DNA purification is a process that can be done by different methods to extract a DNA from any living organism. The aim of this experiment was to purify a DNA from kiwi fruit, and once it’s extracted it can be used for molecular analyses, sequencing and fingerprinting. The main concept of purification a DNA is to isolate it from the cell and separate it from any attached contaminating compound, this happens under a certain steps which is all refer to the same idea. The extracted DNA from the kiwi fruit was formed a white precipitate of DNA, which is agree with the literature review.
Introduction
Deoxyribonucleic Acid (DNA), is the fundamental genetic material in all living organisms that stores instructions of growth and development needed for an organism.
DNA is made up of two polynucleotide chains joining together by a hydrogen bond, This Polynucleotide made up of lots of monomers (small units) called nucleotide which is a type of biological molecules that contains a phosphate group, pentose sugar group (deoxyribose) and a nitrogen base that can vary, the sequence of these bases determines the genetic code.
The DNA found within both eukaryotic and prokaryotic cells. in the eukaryotic cells the DNA organized into protein-DNA complexes (chromosomes) inside the nucleus that located in the central region of the cell and surrounded by organelles that carry out a variety of tasks, and finally a cell membrane that surrounds a cell and made up of double layer of lipids and proteins.
DNA Purification is the process of extracting the DNA from the cell, it begins with the separation of DNA from membrane, proteins and the rest of cellular material. this is done by removing any contaminating compounds using a saturated salt solution such as sodium chloride, this protein salting-out method is safe, fast and inexpensive and produce high quality of DNA.
NaCl do a balance between the charges and prevents the DNA from dissolving in water. in a solution, the NaCl breaks up into Na+ so this positively charge neutralize the negative Po3– on the nucleic acids. Leading the DNA to much less soluble in water.
In order to release a DNA within a nucleus, the cell membrane that surrounds the nucleus must be broken down. This can be done using the detergent to breakdown the cell membrane since it made up of fats and lipids in it is structure.
Once the DNA released outside the membrane, the DNA is no more protected and some enzymes such as DNase can be able to reach the DNA and destroy it, which is something we wish to avoid, so how to prevent this from happening? Basically DNase is an enzyme present in the cytoplasm that catches any viruses exist into the cell and digest it by destroying its DNA. Any enzyme has it’s optimum temperature, if the temperature continues in increasing after this certain point, the enzyme loose their shape, leads the enzyme to be denatured.
Proteins are a long chains of small units called amino acids joining together by a peptide bond. Protease can break down this bond and release small amino acids. This process exactly needed to separate proteins called histones that attached to the DNA.
Now the DNA is released from any contaminating compounds, but it still can’t be visible. This can be solved by a very simple step which is adding alcohol.
How does alcohol work in purification? First to know is DNA soluble in water with the net of negative charge of (PO3-) within the backbone of nucleotides. As mentioned above in the use of NaCl, the positive side in Na interacts with negative side in DNA. But because the polarity and high dielectric constant in water, it hinders in binding between NA+ and PO3-. The Role of alcohol comes here when the OH- within alcohol interacts with the H+ in water. Making everything neutralized, then DNA can be seen as a white precipitate on top of DNA-detergent mixture.
This DNA sample can be analyzed in order to allow scientists to study the genetic causes of diseases and can be used for further investigation.
Materials & methods
You will need a kiwi fruit( onion or tomato will do if kiwi isn’t available), a 10 ul protease enzyme, 2mls ethanol and a 50 ml solution of detergent(washing up liquid or hand soap will do), salt( sodium chloride) and distilled water. Prepare a water bath at 60C degree and an ice bath.
The kiwi cells were broken by a blender for 10 minutes,The broken-up cells were added to a beaker containing 50 detergent solution in order to breaks down the cell membranes, and the salt binds to the DNA causes it to clump,Then this beaker were incubated in a water bath at 60 C degree for 15 minutes, in this step all the DNase enzymes was destroyed. After incubated, the beaker were putted in an ice bath to cool the mixture down for 5 minutes. After it’s cooled, in a flask, the mixture were filtered using a gauze and a funnel. Then, 4mls of the filtered sample was transferred to a clean boiling tube & the contents of the filter paper was discarded. In the boiling tube, 10 ul of protease enzyme were added to the filtered mixture. Note to do this step after you have incubate the beaker in a water bath at 60 degree, if you add the protease first, the enzyme is denatured due to the high temperature. After the protease were added, in an ice-bath, 2mls of cold ethanol were slowly dribble down the side of the tube, it forms a layer on top of the DNA-detergent mixture. The cold ethanol helps the DNA to precipitate more quickly, it must be added slowly, allow for the reaction to subside after each addition.
Discussion
The aim of this experience was to purify a DNA from kiwi fruit in order to be able to see visible strands of DNA, it has been found that the DNA precipitate on Top of the tube near to alcohol, as expected because DNA cannot be dissolve in a non-polar such as alcohol.
The DNA observation was with a fairly amount of strands, as the expected amount of DNA to be purified from the sample depends on the amount of DNA in particular fruit and the number of cells in the fruit as well as the downtown stream application.If theres enough DNA in the sample A stringy white mass will be observe.
In addition, the DNA observation were not able to see in any of the steps, unless alcohol were added to the mixture.
There could have been problems with the experiment due to human error, it could be caused by the water bath being not hot enough to damage the DNase enzyme, or another problem could have by the protease enzyme being added before incubate the beaker in a water bath with high temperature, this cause the protease to denature. If any of these suggestions were a problem in this experiment, the experiment could be repeated, to make sure of the accuracy of the results.
Further experiments could be carried out to purify DNA from other fruits, and with different detergents in order to distinguish between products in which is more readily dissolves the lipids within the cell and nuclear membrane. Also, with a variation in the amount and timing of The salt added. As shown from the results, the aim of this experiment was successfully achieved.
References
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