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Optimization of DNA Extraction from Medically and Environmentally important Fungi for Polymerase Chain Reaction

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Introduction

Qualification BTEC Higher National Diploma in Biomedical Science Level 5 Module Name Project Module Number Module 6 Title of practical Optimization of DNA extraction from medically and environmentally important fungi for polymerase chain reaction Name of Candidate Thevaraja Nirojith Optimization of DNA Extraction from Medically and Environmentally important Fungi for Polymerase Chain Reaction Investigator:Mr.ThevarajaNirojith Principle supervisors - Prof. R .S. Dassanayake Dr. O. V. D. J. Weerasena Co-supervisor – Mr. Mohan Geekiyanage Durdans Molecular Diagnostic Laboratory Durdans Hospital, 3, Alfred Place, Colombo 03. Declaration: I ………………………………………………. confirm that I have read and understood the Institute regulations concerning plagiarism and that the work contained within this project report is my own work within the meaning of the regulations. Signature: Date: Acknowledgment My sincere thanks to Prof. R. S. Dassanayake, Dr. O. V. D. J. Weerasena, (ceygenbiotecdurdans hospital) for granting me a place to do my research. My thanks to Mr. MohanGeekiyanage, Miss. Pushpamali Silva they helped in each and every practical. Finally my thanks to Dr.Sajani( lecture of BMS), Mr. NisamRasak ( Director of BMS), for helping me in finding a research project. Abstract Fungi are eukaryotes that have cell walls composed of chitin with or without cellulose. They are ubiquitous and comprise an estimated 250,000 species, of which only about 150 have been shown to cause disease in humans. PCR-based fungus speciï¬c protocols have been worked out for the medically and environmentally important fungi responsible for the detection using DNA extraction. Following PCR procedures, gel loading was done to observe the band. Spectrophotometric calculation was also carried out to find the amount of extracted quantity of DNA. Introduction Fungi are organisms which are neither plants, nor animals. They are one of the most important group of organisms on this planet, some of the world's largest and possibly oldest individuals, silent killers with deadly poisons, vital ingredient in beer and bread, decomposers, essential for natural recycling, helping to guarantee life on earth, miracle cures for disease and indispensible partners for many plants. ...read more.

Middle

Then the nuclei of both cell fuses together and this cell is now the zygote. These diploid cells can go through mitosis, which they call budding, and four more zygotes or they can undergo meiosis and from an ascus which will split into four ascospores. These haploids can then undergo germination and become haploid yeast again. (Madigan, 457) Saccharomyces cerevisiae is one of the most important fungi in the history of the world. This yeast is responsible for the production of ethanol in alcoholic drinks and is the reasons bread dough rises in the pan. That is where the names brewer?s and baker?s yeast come from. The process in which it produces ethanol is one way this yeast converts glucose into energy. There are two ways Saccharomyces cerevisiae breaks down glucose. One way is through aerobic respiration. This process requires the presence of oxygen. When oxygen is not present the yeast will then go through anaerobic fermentation. The net result of this is two ATP, and it also produces two by products; carbon dioxide and ethanol. So if this yeast is allowed to grow in a container lacking oxygen it will produce ethanol (alcohol). Humans have been isolating this process since the beginning of history. The yeast helps in the rising of bread with its other by-product carbon dioxide. The gas that is produce inside the dough causes it to rise and expand. Both of these processes use the haploid of this yeast for this process. In industry they isolate one strain, either a or ά, of the haploid to keep them from undergoing mating. (Madigan, 457) In the baker?s yeast they have a strain were the production of carbon dioxide is more prevalent then ethanol and vice versa for brewing. (Tomvolkfungi.net) Another importance is that ?live yeast supplementation to early lactating dairy goats significantly increased milk production? (Stella, A.V.1). Mushrooms have been recognized as most loved vegetarian food, rich in nutrition, particularly protein. ...read more.

Conclusion

And also destroyed polysaccharide may interfere with extraction and purification of genomic DNA, which subsequently influence DNA restriction, amplification, and cloning. For example; the presence of polysaccharides makes genomic DNA highly viscous with glue like texture that renders it unmanageable in pipetting and unsuitable for PCR by inhibiting Taq polymerase activity. For the future we would suggest further investigation and prevention of contamination of the DNA extraction as a main priority. In here we got band in negative control, so there was a contamination in the practical. The extracted DNA was enough to perform PCR- based reactions and also used in spectrophotometric detection and semi-quantitative determination of the amount of DNA by staining with Ethidium bromide and evaluation on agarose gel. The purity of a solution of DNA can be determined using a comparison of the optical density values of the solution at various wavelengths. For pure DNA, the observed 260/280 nm ratio will be near 1.8. Elevated ratios usually indicate the presence of RNA, which can be tested by running the sample, ~1µg, on an agarose gel. 260/280 ratios below 1.8 often signal the presence of a contaminating protein or phenol. Alternatively, protein or phenol contamination is indicated by 230/260 ratios greater than 0.5. Once you are sure your sample contains pure DNA, an accurate determination of the concentration of DNA can be made. The ratio was not similar as mention above, that have some difference in the amount. In this extraction we did not get proper band in gel picture, but the extracted DNA was confirmed by the spectrophotometric results. Conclusion In this research some of medically and environmentally important fungi DNA extracted. The spectrophotometric results and gel images shows that DNA has extracted. So the extraction method is correct using MycoSpin DTM column and specific buffers. There is no band observed in the post PCR gel image. So we can conclude that the PCR amplification has not happened properly. Therefore we can conclude that there is a problem with PCR primers. However Molecular Method is the most accurate and sensitive test for the identification of fungal diseases. ...read more.

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