Acid Hydrolysis of DNA Isolated from Allium cepa and Analysis of DNA Components Using Qualitative Color Reaction

Test Anselme W. Ang, Kirk Valentin P. Apara, Ma. Cyvigne M. Avila*, Maria Amabelle Christine M. Biene College of Science, Department of Biological Sciences University of Santo Tomas,Espaa, Manila Abstract
The DNA of Allium cepa (onion) was isolated and the absorbance was measured under the wavelengths 260 nm and 280 nm. The absorbance reading was 0.536 and 0.441 respectively. The absorbance ratio was calculated to be 1.26. the DNA was subjected to acid hydrolysis after which was used in various qualitative color reaction test: Dische test for deoxyribose, Test for phosphate, Murexide test for purines and Wheeler-Johnson test for pyrimidines.

INTRODUCTION Nucleic acids are the building blocks of DNA and its molecular cousin, RNA. The DNA is consist of a phosphate group, sugar deoxyribose and nitrogenous base pairsthe purine bases adenine (A) and guanine (G); the pyrimidine bases cytosine (C) and thymine (T). The other pyrimidines are each restricted to specific type of nucleic acid: Uracil (U) can only be found on RNA, while, thymine (T) is restricted to DNA. The bonding of the base pairs is specific. Adenine always bond to thymine (and vice versa) and guanine always bond with cytosine (vice versa). The two strands of DNA hydrogen bond with each other to form a twisted structure. Both chains are right handed, however, each strand has a free 5 hydroxyl group at one end and 3 hydroxyl group on the other end that gives each strand polarity. The polarity of each strand is in opposite directions, thus, the DNA is described as anti-parallel structure. The process of extracting DNA is to separate DNA from the unwanted substances of the cell gently enough so that the DNA is not broken up. This means that DNA extraction is the removal of deoxyribonucleic acid (DNA) from the cells or viruses in which it normally resides. (Rice, 2010). The objectives of this experiment is to isolate DNA from onion, to determine the concentration and purity of the DNA, and to analyze the DNA components after acid hydrolysis using various chemical tests. MATERIALS AND METHOD PRELIMNARIES The tip of a pipet was heated in a Bunsen flame until it the end can be bend back up towards the top forming a hook. This was used to spool and hook the DNA. 50mL of 95% ethanol was cooled in an ice bath.50mL of the homogenizing solution was placed into a 250-mL Erlenmeyer flask and was heated in a water bath until the solution reaches 60C. The first few layers of the onion was removed. The onion was minced and 25g of the minced onion was weighed. The onion was added to the homogenizing solution. The solution was stirred and was placed in the water bath for 5 minutes with occasional stirring. 1.5g of crude papain powder was added and was kept in the water bath for another 10 minutes and was stirred every 2 minutes. The flask was immediately transferred into an ice bath for 5 minutes and it was swirled gently to allow even cooling. The contents were quickly poured into the blender and was blended for 45 seconds. The homogenate was filtered through 4 layers of cheesecloth into a clean 100-mL graduated cylinder. The volume of the filtrate was measured. The filtrate was transferred to a 250-mL beaker and was cooled on ice. The beaker was tilted into a 45 angle and the ice-cold 95% ethanol (2x of the homogenate volume) was poured gently and slowly down the inner walls of beaker. The mixture was allowed to sit undisturbed for 2-3 minutes until the bubbling stops. The DNA spooler was placed into the beaker so that the end of it was just below the upper layer of the liquid. The spooler was twirled in and out of the two layers in one direction gently and quickly. The precipitate was air dried. The air-dried precipitate was weighed.

B. Determination of DNA concentration and Purity 1.0mg of DNA was dissolved in 3.3mL of SSC solution. Its absorbance was read at 260nm and at 280nm. The protein concentration and nucleic acid concentration was determined in the DNA solution. the absorbance ratio of the DNA solution was calculated. From this ratio, the purity of the isolated DNA was rationalized. C. Acid Hydrolysis of DNA The rest of the isolated DNA with 1.0mL of 1M HCl was mixed in a medium-sized test tube. The test tube was covered with a marble and the mixture was heated at 100C for 60 minutes with occasional agitation. The test tube was removed from the water bath and was cooled at room temperature. 2.5mL of distilled water was added and the hydrolyzate was neutralized with NaOH. The hydrolyzate was filtered and 3mL of the filtered hydrolyzate was made up with distilled water. D. Chemical Characterization of DNA D.1 Test for Deoxyribose or Dische Reaction 1.5mL of diphenylamine reagent was added to 0.5mL of the DNA hydrolyzate. The solution was heated for exactly 10 minutes in a boiling water bath. The heated solution was cooled immediately. The procedure was repeated for D.2 Test for Phosphate 1mL of the concentrated H2SO4 was added to 1mL of the DNA hydrolyzate. 0.5mL of concentrated HNO3 was added the solution. 1mL of distilled water was added and the solution was heated in a boiling water bath for 5 minutes. The solution was allowed to cool and then 1mL of ammonium molybdate solution was added. The solution was mixed and was diluted to 10mL water. The color of the solution and the precipitate formed was observed. The procedure was repeated for the standard phosphate solution. D.3 Test for Purines or Murexide Test A little amount of DNA hydrolyzate was placed in a small evaporating dish. A few drops of concentrated nitric acid was added and the solution was evaporated to dryness very carefully on a water bath in the fume hood. The yellow residue turned to red upon moistening with 10% KOH. A few drops of water is added. The same procedure was performed using the standard guanine solution. D.4 Test for Pyrimidines or Wheeler-Johnson Test 0.5mL of DNA hydrolyzate was treated with an excess of bromine water until the solution turned to yellow. The excess bromine was removed by boiling the solution until the solution turned to yellow or colorless. Barium hydroxide was added in excess (tested with litmus paper). The procedure was repeated for the standard cytosine solution. RESULTS AND DISCUSSION A. Isolation of DNA from Onion The homogenizing solution was used because it destroys the cell and nuclear membranes of the onion cells. There are four important components of the homogenizing solution: Sodium Dodecyl Sulfate (SDS) is an anionic biological detergent that breakdown and emulsifies the lipid and protein components of the cell; Sodium citrate is the chelating agent that causes cellular debris to precipitate for easy filtration; Ethylenediamine tetracetic acid (EDTA) is another chelating reagent that binds to Mg which is needed for DNAse activity; and Sodium chloride that helps the DNA to precipitate out of the solution and shields the negative charges of the phosphate group in the DNA backbone.

Heating the onion soften the lipid in the cell membrane and denatures the DNAse enzymes which cuts the DNA into small pieces so that it will not be spooled. The next step is the adding of papain powder in the homogenized solution. This initiates the protein denaturation of because the protease enzyme (papain) breaks the peptide bond attached to the DNA. During the deproteination process, the temperature was kept at 60C because the maximum activity of the enzyme papain occurs at this temperature and the DNA will be degraded beyond this temperature (up to 75C). Blending the homogenized solution further breaks down the cell membranes. The solution was blended for 45 seconds only to prevent further destruction of DNA molecules. After blending, there is a need to filter the homogenized solution with four-layer cheesecloth so that the soluble DNA pass through while the precipitated cell debris will be left in the cheesecloth. The filtrate was cooled to slowdown the degradation of the DNA and it was swirled to allow even cooling of the solution. Ice-cold 95% ethanol was added to precipitate the DNA and to keep the other components of the filtrate to stay in the solution.

B. Determination of DNA Concentration and Purity

Absorbance reading 260nm 0.536

Absorbance reading 280nm 0.441

Absorbance 260nm/280nm 1.215

ratio Protein Concentration (mg/mL)

Nucleic Concentration (g/mL)

Acid

Table 1.1 Data for the determination of DNA concentration and Purity

The measurement of the absorbance determines the DNA concentration and the purity of the sample. At 260nm, the DNA absorbs light the strongest so the absorbance value at this wavelength was used to estimate the DNA concentration. Also, in this wavelength purines and pyrimidines are detected. The absorbance at 280nm was used as an indicator of protein contamination. The presence of aromatic amino acids such as tyrosine, tryptophan and phenylalanine is detected on this wavelength. The ratio of absorbance was calculated using the formula:

The calculated ratio of absorbance is 1.215. Ratio showed that the sample is contaminated by protein because it is lower than the expected range 1.7-2.0 which indicates a good sample. Computed values higher than the expected range is RNA contaminated. C. Acid Hydrolysis of DNA The addition of 1M HCl and heating the homogenate to 100C breaks down the double helix structure of DNA. Strong acids (such as HCl) at high temperature break down phosphodiester bonds that separate phosphate group from deoxyribose group and nitrogenous base complex; -N-glycosidic bonds that separate nitrogenous bases from deoxyribose sugar; and hydrogen bonds that separate nitrogenous base pairs. The acidified homogenate was neutralized with 1M NaOH because the acidic condition affects the result of the characterization tests.

D. Chemical Characterization of DNA D.1 Dische Reaction (test for deoxyribose)

Deoxyribose Standard Solution Before heating After heating
TABLE 1.2 Results for the Dische reaction

DNA Hydrolyzate Clear colorless solution Clear light blue solution

Clear Solution Dark blue solution

The positive result for this test is a blue colored solution.The reaction depends on the conversion of the pentose to whydroxylaevunilic aldehyde which then reacts with diphenylamine to give a blue-colored complex.

FIGURE 1. Dische Test Reaction

D.2 Test for Phosphates DNA Hydrolyzate Light yellow precipitate with yellow precipitate
TABLE 1.3 Results for the test for Phosphates

Phosphate Standard Solution Light yellow solution on top with yellow precipitate

The observable yellow precipitate which is the phosphoammonium molybdate was formed when the ammonium molybdate formed a complex with phosphate.

D.3 Murexide Test DNA Hydrolyzate Yellow residue

TABLE 1.4 Results for Murexide Test

Guanine Standard Solution Red residue

A positive result for this test is the formation of red precipitate. The condensation of purine by concentrated formeda dialuric acid and alloxan which is the yellow residue. The condensation of alloxan leads to the formation of alloxanthine which reacted with 10% KOH that formed murexide, the observable red precipitate. D.4 Wheeler-Johnson Test DNA Hydrolyzate yellowish solution with white precipitate
TABLE 1.5 Wheeler-Johnson Test results

Cytosine Standard Solution Light purple solution

A positive result for this test is the formation of purple color. The bromination of pyrimidine at C5 produced dibromohydroxyuracil which is the observable yellow color in the solution. The addition of Ba(OH)2 caused the alkaline hydrolysis that produced isodialuric acid. The rearrangement of isodialuric acid dialuric acid and its barium salt which is the purple precipitate. CONCLUSION Certain measures have to be taken to completely isolate the DNA from onion. The purity of the isolate can be determined by measuring the absorbance of the sample solution at 260nm for its DNA concentration and 280nm for protein absorbance. Various test can be used to determine the different presence of DNA molecule in the sample.

Rice, G. (2010, August 27). DNA Extraction . Retrieved August 1, 2010, fromMicrobial Resources:http://serc.carleton.edu/microbelife/research_methods/genomics/dnaext.html3. Boyer, R. (2006). C oncepts In Biochemistry (3rd ed). John Wiley (Asia): Wiley Asia S tudent Edition4. DNA and Gene . (2008, June 30). Retrieved August 3, 2011, from Biotechnology O nline:http://www.biotechnologyonline.gov.au/biotechnologyonline/biotec/dnagenes.htm

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