The experiment was about the isolation and characterization of DNA. The DNA was isolated from the onion. The mass of the isolated DNA was 15.11 g. The purity of isolated DNA was estimated by calculating the ratio based from the absorbance at 260nm and 280nm resulted to 0.671 meaning more protein was absorbed. Meanwhile in denaturation of DNA, the initial absorbance at 260 nm was 1.304 higher than the absorbance at 260 nm after heating which was 1.095.
Deoxyribonucleic acid (DNA) is the genetic material in humans and all other organisms. DNA isolation is the removal of DNA from the cell which it normally resides. Isolation is the removal of DNA from the cell in which it normally inhabits. (1)
Onions are used since it contains little amount of starch which allows the DNA to be more visible. The filtrate is made up of onions treated with salt, distilled water and detergent collectively called as lysis solution. DNA purification is done by enzymatic degradation of contaminating proteins with ethanol. A spectrophotometer is used in determining the concentration and purity of the proteins. (2)
MATERIALS AND METHODS
Isolation of DNA from Onion
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The peeled onion bulb was chopped and measured homogenized. The sample was placed in a blender added with an ice-cold lysis solution then for 45 seconds at low speed. Meanwhile, the lysis solution used was prepared beforehand by mixing 5.00 ml of liquid detergent, 5.00 ml of 0.500M EDTA, 10.0 ml of 50% Na Cl solution, and 80 ml of distilled water and placed in an ice bath. After homogenizing, the sample was filtered through the cheesecloth and the collected filtrate was placed in a 250-ml beaker. A 10.0 ml of 5% pepsin solution was added to the filtrate and placed on an ice bath for 10 minutes with occasional stirring. Ice cold 30.0 ml of 95% ethanol was pipette to the side of the beaker containing the sample and stand for 10 minutes on ice bath. Once the DNA precipitates appeared at the interface of the solution, the DNA was already ready for isolation. The spooled DNA was transferred immediately to a pre-weighed 100-ml beaker to determine the mass and percent yield of the sample. The isolated DNA was added with 10.0 ml of 95% ethanol then covered with aluminum foil and refrigerated in preparation for the next laboratory procedure.
Characterization of DNA.
Little amount of DNA sample was placed in a test tube added with 1.00 ml of 20% TCA followed by heating the sample for 10 minutes in water bath with 1.00 ml distilled water. A 2.00 ml of diphenylamine solution was added then heat again in a water bath for 10 minutes. The color change was observed and the absorbance of the sample from 400 nm to 700 nm was scanned to determine the wavelength of maximum absorption. Mean while, little amount of the DNA sample was placed in a separate test tube filled with 5.00 ml distilled water and scanned to read the absorbance at 260 nm then at 280 nm. After determining the A260/A280 value, the sample was heated to boil for 5 minutes and read the absorbance adain at 260 nm.
RESULTS AND DISCUSSIONS
The mass of the raw sample gathered from onion is 30.4 g. After homogenization and adding of pepsin solution and ethanol, DNA precipitates were became visible and transferred to another beaker. The isolated DNA measures 23 g.
Mass or raw sample: 30.4 g
Mass of isolated DNA: 15. 11 g
Percent Yield: 49.70%
Characterization of DNA
Reaction with Diphenylamine
Color of formed solution: green
Theoretical Û¸ max:
Absorbance at 250 nm: 1.304
Absorbance at 280 nm: 1.942
A260/ A280 value: 0.671
Denaturation of DNA
Initial absorbance at 260 nm: 1.304
Absorbance at 260 nm after heating: 1.095
Percent increase in absorbance: 8%
Calculation 1. Solution for Percentage Yield Determination
The calculated percentage yield was quite high. However, still some sources of error was done while conducting the experiment, the sample with DNA precipitates was disturbed while transferring the DNA. The accumulated DNA precipitates is enough for the next procedure which is characterization.
Figure 1. Absorbance at 700 nm
Heat denaturation of DNA, causes the double helix structure to unwind and form single stranded DNA. Thus, the bases unstacked and can absorb more light causing an increase after denaturation. But based on the results gathered, the initial absorbance at 260 nm was 1.304 then was decreased after heating which was 1.095. The calculated percent increase in absorbance was 8%. This error is maybe, due to the heating process. The DNA acquired was quite greater and was not totally heated afterwards causing double helix structure not to unwind and form a single stranded DNA.
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The filtrate gathered from this experiment was made of onions and lysis solution. Onion was used in this study due to low starch content, allowing the DNA to be more visible considering the onion as one of the best source of DNA. (4)
The used of lysis solution was to separate the DNA from extra cell components and to keep the location in which the DNA will not be tainted. The NaCL provides NA+ ions that will obstruct the negative charge as of phosphate ends of DNA. Permitting these ends to come nearer so they can precipitate out of a cold solution. The detergent causes the breaking down of the cell membrane by emulsifying the cell proteins and lipids. Also, disrupting the polar connections that collectively holds the cell membrane. The complexes formed with these lipids and proteins causes the precipitate out of solution. Meanwhile, the purpose of EDTA is to chelates metal ions. (5) A Pepsin solution was used for purification via enzymatic degradation.
DNA is polar due to its extremely charged phosphate backbone which makes it soluble in water. Thus DNA is insoluble in ice cold ethanol, as a result when the cold ethanol was added, it causes stable ionic bonds to form and precipitate the DNA.
Heating the sample is the one responsible for the formation of the observed color of DNA with diphenylamine. When the DNA is heated with acid, the 2-deoxyribose is converted to w-hydroxylaevulinic aldehyde, which reacts with the compound diphenylamine. Through this, a blue-colored compound supposed to produce. In our sample the color observed was green possibly because of the DNA concentration.
The ratio of absorptions at 260 nm vs 280 nm is frequently used to evaluate DNA contamination of protein solutions. The nucleic acids, DNA and RNA, absorbs at 260 nm and proteins absorb at 280 nm. Based on the results, the rate ratio of absorptions at 260 nm vs 280 nm is 0.671. Since proteins absorb light at 280 nm, the ratio is low meaning there is a lot of protein absorbed at 280nm.