DNA ISOLATION FROM ONION, ULTRAVIOLET MEASUREMENT OF ISOLATED DNA AND CHEMICAL CHARACTERIZATION OF DNA Maria Feliza C.

Abesamis, Marie Em Clarisse P. Acosta, Francheska M. Agustin, Mary Christelle G. Aquitania and Marilu Jane H. Bagsican Group 1 2E Medical Technology Biochemistry Laboratory ABSTRACT
The experiment was about the Isolation of DNA from onion, ultraviolet measurement of isolated DNA and chemical characterization of DNA. DNA was isolated from the plant source (onion). The absorbance of the DNA solution was determined with the use of spectrophotometer. The ratio of absorbance was computed. DNA was hydrolyzed with hydrochloric acid. After, it was subjected into chemical characterization with the use of different tests. DNA was characterized with the use of Test for Deoxyribose, Test for Phosphate, Test for Purines named as Murexide Test and Test for Pyrimidines named as Wheeler-Johnson Test. The test for Deoxyribose produced a brownish solution while the test for ribose resulted to a yellow turbid solution. The test for phosphate gave a colorless solution while the test for purines formed a yellow precipitate. Lastly, the test for pyrimidines and the test for uracil produced a white precipitate and gave no change of color for the litmus paper which was a negative result.

INTRODUCTION
A nucleic acid is a macromolecule composed of chains of high molecular weight biopolymers of monomeric nucleotides. In biochemistry these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are universal in living things, as they are found in all cells and viruses. They function mainly in the storage and expression of genetic information. DNA, or deoxyribonucleic acid, is the hereditary material in humans and almost all other organisms. Nearly every cell in a persons body has the same DNA. Most DNA is located in the cell nucleus (where it is called nuclear DNA), but a small amount of DNA can also be found in the mitochondria where it is called mitochondrial or mtDNA. The information in DNA is stored as a code made up of four chemical bases: adenine (A), guanine (G), cytosine (C), and thymine (T). Human DNA consists of about 3 billion bases, and more than 99 percent of those bases are the same in all people. The order, or sequence, of these bases determines the information available for building and maintaining an organism, similar to the way in which letters of the alphabet appear in a certain order to form words and sentences. DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix. The structure of the double helix is somewhat like a ladder, with the base pairs forming the ladders rungs and the sugar and phosphate molecules forming the vertical sidepieces of the ladder. An important property of DNA is that it can replicate, or make copies of itself. Each strand of DNA in the double helix can serve as a pattern for duplicating the sequence of bases. This is critical when cells divide because each new cell needs to have an exact copy of the DNA present in the old cell. Nucleic Acid Isolation procedure usually consists of three steps: (1) disruption of cell membranes with the cell wall in some organism and the membrane of the subcelullar nucleus to release the nucleic acids, (2) dissociation from nucleoproteins and denaturation of protein (3) separation of DNA from other soluble cellular components.

Figure 1. Structure of DNA

The process of isolating DNA from a cell is the first step for many laboratory procedures in biotechnology. The scientist must be able to separate the DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up or shredded.

determine the purity of DNA isolated, (3) to characterize DNA following acid hydrolysis

EXPERIMENTAL A. COMPOUNDS TESTED USED)/ DEVICE USED (SAMPLES

1. DNA ISOLATION FROM ONION

The following were used for the isolation of DNA from onion: 2 yellow onions, Homogenizing solution: 5% SDS (sodium dodecyl sulfate or sodium lauryl sulfate), 0.15M NaCl, 0,15 sodium citrate and 0.001M EDTA Tris-EDTA buffer (TE buffer), pH 8.0: 0.1M Tris HCl and 0.015M sodium citrate Commercial papain or meat tenderized (6% in water), Ice-cold 95% ethanol.

Figure 2. Formation of DNA strands

2. Ultraviolet Measurement of Isolated DNA

The following devices were used to ultraviolet measurement of isolated DNA: Quartz Cuvets, UV-Vis Spectrophotometer

Ultraviolet DNA

measurement

of

isolated

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 always possible. To determine the concentration and purity of the DNA solution, the absorbance of UV light is measured in a spectrophotometer.

3. Acid Hydrolysis
The following were used for DNA hydrolysis: DNA sample, 1N HCL

4. Test for Deoxyribose

The following were used for the test for deoxyribose: 3.5ml diphenylamine reagent, 1.5ml hydrolyzed DNA solution, 0.5ml standard deoxyribose

Acid Hydrolysis
DNA is stable to bases (alkaline hydrolysis). This is one of the features which distinguish DNA from RNA (RNA is not stable under alkaline conditions). Strong acids at a high temperature, are capable of breaking the DNA molecule into its components. These conditions break both of the phosphate ester bonds and also the N-glycosidic bond between the deoxyribose and the purines and pyrimidine bases. The products of this mixture are the 4 bases, phosphoric acid, and deoxyribose which then polymerises to produce a brown sticky tar. The objectives of the experiment are as follows: (1) to isolate DNA from plant source, (2) to

5. Test for Phosphate

The following were used for the test for phosphate: 1ml conc. H2SO4, 1ml nucleic acid solution, 1ml standard phosphate solution, 0.5ml conc. HNO3, 1ml water, 1 ml ammonium molybdate solution, 10 ml water

6. Test for Purines (Murexide Test)

The following were used for the test for purines: 5-10 drops nucleic acid solution, HNO3, 10% KOH, water

7. Test for Pyrimidines Johnson Test)

(Wheeler
bromine

0.5ml nucleic acid solution, water, barium hydroxide

B. PROCEDURES: 1. DNA Isolation from Onion

A 50 ml of homogenizing solution was placed in a 125ml Erlenmeyer flask and was heated in a water bath until the solution reached 60 C. When the solution reached 60 C, 25g minced onion was added to the pre-heated homogenizing solution. It was then placed in a water bath for 5 minutes with constant stirring. After 5 minutes, 1.5 papain (or 4ml meat tenderizer solution) was added and it was left in the water bath for 10 minutes more. Then 10 minutes after, the flask was placed immediately in an ice bath and was left there to cool down for 5 minutes. The solution was swirled gently which allowed even cooling. Then contents of the flask were homogenized by quickly transferring it into a blender and blended for 45 seconds. The homogenate was filtered through a layer of cheesecloth into a 250mL beaker. Solution was cooled into ice. Then 15-20 ml of ice-cold ethanol was added immediately to the test tube by slow pipetting the ethanol down the side of the tube. A clear layer of ethanol was formed on top of the onion filtrate. The tube was left standing for 3-5 minutes without disturbing it. Bubbles formed and DNA precipitated out of the solution. DNA became visible as white strings in the ethanol layer. The DNA was spooled out by snagging it with a Pasteur pipet with a hook bent into the tip. Afterwards, the DNA was transferred into a clean test tube and re-suspended in TE buffer or SSC solution.

Figure 4. Spooling out DNA with metal hook 2. Ultraviolet Measurement of Isolated DNA
A 0.5 aliquot of DNA solution was dissolved in 4.5ml SSC solution or TE buffer. The solution was then transferred to quartz covet and absorbance was determined at 260nm and 280nm. The ratio was calculated by A260/A280.

3. Acid Hydrolysis
DNA sample was mixed with 1N HCl. (1015 mg sample with 1ml of acid). The mixture was then heated at 100 C for 60 minutes. Tubes were covered with marbles. After heating, tubes were carefully cooled with running water. It was neutralized with 1N KOH. The pH was adjusted to the range of pH 4-6 with glacial acetic acid using pH paper. It was then subjected to centrifugation and afterwards decanted into clean tubes for chemical characterization.

4. Test for Deoxyribose

Diphenylamine reagent with a volume of 3.5ml was added to 1.5ml hydrolyzed DNA solution and 0.5 ml standard deoxyribose solution. It was subjected to heat for 10 minutes in boiling water bath and immediately cooled.

5. Test for Phosphate

A 1mL conc. H2SO4 was added to 1 ml nucleic acid solution and to 1 ml standard phosphate solution. The mixture was heated over a small flame, shaking frequently until the contents of the tube turned brown. After, it was cooled down and 0.5ml of conc. HNO3 was introduced. Heating was continued until white fumes appeared. When the solution didnt turn colorless, 0.5ml of conc. HNO3 was again introduced. Heating was continued until white fumes re-appeared. When liquid turned colorless,

Figure 3. Precipitation of DNA strand in alcohol layer

1ml water was added and it was subjected heat for 5 mins. It was cooled and ammonium molybdate solution was added. It mixed well and diluted with 10ml water. It left standing for 5 minutes and color formed observed.

into 1ml was was was

There are three basic steps in the procedure. Homogenization, deproteinization, and precipitation. The homogenization step involves the heating and blending of the onion tissue in order to break down the cells. The heat treatment softens the phospholipid in the cell membrane and denatures the deoxyribonuclease enzymes, which if present will cut the DNA into small fragments that will not spool. The onion tissue is mixed in a blender with the homogenization medium, which breaks down the cell wall, cell membrane, and nuclear membrane. Deproteinization involves adding the protease enzyme papain. Papain is a common protease used to remove proteins from soft contact lenses. This denatures the proteins clinging to the DNA, thus making the molecule flexible and easy to spool. Precipitation of DNA is facilitated by adding ethanol which causes all the components in the filtrate to stay in solution except DNA. The DNA will gather at the interface of the filtrate and ethanol and then is easy to spool out with a glass rod. The homogenization medium contains three important chemicals that allow the release of DNA. The first is sodium dodecyl sulfate or SDS. It is a detergent that helps to break down the cell membrane. It emulsifies the lipids and proteins of the cell by disrupting the non-covalent interactions that hold the cell membrane together. SDS also forms complexes with these lipids and proteins causing them to precipitate out of solution. Ethylenediamine tetracetic acid or EDTA weakens the cell by complexing with the divalent cations, Mg+2 and Ca+2, which are needed for membrane stability. Therefore, EDTA also aids in the lysing of the cells. Lastly, sodium chloride enables the DNA to precipitate out of an alcohol solution because it shields the negative charges on the phosphate groups of DNA. Thus, the strands will not electrostatically repel and will coalesce.

6. Test for Purines (Murexide Test)

10 drops of nucleic acid solution were placed in a small evaporating dish. Few drops of conc. HNO3 were added. It was evaporated to dryness carefully on a water bath. The residue formed was moistened with 10% KOH and it was further heated. Color changes were observed upon the addition of KOH and upon further heating. Few drops of water were added and were warmed. A yellow solution resulted which yielded a red residue upon evaporation.

7. Test for Johnson Test)

Pyrimidines

(Wheeler-

Nucleic acid solution with a volume of 0.5ml was treated with excess bromine water until the solution turned yellow. Excess was removed by boiling the solution until the solution turned light yellow or colorless. Barium hydroxide was added in excess.

RESULTS AND DISCUSSION: DNA Isolation from Onion A. Isolation of DNA and UV Measurement of DNA from Onion
Table 1. Actual results for the Isolation of DNA and UV Measurement of DNA from Onion DNA Physical Description Thick, stingy white mass precipitate UV Measurement (A260/ A280) A260= >3A A280= >3A 1.00

Plant DNA

Ultraviolet DNA

measurement

of

Isolated

Ratio of Absorbance

= 1.00

The ratio is the absorbance of 260 nm wavelength (from the light spectrum) to the absorbance of 280 nm wavelength. The first is the wavelength that is in the center of the bell curve of absorbance for nucleic acid, and the second is the wavelength that is in the center of

the bell curve of absorbance for Accordingly, a pure sample of DNA,

proteins.

Substantially free of protein, will have a dramatically high 260/280 ratio, in that the absorbance at 280 will be a small number. In contrast, a DNA preparation that is heavily contaminated with protein will have a lesser 260/280 ratio.

B.

Chemical Characterization

Acid Hydrolysis
Strong acids at a high temperature are capable of breaking the DNA molecule into its components. These conditions break both of the phosphate ester bonds and also the N-glycosidic bond between the deoxyribose and the purines and pyrimidine bases. The products of this mixture are the 4 bases, phosphoric acid, and deoxyribose which then polymerises to produce a brown sticky tar.

The test for Deoxyribose produced a brownish solution, a positive result for deoxyribose. The test for ribose resulted to a yellow turbid solution which was also a positive result for ribose test. The test for phosphate gave a solution which is slightly turbid with few yellow precipitate, this was also a positive result for phosphate. The test for Purines formed a yellow precipitate which was also a positive result for the test for purines. The Test for Pyrimidines and Uracil produced a white precipitate and there was change of color in the Litmus paper. The test for Pyrimidines and Uracil yielded negative results. The source of error is the use of incorrect or wrong reagents during the experiment.

REFERENCES: BOOKS Bettelheim,F.A., March,J. (1990). Introduction to organic and biochemistry. Philadelphia: Saunders College. Lehninger, A.L. (2008). Legninger Principles of Biochemistry. New York: W.H. Freeman. McKee. (2003). Biochemistry: The Molecular Basis of Life. Boston: McGraw-Hill.

Table 2 Actual results for the chemical characterization of DNA

Chemical Test Test for Deoxyribose Test for Ribose Test for Phosphate Description (Std.) Two layered liquid: black brown layer Blue green colored solution Yellow crystalline precipitate DNA from Onion Brownish solution Yellow turbid solution Turbid solution with yellow precipitate Yellow precipitate White precipitate / Litmus paper no change of color (-) White precipitate / Litmus paper no change of color (-)

WEBSITES: DNA Process Model http://promotingresearch.com/blog/2008/0 2/28/dna-process-model/ Methods of DNA Isolation. http://www.genome.ou.edu/protocol_book/ protocol_partIII.html#III Retrieved: February 8, 2010 Medpedia http://wiki.medpedia.com/DNA Nucleic Acid http://en.wikipedia.org/wiki/Nucleic_acid Enzymatic Synthesis of Deoxyribonucleic Acid http://www.jbc.org/content/244/11/3029.fu ll.pdf+html?sid=db938064-fe02-4399ad92-4fda354e9ae4 Retrieved: February 8, 2010 Evaluation of Deoxyribonucleic Acid (DNA) Isolated from Human Bloodstains Exposed

Test for Purines Test for Pyrimidines

Red precipitate turned brown Red litmus paper turned into blue; Purple precipitate Red litmus paper turned into blue; Purple precipitate

Test for Uracil

to Ultraviolet Light, Heat, Humidity, and Soil Contamination http://www.nsftc.org Retrieved: February 8, 2010