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ISOLATION AND CHARACTERIZATION OF NUCLEIC ACIDS: DEOXYRIBONUCLEIC ACID (DNA) FROM ONION

Ralph Timothy Maguyon, Majeya Manalastas, Ciro Aloysius Mendoza, Lea Mendoza and Lara Montano Group 5 2E Medical Technology Biochemistry Laboratory

ABSTRACT
The separation of DNA from cells is called isolation. There are various methods and technologies available for the isolation of DNA. In general, all of these methods involve the breaking of the cell's membrane by mixing the cells with detergents, adding of proteases that homogenates the solution and combining with a salt solution to separate protein molecules from DNA. In this experiment, DNA was isolated from Onion. After which the DNAs absorption at A260 was measured against Protein at A280 by using UV Spectrophotometry. A ratio of 0.898 was recorded, which indicated that the DNA sample was protein contaminated. Several chemical characterization tests were also performed on the isolated DNA such as Acid Hydrolysis, Test for Deoxyribose or Dische Test, Test for Phosphate, Test for Purines or Murexide Test and Test for Pyrimidines or Wheeler-Johnson Test. These tests were performed along with standard solutions like Deoxyribose solution, standard adenine or guanine solution and standard cytosine or uracil solution for comparison.

INTRODUCTION
The isolation of DNA is usually one of the first step in many diagnostic processes used to detect bacteria and viruses in the environment as well as diagnosing disease and genetic disorders. [1] DNA isolation can be divided to 4 stages: 1.)Disruption of cell/nuclear membrane 2.)Lysis 3.)Removal of proteins and contaminants 4.)Recovery of DNA Stages 1 and 2 are often combined. While in
some cases, determination the purity of DNA is also performed using a spectroscopic analytical procedure to quantify the concentration of DNA against protein in a solution. Once isolated, the DNA is characterized to be able to determine its components and structure.

detergent which has the ability to break the cell wall and nuclear membrane of the cell. 0.15 M NaCl, 0.15 M Na3C6H5O7 and 0.0001 EDTA) was placed in an Erlenmeyer flask and was heated in a water bath until 60 C. The onion was stirred into the pre-heated homogenizing solution then was placed in a water bath for 5 minutes. 4 mL meat tenderizing solution was then added to the solution. This procedure using meat tenderizing solution cuts the proteins from DNA. The mixture was left to soak in the water bath for 10 more minutes. Immediately after, the flask was placed in an ice bath for 5 minutes, periodically swirled for even cooling. This prevents the breakdown of DNA. The mixture was placed in a blender and was homogenized for 45 seconds. The homogenate was filtrated through 4 layers of cheesecloth. The filtrate was collected and cooled on ice. Ice cold ethanol was added to the solution, to precipitate DNA out. Once a white fibrous precipitate formed, it was snagged from the solution with a Pasteur pipette and was transferred in a testube filled with TE buffer.

EXPERIMENTAL A. Compounds Tested


Onion

B.-Procedure 1. DNA Isolation from Onion .


Onion, the specimen to be used for DNA isolation, was minced and weighted to 25 g. To separate the DNA from proteins, a homogenizing solution was prepared. 50 mL of homogenizing solution (made up of 5% SDS or sodium dodecyl sulphate or sodium lauryl sulfate, a known

2. Ultraviolet Measurement of Isolated DNA


A pinch of DNA was dissolved in TE buffer to thoroughly dilute the mixture, another 0.5 mL of DNA aliquot was introduced to the solution along with 4.5 mL SSC solution. The solution was transferred to a cuvette and its absorabance

along with a blank cuvette was read on a UV-Vis Spectrophotometer on A260 and A280 nanometer wavelength due to the fact that DNA absorbs light best at A260 while protein absorbs light best at A280. To determine the purity of DNA against protein, the absorbance at A260/ A280 was calculated. The concentration of DNA and protein was determined using a monograph as shown in figure 1.

for comparison. Both were placed in a waterbath and their results recorded.

5. Test for Phosphates


1 mL conc H2SO4 was added to 1 mL hydrolysed DNA solution. The same was done to a standard phosphate solution for comparison. Both tubes were placed in a waterbath until the contents turned brown., 0.5 mL of conc. HNO3 and 1 mL of water was added as the solution heated. Once the mixture cooled, 1 mL 10% (NH4)2MoO4 was added. The mixture was diluted with 10 mL water and was let stand for 5 minutes

6. Test for Purines (Murexide Test)


10 drops of DNA hydrolysate was placed in a testube along with 5 drops of conc HNO3. The same was done to a standard adenine solution for comparison. The mixture was evaporated in a water bath then the residues was moistened with 3 drops of 10% KOH and was once again heated. 3 drops of water were added and the solution was evaporated.

7. Test for Johnson Test)

Pyrimidines

(Wheeler-

Figure 1. Monograph of Absorbance Concentration of Protein and DNA.

and

3. Acid Hydrolysis
10 mg of DNA sample was mixed with 1 mL of 1 M HCL. The mixture was heated at 100 C for 30 minutes. The mixture was cooled and neutralized by addition of 1 M KOH its pH tested using litmus paper. After neutralization, the solution was made to be acidic by the addition of glacial acetic acid, once again its pH was tested by litmus paper. 10 mL of distilled water was added then the solution was decanted into a test tube.

Bromine water was added to 3-5 drops of DNA hydrolysate until it turns yellow. The same was done to a standard uracil for comparison. The solution was placed in a water bath until the solution turns colourless. It was turned basic by the addition of Ba(OH)2 solution, the pH tested by litmus paper.

RESULTS AND DISCUSSION

DNA

Physical Description

UV Measurement ( A260/A280)

Concentration Protein (mg/mL ) 0.180 DNAZ (mg /mL) 3

Plant DNA

White fibrous ppt

0.898

4. Test for Deoxyribose (Dische Test)


3.5 mL diphenylamine reagent was added to 1.5 mL hydrolysed DNA solution. The same was done to a 1.5 mL standard deoxyribose solution Figure 2. Table of DNA and UV Measurement results The ratio due to the fact that it is <1.8 indicates that the DNA sample is protein

contaminated. Futher established by the estimated amount of protein concentration is 0.180 there is a considerable amount of protein in the isolated DNA sample.
Chemical Test Test for Deoxyribose DNA from Onion

species and wavelength, is a constant known as the molar absorptivity or extinction coefficient.

REFERENCES:
[1] Overview Of Dna Manipulation, retrieved from:http://www.accessexcellence.org/AE/AEPC/ WWC/1993/overview.php (1-25-2014) [2] DNA Extraction retrieved from: http://serc.carleton.edu/microbelife/research_m ethods/genomics/dnaext.html (1-25-2014) Plasmid DNA Purification, retrieved from: http://www.taq-dna.com/plasmid-dnapurification-_400.html (1-25-2014) DNA Extraction retrieved from: http://learn.genetics.utah.edu/content/labs/extr action/ (1-25-2014) DNA isolation methods retrieved from: http://science.marshall.edu/murraye/links%20fo r%20students/samantha%20qiagin%20method. pdf (1-25-2014) How to Extract DNA from Anything Living retrieved from: http://learn.genetics.utah.edu/content/labs/extr action/howto/ (1-25-2014)

Test for Phosphates

Test for Purines

Test for Pyrimidines

Standard: dark brown solution DNA: clear light brown solution Standard: clear colorless solution DNA: clear colorless solution Standard: clear colorless solution DNA: pale yellow to clear solution Standard: purple solution DNA: clear colorless solution

Figure 3. Results of Chemical Characterization

Test for Deoxyribose Test for Phosphates Test for Purines Test for Pyrimidines

UV Spectrophotometry was based on BeerLamberts Law which relate the absorbance of a solution to the concentration of a particular solute to in a solution, as shown in the formula in Figure 3.

[ ]
Figure 3. Beer-Lambert Law Where A is the measured absorbance, I0 is the intensity of the incident light at a given wavelength, I is the transmitted intensity, L the pathlength through the sample, and c the concentration of the absorbing species. For each

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