Agarose Gel Electrophoresis for the Isolation of Onion DNA.

Abstract

Agarose Gel Electrophoresis method was applied on an onion sample to isolate onion DNA. The objective of this experiment was to be able to extract the DNA of an onion in the presence of Ethylenediaminetetraacetic acid. The A260A280 absorbance ratios of isolated DNA were around 1.0593, suggesting that the DNA was not pure and could not be used for further analysis. Moreover, the A260A280 values were lower than 1.8, suggesting that there was a contamination by proteins. The DNA isolated by this protocol was not of sufficient quality. The integrity of the It was concluded that the DNA was not pure due to protein and aromatic contaminations.

Introduction

Nucleotides play an important role in the cell, either as energy carriers, as coenzymes or as building blocks for nucleic acids. Nucleotides consist of purine bases, adenine ad guanine and pyrimidine bases, cytosine, uracil, or thymine. (Wink, 2006). Deoxyribosenucleic acid is the cells master repository for genetic information. It is a negatively charged, macromolecule consisting of two strands of linked nucleotides, each of which are made up of a deoxyribose sugar residue, a phosphate group and one of bases; thymine, adenine, cytosine or guanine. (Voet and Voet, 2005). It is found coiled up around basic proteins called histones. (Bettelheim and Landesberg, 2009). DNA does not exist as a free molecule in a cell, but instead is associated with proteins and RNA.

DNA isolation is the removal of deoxyribonucleic acid from cells in which it normally resides. The use of DNA for analysis or manipulation usually requires it to be isolated and purified. This is done in a very gentle way so as to prevent it from fragmenting. This is done in the presence of Ethylenediaminetetraacetic acid (EDTA) which chelates the Mg2+ needed for enzymes do degrade DNA. (Wilson and Walker, 2005).

Electrophoresis is the procedure used to extract and isolate DNA. It is either done in agarose or polyacrylamide gel added to an electrophoresis buffer. Agarose gel electrophoresis is the standard procedure to separate DNA fragments that vary in size; this is followed by staining with ethidium bromide. This is the most widely applicable and easiest method. (Wilson and Walker, 2005). The speed at which the DNA fragments migrate through the gel, in an electric field, depends highly on the size of the DNA fragments. Other factors such as buffer conditions, molecule shape and gel concentration can also affect the migration of the NDA through the gel. When precipitating with alcohol, the DNA molecules precipitate as large, thread-like aggregates that can physically be seen and fished from the solution with a glass rod or pipette. (Chirikjian,)

The aim of this experiment was to isolate the DNA from an onion sample and to discover the fundamental properties of DNA and also the methods used to isolate it.

Methods and Materials

80 ml of 50 mM of Tris-HCl pH 7.5 was placed into a beaker with 10g of NaCl. Then 50g of finely grated onion was added and then incubated in a water bath of 60°C for 15 minutes. The homogenate was then filtered through four layers of cheese cloth and the filtered solution saved. One gram of meat tenderizer was added to the strained homogenate then left to stand for 5 minutes at room temperature. The homogenate was then left to cool on ice to 10°C. Then 50 ml of ice cold isopropanol was poured down the side of the beaker containing the homogenate to form two distinct layers with the homogenate at the bottom. White stingy DNA should appear at the interface by gently swirling a glass rod around the interface in a unidirectional way. The DNA appears as a blob of oils on the glass rod. Then lastly re-suspend the DNA in 250 µl of Tris-EDTA (TE) buffer.

A gel comb is pushed into pre-prepared 0.8% Agarose gel and the gel mounted into an electrophoresis tank. Electrophoresis buffer is added to cover the gel to a depth of about 1mm. The DNA is mixed with gel loading buffer (comprised of 0.25% bromophenol blue and 40% sucrose in water), with 10 µl of 6 x DNA loading buffer per 50 µl DNA solution. Then 30 µl of the mixture is slowly loaded into the submerged gel slots using an autopipette. A sample of λ DNA restricted with PstI restriction endonuclease is loaded (10 µl of approximately 1 µl DNA). The lid of the gel tank is closed and the leads attached so as to make the DNA migrate to the anode. A voltage of 1 to 5 V/cm is applied. It is then left to electrophorese until the bromophenol blue has migrated three quarters the length of the gel. Examine under a UV illuminator and photograph with a gel documentation system.

A spectrophotometer is set to a wavelength of 260 nm and zeroed using 600 µl of water. 30 µl of the DNA solution is added to 600 µl of water. Measure and record the absorbance. Then set the spectrophotometer to 280 nm and zero using water. Measure and record the absorbance reading. If the absorbance readings exceed 0.6, the sample should be further diluted. The concentration and purity of the sample is then calculated.

Results Loading wells Clustered DNA
11501 base pairs
5077 base pairs
11963 base pairs
1700 base pairs
Marker (dark bands)
Figure 1: Agarose gel showing onion DNA isolation
Calculation of DNA purity and concentration of sample:
A260 = 0.089
A280 = 0.085
[DNA (ngμl)] = A260nm x 50 ng/µl x (63030) (concentration) = 0.089 x 50 x (63030) = 93.45 ng/µl
Purity of DNA = A260A280 = 0.0890.084 = 1.0593

Discussion

The DNA from this experiment was found to be impure and this resulted in the photograph not being produced. This could have been caused by the sample DNA being too dilute. Also this could have been caused by the sample solution having too many contaminants. The contaminants in this case were proteins. Also the solution may have been left in the water bath for too long causing some of the DNA to break up. The above photograph was taken from another group doing the same experiment.

DNA absorbs light at a wavelength of 260nm. The absorption of the DNA bases in the sample solution was found to be 0.089. This is lower than 0.6. This very low absorption reading means that the sample solution was too dilute. The reading at wavelength of 280nm is the reading at which proteins absorb light. The reading was found to be 0.085. This means that there were almost as many proteins as there was DNA and this is what could have caused the photograph not being produced. The ratio of the DNA to protein in the solution was 1.0593. This is a not good ratio and meant that the DNA was not pure. Pure DNA should give a ratio of more than or equal to 1.80, while a ratio of less than 1.80 means that the preparation was contaminated with proteins and other aromatic substances. (Wilson and Walker, 2005). This could have been corrected by redigesting with the SDS. (Keller and Manak, 1989). The concentration of the DNA in the solution was 93.45 ng/µl.

The clustered DNA in well 5 is due to residual bound proteins that retard migration of the DNA into the gel.
The results were not as expected due to the abovementioned reasons that is that proteins and aromatics contaminated the solution. The purification method was not effective because there was no picture after the gel was taken for analysis. The reason for the photograph not coming up even after the purification could have been due the fact that the onion might have been because it might have too finely chopped up that the DNA was damaged. This method could have been improved by avoiding mechanical force when chopping up the onion and the sample should have been fresh.

Finally, taking all the above into consideration, it can be concluded that our aim was not achieved and the fundamentals of DNA and the methods of isolating it. This being because the sample was too dilute and contaminated with proteins and other aromatic substances.

References

Bettelheim, F.A., and Landesberg, J.M. (2009). Laboratory Experiments for Introduction to General, Organic and Boichemistry, 7th Ed. Experiment 43: Isolation and Identification of DNA from Onion, pp. 515-517, Cengage Learning. United States of America.

Chirikjian, J.G. (1995). Biotechnology: Genetic Engineering Mutagenesis Separation Technology. Unit 1: DNA Isolation. Volume 2, pp. 4-5, Jones and Bartlett Publishers International, England.

Keller, G.H., and Manak, M.M. (1989). DNA Probes, pp 36, MacMillan Publishing Ltd, New York.

Wilson K., and Walker, J. (2005). Principles and Techniques of Biochemistry and Molecular Biology, pp 166, 172-173, 191-195, Cambridge University Press.

Wink, M. (2006). An Introduction to Molecular Biotechnology: Molecular Fundamentals, Methods and Applications in Modern Biotechnology, pp. 29, 33, Wiley-VCH Verlag GmbH & Co. KGaA, Germany.

Tutorial

1. SDS stands for sodium dodecyl sulphate. It is an anionic detergent used before electrophoresis. It is added to the initial solution so as to bind to the protein surface in the solution and cause all the proteins to become negatively charged and dissociates most of the multi-chain proteins. It also breaks down the membrane lipid molecules of the cells and nuclear membranes, releasing the contents of the cells, including the DNA.

2. It is necessary to heat the solution so that the DNA is properly removed from the cells. The heating separates the DNA into single strands, exposing the bases. This process is called DNA melting. The heating of the solution releases further releases the DNA into the solution but also denatures it by separating the two strands.

3. Salt provides the DNA with a favorable environment; it contributes positively charged atoms that neutralize the normal negative charge of DNA. The salt also causes the precipitation of proteins and carbohydrates, further helping to separate the cells.

4. The meat tenderizer acts as a protein and contains proteases, which are enzymes that break down proteins. The proteases break down and cut the protein core, called a histone, on which the DNA is super-coiled around. This allows the DNA to uncoil and stretch out, allowing it to be seen.

5. The integrity of purified DNA can be compromised by nucleases, pH’s below 6.0 and 9.0, UV light and oxidation of free radicals. EDTA is added to chelate divalent cations required for nuclease activity. Tris-based buffers provide a safe pH of 7 to 8 and don’t generate free radicals.