Extraction of DNA from onions

The purpose of the experiment was to experience firsthand the isolation of DNA form a plant tissue without destroying its structure and sequence. A white onion was used for the experiment. After several processes, DNA isolate was the visible result. Different chemical tests were performed on the DNA isolate, namely: Dische Test, Murexide Test, Wheeler-Johnson test and Test for Phosphate. Visible results were then noted.

INTRODUCTION

DNA (deoxyribonucleic acid) was discovered in the late 1800s, but its role as the material of heredity was not elucidated for fifty years after that. It occupies a central and critical role in the cell as the genetic information in which all the information required to duplicate and maintain the organism.

Today, Scientists can analyze DNA from minute samples of blood, hair roots and saliva, but before they can analyze DNA, scientists must be able to extract it. The process of DNA extraction is the first step for many laboratory procedures. Scientists must be able to isolate the DNA from the other unwanted substances of the cell gently enough so that the DNA is not broken up.

For this experiment, an onion was used because of its low starch content, which allows the DNA to be more clearly seen. The salt shields the negative phosphate ends of DNA, which allows these ends to come closer so that the DNA can precipitate out of a cold alcohol solution. The homogenizing solution causes the cell membrane to break down by dissolving the lipids and proteins of the cell, which disturbs the bonds that hold the cell membrane together. The homogenizing solution then forms complexes with the lipids and proteins causing them to precipitate out of the solution.

After the isolation of the DNA from the solution, different chemical tests were then performed on the isolate, which were: The Dische Test, Murexide Test, Wheeler-Johnson Test and Test for Phosphate.

The Dische test is a test done to identify DNA. It is the reaction between Dische reagent & 2-deoxypentose yields a blue colored solution.

The Murexide test a test in which the treatment of a substance, usually urine, with nitric acid and ammonia indicates the presence of uric acid by formation of murexide. Positive results form this test yields Purines form Yellow to Red-Violet.

The Wheeler-Johnson Test is a qualitative test for the pyrimidine bases cytosine and uracil, which produces a green coloration when the sample is treated with bromine water. The addition of barium hydroxide will turn the liquid purple.

The Test for phosphate is used to detect phosphate in the DNA, positive reaction yields yellow precipitate.

RESULTS AND DISCUSSION

The following procedures were done in order to isolate the DNA from the onion. First, the onions were minced and then weighed to 25g. The onions were minced because by mincing the onions, it increase its surface area that helps make the membrane at the surface easier to dissolve, and also allows for more efficient absorption of heat and solutions.

Next it was added to a 50ml homogenizing solution, this was done because the homogenizing medium, made of 5% SDS, 0.15M NaCl, 0.15 M sodium citrate and 0.001 M EDTA, was similar to a detergent. It helped break up the phospholipid bilayer of the cells’ plasma membranes and nuclear envelopes. When in SDS, the proteins and lipids of the membrane, but especially the lipids, are broken down because the SDS causes the bonds holding the membrane together to break. The lipids, which are repelled by the SDS, separate and break up the membrane. This is why detergent is used to remove a grease stain; grease is a lipid. The EDTA helped weaken the membrane as well, making the DNA that was in the nucleus now available for extracting.

Then the solution together with the onion was heated at 60 degrees, this method was used to speed the breakdown process by energizing the molecules, and also, hopefully help to dissolve the phospholipid bilayer by destroying the proteins and breaking the bonds that hold the phospholipids in place. By exciting the molecules, the heat might cause the fluid bilayer to break and become a gas-like substance, no longer serving as a barrier for the DNA. The heat softens the membrane as a whole. This entire step, homogenation, is used to free the DNA from its protective barriers.

Then crude papain was added, because Deproteinization which involves adding a protease enzyme Papain will denature the proteins clinging to the DNA making the molecule flexible and easy to spool. Then the solution was then heated again to 60 degrees for 10 minutes with occasional stirring.

Then it was placed in an ice bath, this is because by cooling the solution, it will help prevent denaturation, which might destroy the DNA if it was exposed to prolonged heating. Swirling was done in order not to “shock” the DNA from the sudden change in temperature.

After putting it in an ice bath, the solution was then placed in a blender and was blended for 45 seconds, after. This was done because the procedure also frees the DNA from another protective barrier, the thick cell wall that all plant cells have. The blending should cause many of the cell walls, containing cellulose, to break, and therefore leave the DNA in the cytoplasm, and no protective thick cellulose wall blocking the cytoplasm from its external environment. We did not need to do this to the bacteria because it does not have a cell wall.

The blended solution was then filtered through 4 layers of cheesecloth leaving the foam untouched. This is because it leaves behind thicker materials, including any parts of the remaining onion itself. The liquid that is filtered through contains the DNA, no longer held behind a plasma or nuclear membrane, and ready to extract. All waste not needed for the experiment is filtered out, such as cellulose of the cell walls, pectins, and excess tissues like the skin sclerenchyma cells of the onion.

Then the volume of the isolate was measured and was then transferred onto a 250mL beaker, and was then placed in an ice bath, then beaker was then tilted to 45 degrees and 95% ice-cold ETOH was added. The analysis of this was that the DNA is polar, but the reaction with the ethanol makes the DNA nonpolar, and therefore resistant to the homogenizing medium that it is in. The DNA forms its own layer, a precipitate between the liquid ethanol and liquid homogenate. Also, the DNA is the only component of the solution that is not soluble in the ethanol. Therefore, it becomes its own clear layer on top of the lighter ethanol, but free of the soluble homogenizing medium. However, the reaction above, with the DNA precipitating to the top of the homogenate because it becomes nonpolar and remaining under the ethanol because it is not soluble only happens at the top of the solution where the DNA is exposed to the ethanol and does not move far to precipitate out. Therefore, the top DNA remains between the two layers.

Lastly, the DNA was spooled and placed in a foil, then it was air-dried for quite sometime, then was prepared for the different colored reactions.

Chemical Tests

Dische Test (Standard: D-Ribose Solution)

Murexide Test

(Standard: Guanine)

Wheeler-Johnson Test

(Standard: Cytosine)

Test for Phosphate

(Standard: Conc H3PO4)

DNA Isolate

Faint blue solution formed

Yellow residue formed

White solution formed; red litmus paper turned blue

Clear, colorless liquid formed

Standard

Dark blue solution formed

Orange residue formed

Violet solution formed; red litmus paper turned blue

Faint yellow precipitate formed

TABLE 1. Results of the DNA isolate and the standards in the different chemical tests

For the Dische test, the reaction depends on the conversion of the pentose to the aldehyde which then reacts with diphenylamine to give a blue-colored solution. The intensity of the blue color is proportional o the concentration of DNA. Dische reagent does not form a blue-colored solution in RNA.

CONCLUSION

After conducting the experiment, the group has concluded that isolating DNA from onions is not that easy. Several precautions like keeping the temperature to 60 degrees must be monitored, and also the blending of the solution limited to 45 seconds. Several steps including placing the placing of the solution in an ice bath is also monitored, likewise with the swirling of the solution. As for the DNA isolate, it reacted mostly the same compared to the different standards used in the chemical tests. It may differ in the intensity of the color from the standards but still, it yielded positive results.

REFERENCES

Books:

[1] Stoker, Stephen. Exploring General, Organic, and Biological Chemistry. Cengage Learning, 2010.

[2] Straumans, Andrei. Biological Chemistry : A guided inquiry. Houghton Mifflin, Boston, MA, 2012.

Internet:

[3] Keusch, Peter. (n.d). _Test for Deoxyribose in DNA_. Retrieved from http://www.uni- regensburg.de/Fakultaeten/nat_Fak_IV/Organische_Chemie/Didaktik/Keusch/p3 1_d_rib-e.htm

[4] _Extraction of DNA from Onion_. Retrieved from http://dwb.unl.edu/Teacher/NSF/C08/C08Links/cpmcnet.columbia.edu/dept/physi o/tchrplan/oniondna.html