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A nucleic acid is a macromolecule composed of chains of monomeric nucleotides. In biochemistry these molecules carry genetic information or form structures within cells. The most common nucleic acids are deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Deoxyribonucleic acid (DNA), is the hereditary material in humans and almost all other organisms. Most DNA is located in the nucleus, but a small amount of DNA can also be found in the mitochondria (mtDNA).
DNA bases pair up with each other, A with T and C with G, to form units called base pairs. Each base is also attached to a sugar molecule and a phosphate molecule. Together, a base, sugar, and phosphate are called a nucleotide. Nucleotides are arranged in two long strands that form a spiral called a double helix.
Ribonucleic acid (RNA) is a biologically important type of molecule that consists of a long chain of nucleotide units. Each nucleotide consists of a nitrogenous base, a ribose sugar, and a phosphate. RNA is single-stranded. RNA nucleotides contain ribose while and has the rather base uracil. There are 3 types of RNA , mainly Messenger RNA(mRNA) , Ribosomal RNA(rRNA) and transfer RNA(tRNA). (refer to Appendix A for more information).
White blood cells or leukocytes , are cells of the immune system involved in defending the body against both infectious disease and foreign materials. Two groups of leukocyte exist, granulocytes and agranulocytes. Granulocytes, have cytoplasm which has a grainy appearance and they have multi-lobed nucleus . Agranulocytes single, large nucleus . (refer to Appendix B for more information.)
Methyl Green Pyronin(MGP) was used for the experiment. Methyl Green has a preferential affinity for Deoxyribonucleic acid , which stains it Green in colour. Pyronin has a preferential affinity for Ribonucleic acid , which stains it Rose red in colour. The higher the intensity of Rose red and green colouration , the higher the concentration of RNA and DNA respectively.
MGP stained the nucleus , making it more visible. Presence of multi-lobed nucleus indicates that it is a granulocyte and presence of single , large nucleus indicates that it is an agranulocyte.
The aims of the experiment was to prepare blood smear , classifying the white blood cell into 2 groups , locating the DNA and RNA in white blood cell and observing the effects of RNase on white blood cell.
The hypothesis was that there were 2 groups of white blood cell found , the slide which was treated with MGP would be stained both rose red and green in colour and the slide which was treated with MGP and RNase would be stained green in colour only.
The results obtained from the experiment was not desirable. These could be due to several factors.
Dryness of the blood sample would affect the concentration of DNA and RNA observed under the microscope. During the air drying process , the samples were not completely dried and was henced placed in solutions such as Carnoy fixative , Ethanol , Distilled water , Methyl Green Pyronin and RNase. The solution would have a high chance of washing the blood sample off from the 2slides.
Hair dryer was used during this experiment to speed up the evaporation rate. However , this speeding up of reaction also caused the inaccuracy of results obtained. Due to the high amount of heat produced by the hair dryer , the cells would not be able to withstand it. This would lead to the cracking of cells.
Instead of using just 95% of ethanol for this experiment to dehydrate the blood sample , increasing percentage of ethanol should be used. This would ensure the blood sample to be much more dehydrated.
Amount of Strength used when smearing the blood could affect the concentration of DNA and RNA observed under the microscope. If too much strength was used during the blood smearing process , the cell could be broken apart. The cell contents would be flowing out from the cell , causing the inaccurate amount of DNA and RNA observed.
Theoritically , xylene could make the background of the microscopic slides transparent. For this experiment , xylene could be used after staining the slides with Methyl Green Pyronin. The colour of stain would be intensified greatly , making it easier to be observed under the microscope.
Comparing Hypothesis and Results , it is exactly the same. Thus the hypothesis was supported. The experiment was successful.
Based on the experiment , the slide treated with MGP only was stained Green and Rose-red in colour ,showing the presence of both DNA and RNA respectively. The slide treated with MGP and RNase was stained Green in colour , showing the presence of DNA only. RNA was seen around the cytoplasm due to the Rose-red colouration seen while DNA was seen around the nucleus area due to the green colouration seen. RNA were found in cytoplasm because protein synthesis was synthesized there and different types of RNA such as mRNA , tRNA , rRNA were present. Apart from colour seen , there were 2 groups of white blood cell observed(Figure 3). From Figure 4 , it shows that there were more granulocytes(67%-75%) than agranulocytes(23%-33%).
To improve the accuracy of the results , hair dryer should not be used to speed up the reaction. Instead , the blood sample should be left on the bench to let it evaporate naturally. The dehydration process by using ethanol should be in increasing percentage such as 50% ƒ 70% ƒ 75% ƒ 90% ƒ 95% ƒ 100%.This would ensure the blood sample to be fully dehydrated. Amount of strength used during the smearing process should be controlled to prevent the cells from rupturing. Xylene could be used to intensify the colouration observed under the microscope.
In conclusion , the results observed for staining of DNA and RNA was not very desirable and the results observed for white blood cell was that agranulocytes and granulocytes was found in both RNA and DNA. Location of DNA and RNa was located using MGP ; effects of RNase was known. The results can be improved if the following factors such as dryness of blood sample , amount of strength used and using of xylene were taken into consideration. Results were the same as hypothesis ; aims were met ; the experiment was successful.