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Biochemistry Laboratory (2012 2013) 2A-BC Group 3 Experiment 4

Isolation, Acid Hydrolysis and Qualitative Color Reaction of DNA from Onion.
Kenneth Dy, Julius Gabriel Fondevilla, Mark Ledge Gerona, Erick Guerrero, Danniel Ileto Department of Biochemistry, Faculty of Pharmacy University of Santo Tomas, Espana Street, Manila 1008
Date Submitted: February 11, 2013

DNA, also known as deoxyribonucleic acid is one of the essential biochemical molecules of an organism that serves as the repository of genes. The objectives of the experiment are to isolate DNA from the onion sample, to subject it to acid hydrolysis and to perform qualitative color reactions. DNA was isolated from onion by disrupting its cell membrane and other cellular components. From the experiment, the isolate was found to have a thread- like structure.. Spectroscopy was applied to test the purity of the DNA sample. In the experiment, the DNA isolate had an absorbance of .607/ .282 which indicates that the isolate was pure. The sample was subjected to acid hydrolysis and qualitative color reaction test that includes test for deoxyribose, test for phosphate, test for pyrimidine and test for purines. In the experiment, the hydrolysate produced dark brown and colorless layer which is a positive result for test for deoxyribose,, clear colorless solution with yellow precipitate for test for phosphate which is also a positive result, and yellow residue for test for purine which indicates the presence of adenine however it produced a white cloudy solution for test for pyrimidines which is a negative result.

Introduction Nucleic Acid is one of the essential biochemical molecules present in an organism. It functions for endcoding, transmitting, and expressing the genetic information. It has two types namely the DNA (deoxyribonucleic acid) and RNA (ribonucleic acid). DNA was the main focused on the experiment.

Nucleic Acid contain a nitrogenous base (purine or pyrimidine) covalently bonded to a 5- carbon sugar (ribose) on 1 and the sugar is covalently bonded to phosphate groups on their 5. Nitrogenous bases can be purine or pyrimidine. Purines are basically Adenine and Guanine. They covalently bond with sugars on N9. Pyrimidines on the other hand are Cytosine, Guanine, Uracil ( for RNA) and deoxythymine ( for DNA). They covalently bond with sugars on N7.

DNA (deoxyribonucleic acid) on the other hand contains the genetic information of an organism specifically; sequences. It is also capable of replication which means that it can make copies which used for reproduction purposes. Figure 1. Structure of nucleic acid

Figure 2. Structure of DNA DNA structure differs from RNAs in many ways. Firstly, DNA is double stranded which means that it has two strands that goes into antiparallel direction while RNA has only one strand. Second is that RNAs sugar contains an OH bonded to its 2 C while DNA has only H. Third is that in complementary base pairing, in DNA, Adenine bonds with Thymine while in RNA, Adenine bonds with Uracil. The objectives of the study are to [1] isolate DNA from Onion and perform purity test using UV spectroscopy, [2] subject the isolate to acid hydrolysis, and [3[ to do qualitative color reaction test to the hydrolysate namely, test for deoxyribose, phosphate, purines, and pyrimidines. Methodology A. Compounds/ Sample used

was subjected to water bath until it reached 60 degree- celcius. 25g of minced onion was added on the pre heated homogenizing solution and was stirred and let stood in a water bath for 5 minutes. 1.5 g. of papain was added and was kept again in water bath with 60 degree celcius for another ten minutes. After the water bath, the flask was placed in an ice bath immediately and was swirled to impede the breakdown of DNA by deoxyribouncleases. After the ice bath, the contents of the flask was placed in a blender and was homogenized for 45 seconds. The homogenatae was the filtered using a 4 layered cheesecloth into a 250 mL- beaker and was cooled off using ice. 15 mL. of ice cold ethanol was added by dripping it down to the side of the tube so that it would produce a clear layer on the top of the ethanol onion filtrate. After doing this, DNA was precipitated because all of the contents of the onion filtrate are soluble to ethanol while DNA is not. The solution was let stand for 5 minutes. Bubbles was formed and DNA was precipitated out of the solution. It became as visible thread-like structure in the ethanol layer. DNA was then spool and was transferred in to a clean test tube. 2. UV spectroscopy The DNA isolate was resuspended in a TE buffer and was subjected to UV spectroscopy to get its absorbance and to test its purity. 3. ACID HYDROLYSIS

Onion, homogenizing solution, papin ( or meat tenderizer solution), ice cold ethanol, TE buffer, diphenylamine, conc. H2SO4, conc. HNO3, 10% (NH4)2MoO4 solution, 10 % KOH, Bromine water, Ba(OH)2 solution. B. Procedure 1. Isolation of DNA from onion 50 mL of homogenizing solution was placed in a 125- mL Erlenmeyer flask nd

5 drops of the DNA isolate was mixed with 1mL of 1M HCL. The mixture was heated at 100 degree- celcius for 60 minutes with marbles as its tubes cover. After 60 minutes, the tubes were cooled off in a running water and was neutralized with 1M KOH. The pH was adjusted to pH 4 with glacial acetic acid using pH paper. The sample was then centrifuge and decant into clean test tubes for qualitative color reaction tests.

4. Qualitative Color Reaction Tests Test for deoxyribose: 3.5 phenylamine was added to 1.5 mL of the hydrolyzed DNA solution and was placed in a boiling water bath for ten minutes. After the water bath, the tube was cooled off and the result was observed. Test for phosphate: 1 mL of conc. H2SO4 was added to mL of nucleic acid solution and was heated in a small flame and was mixed until the contents turned brown. The mixture was cooled off and was added with 0.5 mL of conc. HNO3 and was heated again until white fumes appeared and it turned colorless. 1 mL of water was added to the colorless liquid and was heated in a boiling water bath. After water bath, the tube was cooled off and 1 mL of 10% (NH4)2MoO4 solution was added. The solution was mixed well and was diluted with 10 mL of water. The solution was let stood for 5 minutes and the formation of precipitate was take note. Test for Purines (Murexide test): 10 drops of nucleic acid was placed into a small evaporating dish and was added with 5 drops of conc. HNO3 . The solution was evaporated using water bath until it became dry. The residues was moistened with 10 % KOH and was heated again. The change in the color upon addition of KOH and heating was take note. Few drops of water was added and was placed in a water bath. The solutions color was observed and was then again evaporated. The result was observed. Test for Pyrimidines ( WheelerJohnson Test) : 0.5 mL of the

nucleic acid solution was treated with excess bromine wate until the solution turned yellow. The excess was removed by boiling it until it turned light yellow or colorless. Excess Ba(OH)2 was added and was tested with litmus paper. The color was noted. Results and Discussion A. Isolation DNA from onion After homogenizing the sample and after putting ice cold ethanol, a thread- like structure was formed in the ethanol layer. This was basically the DNA isolate. Bases from the mechanism, the onion filtrate contains the DNA and other impurities. Other impurities are soluble to ethanol while DNA is not. Ethanol was added to mixed with the other impurities and to precipitate out a pure DNA. B. UV spectroscopy Table 1. Isolation and UV measurement DN A Sam ple Physic al descri ption UV measur ement (260/280 ) Protein concent ration (mg/mL) Nucleic acid concentr ation (microgr am/mL 22 ug/mL

Onio Thread .607/.28 n - like 2 DNA (fibrous )

0.07 mg/mL

The DNA isolate was resuspended to TE buffer to test its purity through UV spectroscopy. This method will give its absorbance that will be used to compare to the standard absorption of the DNA to test its purity. A positive result of the isolate should show an absorbance ration ( 260/280) of 1.8 which indicates a pure DNA isolate but if it iss less than 1.8, it contains protein contamination. In the

experiment, the samples absorbance was .607/.282. It indicates that the sample is not less than 1.8 so the sample was pure DNA with ver minimum amount of protein contamination. After getting the absorbance, the protein concentration was plotted and was found out with 0.07 mg/mL. The nucleic acid concentrations was plotted too and was found out to contain 22 microgram/ mL which indicates a good isolate. C. Acid Hydrolysis and Qualitative color reaction tests. Table 2. Qualitative color reaction test results Chemical test Standard DNA from onion Black Upper brown w/ layer; yellow dark layers brown, lower layerclear colorless Yellow Clear ppt. colorless with yellow ppt. Purple White soln, red- cloudy blue solution litmus paper Adenine= Yellow yellow residue Guanine= red layer Figure 3. Test for deoxyribose reaction mechanism Test for deoxyribose: The reaction between the dische reagent and 2deoxypentose results in the development of a blue color. The reaction depends on the conversion of the pentose to hydroxylaevunilic aldehyde which then reacts with diphenylamineto give a blue colored complex. The intensity of the blue color is proportional to the concentration of the DNA. The sample produced a positive result.

Test for deoxyribose

Test for phosphate

Test for Pyrimidines

Test for phosphate: Ammonium molybdate reacted woth the sample wich yields yellow crystals forming phosphoammonium molybdate which is a positive result. Test for purines: DNA reacted with nitric acid since purines is known to be readily soluble in diluted acid. Nitric acid oxidized it leaving a yellow precipitate. The sample produced a yellow precipitate which indicates a positive result. Test for Pyrimidines: Bromine water reacted with the sample to form 5- bromo6- hydroxyhydroxo derivative which provides a green coloration. Upon addition of Ba(OH)2 will give a result of purple precipitate. The sample produced a white cloudy solution which is a negative result.

Test for Purines

References [1] Boyer, R. (1993). Modern Experimental Biochemistry. 2nd ed. The Benjamin/ Cummings Publishing Company Inc.

[2] Ryden, L. (1998). Nucleic Acid Purification. 2nd ed. New York: John Wiley & Sons Inc. [3] Switzer, R. (1999). Experimental Biochemistry. New York, U.S.A.: WH Freeman and Company. [4] Tijsen, P. (1993).Laboratory Techniques in Biochemistry and Molecular Biology. Amsterdam, Netherlands: Elsevier Science Publishers B.V. [5] Walker, J. (1994). DNA Protocols. Totowa, New Jersey: Humana Press Inc. [6] Wilson, K. (1987). A Biologists Guide to Principles and Techniques of Practical Biochemistry. 3rd ed. Bedfood square, London: Edward Arnold (Publishers) LTD. [7] Dischei test- Qualitative Analysis of deoxyribose http://www.protocolpedia.com/sobi2.html?so bi2Task=sobi2Details&catid=4&sobi2Id=174 1 [8] Qualitative Testing for Nucleic Acids http://labopslton.wikispaces.com/file/view/Q ualitative+Testing+for=Nucleic Acid+%26+Proteins.pdf [9] Nucleic Acid Tests. http://forum.daffodilvarsity.edu.bd/index.php ?topic=9968.0