DNA ISOLATION FROM ONION, ULTRAVIOLET MEASUREMENT OF ISOLATED DNA AND CHEMICAL CHARACTERIZATION OF DNA Mendez, H.C.A.

, Miranda, M., Maravillias, M., Manlutac, D., Navarro, M.A., Panaligan, A, Pingol, E., Pique, V.M., Group 5-2B-Pharmacy ABSTRACT The Experiment was about the isolation of DNA from onion, Ultraviolet measurement of isolated DNA and Chemical characterization of DNA. DNA was isolated form the plant source (onion). The Absorbance of the DNA was then Determined with the use of a Spectrophotometer, the ratio of absorbance was then computed. The DNA was then hydrolyzed with hydrochloric acid. The DNA was characterized with the use of Tests ford Deoxyribose or The Dische reaction test, Test for Phosphate, Test for Purines a.k.a murexide Test and Test for Pyrimidine namely as Wheeler-Johnson Test. The test for Deoxyribose produced a brownish solution while the test for ribose produced a yellowish solution. The test for phosphate gave a colorless with crystals solution while the test for purines formed a clear solution with yellow precipitate/crystals. Lastly, the test for pyrimidine gave a purple solution.

INTRODUCTION
Deoxyribonucleic acid is a nucleic acid containing the genetic instructions used in the development and functioning of all known living organisms. DNA is made of chemical building blocks called nucleotides. These building blocks are made of three parts: a phosphate group, a sugar group and one of four types of nitrogen bases. To form a strand of DNA, nucleotides are linked into chains, with the phosphate and sugar groups alternating. The four types of nitrogen bases found in nucleotides are: adenine (A), , thymine (T), guanine (G) and cytosine (C). The order, or sequence, of these bases determines what biological instructions are contained in a strand of DNA. DNA contains the instructions needed for an organism to develop, survive and reproduce. To carry out these functions, DNA sequences must be converted into messages that can be used to produce proteins, which are the complex molecules that do most of the work in our bodies. DNA is found inside a special area of the cell called the nucleus. Because the cell is very small, and because organisms have many DNA molecules per cell, each DNA molecule must be tightly packaged. This packaged form of the DNA is called a chromosome.

DNA spends a lot of time in its chromosome form. But during cell division, DNA unwinds so it can be copied and the copies transferred to new cells. DNA also unwinds so that its instructions can be used to make proteins and for other biological processes. Certain tests will be used in order to isolate and classify the DNA ; Isolation of DNA from onion, Ultraviolet measurement of Isolated DNA, Acid Hydrolysis, Dische Test, Test for Phosphates, Test for Purines, and Test for Pyrimidine.

DNA ISOLATION FROM ONION

Isolation of the DNA is the first step in the experiment, The Raw Minced onion is then added with the Homogenizing solution, which involves breaking down and removing cell walls and membrane.

ULTRAVIOLET MEASUREMENT OF ISOLATED DNA

When DNA is isolated from organisms, frequently there remains protein present in the DNA solution; protein is tightly bound to DNA and complete removal of protein is not possible. To determine the concentration and purity of the protein it is therefore subjected for analysis in a spectrophotometer.

ACID HYDROLYSIS
DNA is stable to bases, these is one of the features that distinguish DNA from RNA. Strong acids at high temperatures are capable of breaking down DNA molecules onto its basic components, at these conditions both of the phosphate ester bond and the Nglycosides bends between the Deoxyribose, the Purines and the pyrimidine bases.

Test for Pyrimidines Lastly the following were used in the test: 0.5ml Nucleic Acid solution, Bromine Water, Barium Hydroxide

DISCUSSION
Isolation of DNA from Onion. A 50 ml Homogenizing solution is then prepared in a 125 ml Erlenmeyer flask and was heated in a water bath until the solution reached 60 deg C, 25 g minced onion was added to the preheated water bath. A 1.5g papain was then added and was then kept for 10 minutes. After heating place the Erlenmeyer to an ice bath for about 5 minutes, after the cooling process, place the solution to in a beaker to be homogenized; after the homogenization process the homogenate was then filtered using a cheese cloth into a 250ml beaker and cool it after wards. The cooled solution was then added with 15-20ml of ice-cold ethanol, place it on the sides and allow it to drip downwards, a clear layer of ethanol should form on top of the solution, DNA is not soluble in ice-cold ethanol, when ethanol is added to the mixture, all the components in the solution except for the DNA stay in the solution, while the DNA precipitates out. Let the mixture stand for 3-5 minutes without disturbing, the DNA can be perceived by a white precipitate/ strings. Spool the Formed DNA by using a pre-bent glass pipette, the obtained specimen was then added with TE buffer solution. Ultraviolet Measurement of Isolated DNA The obtained DNA solution (0.5ml) is then added to a 4.5 SSC or TE buffer solution. It is the transferred to a quartz cuvette to determine the absorbance at the range of 260nm-280nm. The obtained results are then calculated for the ratio to determine the concentrations of protein and nucleic acid. Acid Hydrolysis Mix the DNA sample with 1M HCl, then heat it up to 100deg C for 60 minutes with occasional agitation, cover the tubes with marbles and placed under a running water to neutralize the temperature and add

EXPERIMENTATION
Compounds Tested; Samples used and Devices used DNA isolation from Onion The following were used for the isolation of DNA; 2 White/yellow Onions, Homogenizing Solutions; 5% SDS( Sodium Dodecyl Sulfate), 0.15 M NaCl, 0.15 M Na3C6H5O7 and 0.001 M EDTA, Ice-Cold 95% ethanol. Ultraviolet Measurement of DNA The Following were used in the Measurements: Quartz Cuvettes, UV-VIS Spectrophotometer. Acid Hydrolysis The Following were used in the Acid Hydrolysis: DNA Sample, 1M HCl Test for Deoxyribose The following were used in the test: 3.5ml Diphenylamine reagent, 1.5ml Hydrolyzed DNA solution, 0.5 ml Standard DNA, Hot water Bath Test for Phosphate The Following were used in the Test for phosphates: 1ml conc. H2SO4, 1ml Nucleic Acid Solution, 1ml Standard Phosphate solution, 0.5ml conc.HNO3, 1ml H2O, 1ml Molybdate Solution, 10ml water. Test for Purines The Following were used in the Murexide Test: 5-10GTT Nucleic Acid solution, HNO3,10% KOH, water

1M KOH. Adjust the PH to 4-6 using Acetic Acid. Use PH paper to determine the acidity, Centrifuge and add to clean test tubes for chemical Characterization. Test for Deoxyribose Add 3.5 ml Diphenylamine reagent to 1.5 ml hydrolyzed DNA solution. The same procedure used with the 0.5ml Standard Deoxyribose Solution. Place the prepared mixture in a hot water bath for at least about 10 minutes. Cool immediately and observe the results. Test for Purines 10 drops of nucleic acid solution into a small evaporating dish, a few drops of conc. HNO3 is then added. Carefully evaporate until try in water bath. Moisten the evaporated nucleic acid with some 10% KOH and heat further, note the color changes upon adding the KOH and upon heating. Add a few drops of water then observe the color, then lastly evaporate and note the color changes. Test for Pyrimidine Treat 0.5 ml nucleic acid solution with an excess of bromine water until the solution turns yellow, remove the excess by boiling the solution until it turns yellow, note the changes, add the excess Ba(OH)2 solution, then test with litmus paper. Take note of the appearance of the solution.

Next is the deproteinization, this process involves adding the protease enzyme papain. It is a common enzyme that removes or prevents proteins from clinging to the DNA. DNA Precipitation is done by means of adding ice-cold ethanol. This is done so the DNA will separate from the solution. The homogenizing solution is important in the experiment, it allows the release of the DNA from the clinging proteins, there are 3 chemicals that are used in this process, it is Sodium Dodecyl Sulfate, it is a detergent that helps facilitate in the breakdown of the cell membrane. Ethylenediamine tetra acetic acid or EDTA, it also weakens the cell by destabilizing the cations. And lastly Sodium Chloride Facilitates the DNA to precipitate out of the alcohol solution. Ultraviolet measurement of DNA
Table 1: Isolation and UV measurement of DNA

DNA

UV measurement

PROTEIN Mg/mL 1/0.7=1.43 mg/ml

PLANT DNA

1.o85(260nm)/ 0.801(280nm)= 1.35 ratio

NUCLEIC ACID g/mL 24/18=1.33 mg/ml

RESULTS AND DISCUSSION

Isolation of DNA and Ultraviolet Measurement of DNA Isolation of DNA can be done in three steps which are Homogenization of deproteinization and precipitation. In homogenization the onion is minced, then heated. The heat treatment is done in order to soften the cell wall and for the breakdown of the cell. Heating softens the phospholipids in the cell membrane and denatures the ribonuclease enzymes, which if presents will cause the fragmentation of the DNA and thus prevent it from being spooled.

The chart above shows the ultraviolet measurements of the obtained samples. As shown from above, 1.085 at 260nm and 0.801 at 280nm is obtained from the spectrophotometer. At normal condition it has been observed that the normal range for a good quality DNA sample should have an A260/A280 ratio of 1.7-2.0, but we obtained a ratio of 1.35, it can be inferred that our DNA sample is contaminated or sensitive for it not to be at the normal range. The amount of protein present in the DNA is about 1.43mg/mL, we obtained this by dividing 1 with 0.7 we obtained both data from the given chart of the amount of proteins present. Lastly the amount of Nucleic acid is 1.33mg/ml, we obtained both of the given data from the given chart. Chemical Characterizations Acid Hydrolysis Strong acids at high temperatures are capable of breaking down DNA molecules onto its basic

components, at these conditions both of the phosphate ester bond and the N-glycosidic bends between the Deoxyribose, the Purines and the pyrimidine bases
Table 2: Chemical Characterization

REFERENCES

BOOKS DNA(standard) Biochemistry (3rd Edition) by Christopher K. Mathews. Lehninger Principles of Biochemistry, Third Ed..by David L. Nelson Crisostomo., Laboratory Manual in General Chemistry., 2010. WEBSITES

Chemical Test

DNA(onion)

Deoxyribose Phosphate Pyrimidine Purines

Yellow Solution No result Purple Solution Reddish brown

Brown Solution Clear Solution w/yellowcrystals Purple Solution Yellow residue w/ red Dots

1. http://www.uniregensburg.de/Fakultaeten/n at_Fak_IV/Organische_Chemie/Didaktik/Keusch/p31_d _rib-e.htm

As shown from the chart, there are various results for the characterization of DNA. In the Deoxyribose Test we obtained a Yellow solution in the DNA Of onions while we obtained Brown solution in the standard DNA. The test for phosphates didn t show any results with the DNA in onions while The Standard DNA resulted in a Clear solution w/ yellow crystals, test for Pyrimidine resulted in a Purple Solution in the DNA of the onion, same as The Standard DNA. Lastly the Test for purines Resulted in a Reddish Brown solution in the DNA of the onion, while the standard DNA gave a yellow residue w/ red dots. These tests showed a significant difference between the DNA in onions compared to the Standard DNA, the DNA of the onion may somewhat be a more sensitive or may be more resistive to the test, thus it gave a different result. It can be inferred that in the process of characterization of DNA, the way of preparation of the solution can affect the resulting data, for example, our DNA string were not successfully obtained partially due to a process that was not properly done, we recommend the utmost care in measuring the solutions that will be used, it is also important to properly practice laboratory safety guidelines, for the chemicals that are being dealt with are somewhat volatile and can easily harm the examiner. Proper procedures should also be done in order to achieve accurate data of the results.

2. http://www.medicalhealthtests.com/blog/bl ood-tests/blood-phosphate-level.html

3. http://www.biochemia.amb.edu.pl/pdf/class 9.pdf