps were repeated for all other DNA samples.
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•
2.2.6 Gel Electrophoresis 0.7% agarose 24 was prepared and a gel was poured.
•
30μl of 3x PC was loaded into a well and topped with 2μl loading dye 25.
•
The previous step was repeated for all other DNA samples.
•
Gel was placed in electrophoresis set (See Figure 7), was submersed in 1x TBE buffer26, and was left under current for 2-3 hours.
Figure 7: Electrophoresis set used
•
Gel was removed from electrophoresis set and stained using Methylene Blue27
•
Destaining was carried out until bands and streaks were clearly visible.
24
See Appendix 2 See Appendix 2 26 Buffer consisting of Tris base, Boric acid, and EDTA. Keeps the DNA deprotonated and soluble in water 27 C16H18N3SCl dissolved in water 25
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Chapter 3: Data Collection and Presentation 3.1
Quantitative Raw Data 3.1.1 Establishment of Absorbance-Concentration Relationship
In order to establish whether the absorbance of DNA samples at varying concentrations changes, the UV Spectrometer was used to quantify known concentrations of Lambda DNA. These concentrations of DNA were obtained from a dilution of stock solution of 5μl Lambda DNA in 95μl TE Buffer. The results were as follows: TE Buffer / μl
Lambda DNA / μl
% DNA
Absorbance at
Absorbance Ratio,
Concentration / %
260 nm28
260/280 nm
95.000
5.000
5.00
0.270
1.786
97.500
2.500
2.50
0.169
1.769
98.750
1.250
1.25
0.094
1.696
99.375
0.625
0.63
0.057
1.731
99.688
0.312
0.31
0.026
1.857
99.844
0.156
0.16
0.006
1.778
Table 5: Establishment of Absorbance-Concentration Relationship
It is possible to establish that though the concentration of DNA at 260nm changes as the amount of DNA dissolved in the solution changes, it has no significant bearing on the purity reading of the sample. It can be concluded that DNA concentration increases, the 260nm Absorbance will increase; that the relationship is directly proportional. (See 3.2.1, Standard Calibration Curve for Lambda DNA). It can also be concluded that the concentration of DNA does not affect its absorbance ratio. Hence the extracts obtained in this study are sufficient for obtaining a justified absorbance ratio.
28
Assuming DNA is pure, the concentration at 260 nm should be indicative of the concentration of DNA.
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3.1.2 Establishment of DNA Degradation over Time In order to establish whether the absorbance of DNA samples changes as time passes (degradation), the UV Spectrometer was used to quantify samples of Lambda DNA over a period of two days. The results were as follows: Preparing Lambda DNA TE Buffer / Îźl
Lambda DNA / Îźl
% DNA
Absorbance Ratio,
Absorbance Ratio,
Concentration / %
Day 1
Day 2
95.000
5.000
5.00
1.786
1.753
97.500
2.500
2.50
1.769
1.601
98.750
1.250
1.25
1.696
1.580
99.375
0.625
0.63
1.731
1.667
99.688
0.312
0.31
1.857
1.301(a)
99.844
0.156
0.16
1.778
- (b)
Average
1.770
1.581
Table 6: Establishment of DNA Degradation over Time (a)
Data point was erroneous and was not considered in the calculation of the average.
(b)
Data point was not measured.
There was a large difference between the average absorption ratios on day one and two, and the DNA samples did degrade after being isolated from the stock. It was noted that this degradation was mostly limited to those samples of minute DNA concentration. It can be concluded that the extracted DNA does degrade over time. Thus, the absorbance ratio of extracted samples should be measured immediately after purification.
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Data Presentation 3.2.1 Standard Calibration Curve for Pure DNA
Using the 260nm absorbance readings obtained from the known Lambda DNA concentrations above, a standard calibration curve for pure DNA was created as follows:
Standard Calibration Curve for Pure DNA at 260nm 0.4 0.35
260nm Absorbance
y = 0.0666x R2 = 0.9964
0.3 0.25 0.2 0.15 0.1 0.05 0 0
1
2
3
4
% DNA Concentration of Solution / % `
5
6
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3.2.2 Absorbance Ratio vs Time & Absorbance Ratio vs DNA Concentration
`
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260/280nm Absorbance Ratio Data
Extraction and purification was done on two occasions: Extraction 1 and Extraction 2. All data was obtained using the UV Spectrometer, model Optizen 2120 UV. The recorded data was reported in Table 7 below: Absorbance Ratio, 260/280 nm Dilution Factor 10 fold(a) Sample Type
2 fold(b)
Extraction 1(c)
3x PC
1.789
- (d)
1x PC
1.636
-
PK
1.270
-
PC + PK
1.519
1.671
Unpure (-)
1.479
-
Extraction 2(e) 3x PC
1.759
-
1x PC
1.533
-
PK
1.177
1.146
PC + PK
1.621
1.671
Unpure (-)(g)
1.499
-
Lambda (+)(g)
1.770(f)
-
Table 7: Raw Data collected from UV Spectrophotometer (a)
10 Îźl of extract stock in 90 Îźl of TE buffer. 2 fold dilution of the 10-fold described in (a) (c) Extraction completed September, 2010 (d) Data point was not collected due to some error with Spectrophotometer (e) Extraction completed October, 2010 (f) Taken from the average of the data presented in 3.1.1 (g) Negative and Positive control, respectively (b)
Samples not reflected above were not included because it was found that the obtained Absorbance Ratio was either erroneous or immeasurable by the UV Spectrophotometer.
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Figure 6: Raw Data from UV Spectrophotometer of Absorbance Ratio at 260/280 nm In the preceding table, the varying dilution factors are the result of inconsistent UV Spectrometer readings. If the readings were erroneous, or if the UV Spectrometer gave an error message (sample too concentrated), the sample was diluted until an appropriate reading was obtained.
For further calculation, the above data (3.3) was combined and averages found. The results can be seen in Table 8 below: Absorbance Ratio, 260/280nm Sample Type
Extraction 1
Extraction 2
Average(a)
3x PC
1.789
1.759
1.774
1x PC
1.636
1.533
1.585
PK
1.270
1.162(b)
1.216
PC + PK
1.595(c)
1.646(d)
1.621
Unpure (-)(f)
1.479
1.499
1.489
Lambda (+)(f)
-
-
1.770(e)
Table 8: Calculated averages for 260/280nm Absorbance Ratio (a)
Average calculated using the values from Extraction 1 & 2 Value calculated by averaging values from Extraction 2 (c) Value calculated by averaging values from Extraction 1 (d) Value calculated by averaging values from Extraction 2 (e) Taken from the average of the data presented in 3.1.1 (f) Negative and Positive control, respectively (b)
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3.4
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Qualitative Raw Data 3.4.1 Precipitation:
Proceeding the second precipitation after Phenol Chloroform purification (See Methodology, 2.2.4), the samples were visibly different in their consistencies. It was hypothesized that these differences after the second precipitation would relate to the purity of the sample as measured with the UV Spectrophotometer.
During the second extraction cycle, this phenomenon was observed as follows:
Figure 8: Varying DNA consistencies after second precipitation
It was noted that the precipitated DNA in the 1x PC and PC+PK samples was cloudy and unclear, while the 3x PC sample DNA was very stringy and clear.
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Leo Forster 3.4.2
2213-011 Gel Electrophoresis Results
After obtaining the Absorbance Ratio for each extract sample, gel electrophoresis was done to provide qualitative data and ensure that the samples actually contained DNA (See Methodology, 2.2.6).
The positive control (Lambda DNA) for the Gel Electrophoresis was run separately, and can be seen in Figure 9 below:
Figure 9: Positive Control for Gel Electrophoresis (zoomed in)
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Gel Electrophoresis was performed twice and the results shown in Figures 10 & 11:
DNA Band
Smearing (contaminants)
Figure 10: Results of Gel Electrophoresis 1
Figure 11: Results of Gel Electrophoresis 2 Note that the lanes do not appear in the same order. Page 32 of 42
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Chapter 4: Discussion of Data 4.1
Interpretation of Results 4.1.1
260/280nm Absorbance Ratio (Quantitative)
Assuming that a 260/280nm absorption ratio of 1.8 indicates pure DNA, the data obtained from the UV Spectrophotometer indicates the following:
Unpurified29 With a 260/280nm absorbance ratio of 1.489, unpurified DNA was used as negative control30. Using the table on page in 1.5.3, it was determined that the extracted samples of DNA contained approximately 50% contaminants and 50% DNA. This was reinforced in that samples of undiluted unpurified DNA were very cloudy and discolored, indicating contamination. Hence, the extraction protocol was functional as it gave an acceptable ratio of DNA to contaminants. Had the extraction been of lower quality, it may have been more difficult to produce pure DNA.
1x Pc A single treatment with Phenol Chloroform increased the 260/280nm absorbance ratio to 1.585. Hence, a single treatment with PC is not sufficient to remove all contaminants in a sample, but also shows that phenol-chloroforming is effective. Additionally, during phenol-chloroforming the supernatant after 1x PC was much larger than after 2x or 3x PC. This may indicate that there were too many contaminants in the sample for the volume of PC which had been added to handle â&#x20AC;&#x201C; that maybe a 1x PC done with an increased volume of PC would yield better results.
29 30
DNA taken directly from the onion extract, without any further processing. Indicator for the relative purity of samples.
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3x PC Two further treatments of Phenol Chloroform resulted in a sample with 260/280nm absorbance ratio of 1.774. Thus, the sample can be considered almost perfectly pure and is comparable to the commercially purchased Lambda DNA (with 260/280nm=1.770). It was also demonstrated that further PC trials will increase sample purity as contaminants will continue to be removed.
PK Surprisingly, Proteinase K treatment resulted in a 260/280nm absorbance ratio of only 1.216, indicating a sample composition of almost 100% contaminants. Perhaps too much Proteinase K was added to the sample, such that the volume of enzyme dwarfed the volume of DNA. Hence, the spectrophotometer would measure the enzyme as a contaminant and give an accordingly low absorbance ratio. It was impossible to determine how much of the contaminants were digested by Proteinase K, as the enzyme inhibited the spectrophotometerâ&#x20AC;&#x2122;s measurements.
PK + PC Not surprisingly, a phenol-chloroforming of a PK sample increased its 260/280nm absorbance ratio to 1.621. This means that PC was successful in removing the enzyme; but it was impossible to determine how the increase in ratio was due to enzyme or undigested contaminants being removed. Hence, it was thought that PC was able to bring up the sample purity to a level comparable with 1x PC, and that PC and PK were more or less even in their purifying capability.
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Leo Forster 4.1.2
2213-011 Gel Electrophoresis (Qualitative)
Gel electrophoresis was used to verify the data produced by the UV spectrophotometer.
As smearing during gel electrophoresis signifies contamination, increased smearing indicates less pure DNA [11]. Linear DNA will produce a band in the gel: the visibility/size of this band indicates the amount of DNA in the sample [12]. Hence, the size of the band compared to the amount of smearing gives an indication of the ratio of DNA to impurities within a sample.
In Gel Electrophoresis 1, 3x PC and 1x PC displayed visible bands of DNA with evidence of limited smearing, while PC+PK and PK displayed no visible band and PC+PK in particular was one large smear. Thus, it can be concluded that 3x PC and 1x PC contained DNA as well as some contaminant remnants while PC+PK contained small amounts of DNA and comparatively large amounts of contaminants.
In Gel Electrophoresis 2, there was some error with the loading of 1x PC and so it remained in its well. Conversely, 3x PC showed a visible band of DNA with limited smearing while PK also displayed evidence of a band of DNA. PC+PK was a single large smear of contaminants â&#x20AC;&#x201C; as previously.
Thus, it is not surprising to see that while all other samples showed a very faint or no band with evidence of a large amount of smearing, 3x PC and 1x PC showed evidence of a clear band of DNA with limited smearing. As these two samples were also the samples with the best quantitative results, they were confirmed as being the most pure.
In conclusion, based on collected qualitative and quantitative data, it was found that 3x PC is both the best and most cost effective method of DNA purification.
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Chapter 5: Evaluation 5.1
Limitations and Improvements 5.1.1 Phenol Chloroform Contamination
Small amounts of phenol in a DNA sample can skew the 260/280 absorbance ratio because phenol absorbs at 270nm [13]. Hence, it will raise the 260nm absorbance and lower the 280nm absorbance, and the ratio will go up. Small amounts of DNA with large amounts of phenol may still give a ratio of 1.8. This was observed in samples which were contaminated with phenol, as their ratio was ~2.0.
The phenol contamination can be estimated using the 260/270nm absorbance ratio of the sample. Samples uncontaminated by phenol should have a 260/270nm ratio of 1.2.
5.1.2
Time Frame
Due to physical time constraints only two extractions were done. Thus, the conclusions are not statistically relevant, but give a good general trend for what was being measured. Large amounts of time were spent perfecting the extraction procedure, and so there was not enough time to carry out additional extractions
5.1.3
UV Spectrophotometer
Due to the sensitive nature of the UV spectrophotometer it is possible that, instead of DNA concentration, fluctuations in measurement correspond to varying absorbance ratios. This is unlikely given the consistency between the ratios of similar samples though.
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5.2
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Further Investigation 5.2.1
Solvent
Different, less toxic, organic solvents should be explored so that students might be able to carry out purification in the classroom. Phenol Chloroform is too toxic for classroom use. 5.2.2
230/260 & Other Ratios
For a true indication of sample purity, other absorbing factors should be considered. The 260/280nm Absorbance Ratio does not provide a complete picture of the purity of a given sample: it only accounts for contaminants which absorb at 280nm such as proteins and enzymes.
Other ratios to consider include: 230/260 or 320/260nm [14][15]. The 230/260nm ratio will show contamination by organic compounds, while the 320nm measurement will tell the contamination of the quartz cuvette by dust and other factors.
5.2.3
DNA Source
Extraction could be done from sources such as Lamda-phage, broccoli, wheat germ, or yeast to investigate yield differences and optimize the application of the 3x PC purification across species.
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Chapter 6: Conclusion DNA was extracted from onions and purified using Phenol Chloroform and Proteinase K. Purity was measured with the 260/280 absorbance ratio, using a UV spectrophotometer. The results were compared and it was conclusively proven that Phenol Chloroform produces samples of higher purity at a lower cost, and is therefore optimal for purification of onion DNA â&#x20AC;&#x201C; as was hypothesized.
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Chapter 7: Bibliography 7.1
Citations
[1]
Oswald, Nick. “Determining DNA Concentration & Purity”. BitesizeBio. Available from http://bitesizebio.com/2007/08/22/dna-concentration-purity/. Internet.
[2]
"Proteinase K." Promega Corporation. Available from http://www.promega.com/tbs/9piv302/9piv302.pdf. Internet. 2011.
[3]
Mecadi GmbH. "Isolation of Genomic DNA”. Mecadi GmbH. Available from http://www.proteinasek.com/index.php?id=659&L=1. Internet.
[4]
Sigma-Aldrich. "Analytical Enzymes, Proteinase K". Sigma-Aldrich. Available from http://www.sigmaaldrich.com/life-science/metabolomics/enzymeexplorer/analytical-enzymes/proteinase-k.html. Internet.
[5]
Oswald, Nick. "How Phenol Extraction Works." BitesizeBio. Available from http://bitesizebio.com/2008/02/12/the-basics-how-phenol-extraction-works/. Internet.
[6]
Uregina. "Phenol/Chloroform Extraction and Ethanol Precipitation." Available from http://uregina.ca/~ngdann/Bioc422/proj1.htm. Internet.
[7]
Strauss, William. "Preparation of Genomic DNA from Mammalian Tissue." Available from http://www.nshtvn.org/ebook/molbio/Current%20Protocols/CPI/im1002.pdf. Internet.
[8]
Oswald, Nick. "How Ethanol Precipitation of DNA and RNA Works."BitesizeBio. Available from http://bitesizebio.com/2007/12/04/the-basics-how-ethanolprecipitation-of-dna-and-rna-works/. Internet.
[9][10] “Nucleic Acids Analysis”. Wikimedia Foundation. Available from http://en.wikipedia.org/wiki/Nucleic_acids_analysis. Internet. [11]
Dube, Shanta. " DNA Agarose Gel Electrophoresis". Life Technologies. Available from http://www.bio.davidson.edu/courses/molbio/tips/trblDNAgel.html. Internet.
[12]
Bowen, Robert. "Preparing and Running Agarose DNA Gels”. Available from http://www.vivo.colostate.edu/hbooks/genetics/biotech/gels/agardna.html Internet.
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[13]
Bioteachnology.com. "The Analysis of DNA or RNA using Its Wavelengths: 230 nm, 260 nm, 280 nm." Available from http://bioteachnology.com/dna/analysisdna-rna-wavelengths-230-260-280-nm. Internet.
[14]
Held, Paul. “The Importance of the 240 nm Absorbance Measurement.” BioTek. Available from http://www.biotek.com/resources/docs/PW200240nmAM.pdf. Internet.
[15]
Thermo Fisher. “260/280 and 260/230 Ratios”. Thermo Scientific. Available from http://www.phenogenomics.ca/transgenics/docs/NanoDrop%20Nucleic-AcidPurity-Ratios.pdf. Internet.
7.2
References Anderson, Nadja. "Restriction Enzyme Analysis of DNA." Biotech Project, University of Arizona. Available from http://biotech.biology.arizona.edu/labs/DNA_analysis_RE_student.html. Internet. Applied Biosystems. “Quantitating RNA”. Ambion. Available from http://www.ambion.com/techlib/tn/94/949.html. Internet. Boujtita, Nadia. “Isolating Genomic DNA from Whole Blood”. Cole-Parmer. Available from http://www.coleparmer.ca/techinfo/techinfo.asp?htmlfile=isolating-genomicDNA.htm&id=1108. Internet. Children's Medical Research Institute. "Kitchen Style DNA Extraction, Restriction Enzymes and DNA electrophoresis." Jeans for Genes. Available from www.jeansforgenes.org.au/ArticleDocuments/51/DNAExtraction.pdf. Internet. Chomczynski, Piotr. "Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction". SciVerse. Available from http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6W9V-4DYTRY23W. Internet. Edvotek, Inc. "Isolation of DNA from Onions." Edvotek. Available from www.edvotek.com/pdf/WR2-031.pdf. Internet. Hays, Lana. "Introduction to DNA Extractions." Access Excellence. Available from http://www.accessexcellence.org/AE/AEC/CC/DNA_extractions.php. Internet.
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Kubo, Ken. "What is DNA Fingerprinting? Case of the Bloody Micropipettor." Biotech Project, University of Arizona. Available from http://biotech.biology.arizona.edu/labs/DNA_Fingerprinting_teach.html. Internet. Kubo, Ken. "DNA Extraction from Onion." Biotech Project, University of Arizona. Available from http://biotech.biology.arizona.edu/labs/DNA_extraction_onion_teach.html. Internet. Kuhn, Dwight. "Onion DNA Extraction." Virtual Lab Book. Available from http://classic.sidwell.edu/us/science/vlb5/Labs/DNA_Extraction_Lab/Onion_and _E__coli/onion_and_e__coli.html. Internet. Lawrence Livermore National Laboratory. "Restriction of Lambda DNA." LLNL. Available from http://education.llnl.gov/bep/science/10/sLamb.html. Internet. National Centre for Biotechnology Education. " The Lambda Protocol." University of Reading. Available from http://www.ncbe.reading.ac.uk/ncbe/protocols/PDF/LambdaSG.pdf. Internet. University of Wisconsin. "Extraction of DNA from Onion." UWSP. Available from www.uwsp.edu/chemistry/tzamis/lab/onion_dna_lab.pdf. Internet.
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Chapter 8: Appendices Appendix 1 Materials for preparation of lysis solution: − 12.5g SDS − 2.2g NaCl − 1.1g Sodium Citrate − 0.07g EDTA
Appendix 2 The process uses a gel made of agarose31 of varying concentration. High agarose concentrations resolve small DNA fragments better, while lower agarose concentrations resolve larger DNA fragments better. In this investigation, 0.7% agarose was used, as it is sufficiently low to allow the expected large fragments of DNA to travel in it.
The gel is created by pouring liquid agarose into a gel former; and a well-forming comb is used to ensure that once hardened, the gel will contain wells into which the DNA can be loaded. The gel is placed in a buffer, and the nucleic acid sample is mixed with a sucrose-based loading dye and inserted into the well. The buffer is placed under a current. Due to the negatively charged phosphate backbone of the DNA strands, they will migrate with the current – through the gel. The rate of their migration depends on their size and weight, and so gel electrophoresis is commonly used to separate nucleic acid samples into fragments by size.
Methylene Blue works as a dye because it binds to the DNA molecules, and so they appear darker than the gel background.
31
A polysaccharide extracted from algae and seaweed; used because it does not interfere with the proteins and nucleic acids during electrophoresis.
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