STUDY OF NUCLEIC ACIDS ISOLATED FROM BIOLOGICAL OBJECTS

Аннотация
INTRODUCTION B 4
L
1. LITERATURE REVIEW 8
1.1. RESEARCH BACKGROUND ANDOSIGNIFICANCE 8
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1.1.1. THE IMPORTANCE OF BLOODfflNA «EXTRACTION IN THE
BIOMEDICAL FIELD 9
1.1.2. SHORTCOMINGS IN DATA PROCESSING AND ANALYSIS IN
EXISTING RESEARCH 10
1.2. THE PRINCIPLE OF EXTRACTING DNA FROM BLOOD 12
1.2.1. THE CORE PRINCIPLE OF CHLOROPHENOL METHOD FOR
EXTRACTING DNA FROM BLOOD 12
1.2.2. INTRODUCTION TO THE PRINCIPLES OF OTHER METHODS FOR
EXTRACTING DNA 15
1.3. THE COMPOSITION OF BLOOD AND THE DNA CONTENT IN BLOOD
17
1.3.1. COMPOSITION OF BLOOD 17
1.3.2 FORMS OF DNA IN BLOOD 19
2. EXPERIMENTAL SECTION 22
2.1. MATERIALS 23
2.2. METHODS 23
2.2.1. EXTRACTING DNA FROM BLOOD BY PHENOL-CHLOROFORM
METHOD 24
2.2.2 THE EXTRACTING DNA BY COLUMN METHOD 28
2.2.3. METHOD OF SPECTROPHOTOMETRY 30
2.2.4. STATISTICS 33
3. RESULTS AND DISCUSSION 35
CONCLUSION 45
REFERENCES 46

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Perhaps the most common method in biology is the extraction of nucleic acids. The result of a multi-stage experiment often depends on this first step. The employees of laboratories of different profiles are united by the need to work with nucleic acids. For example, a geneticist from a medical clinic will need to extract DNA from blood cells to conduct testing for a certain syndrome. Whatever the goal of each researcher, the first stage of work will always be one of the most routine - the isolation of nucleic acids. Extraction of DNA is crucial for the success of many experiments. The result of subsequent stages of work - for example, PCR - will depend on the purity and yield of the nucleic acid. It requires DNA of excellent quality: the better, the higher the probability of obtaining a reliable result. The goals of experiments may vary, but all researchers will likely prefer those methods that: they are specific and suit the characteristics of the object from whose tissue the extraction is carried out; time-saving; simple and do not require highly specialized equipment; includes the use of safe reagents; can be modified to suit the methods that will be used in future work; maximum protection from contamination. But time passed, new knowledge about the functions and significance of DNA and RNA appeared - and, of course, other methods of working with them. All together became the reason for the need to optimize the extraction: reducing the time for extraction, increasing the yield and quality of nucleic acids. Scientists around the world began experiments to change the stages of the original method and in more than 150 years have made significant progress. In parallel with approaches to the extraction of nucleic acids, other areas of the molecular biology arsenal, such as sequencing, PCR and electrophoresis, were also developing. It became increasingly clear that pure source material was needed for high-quality results, and the evolution of nucleic acid extraction methods continued. The first methods did not fade into the shadows, they were modified, improved and only for some specific tasks are inferior to more modern ones in the purity of the final product and productivity. The phenolchloroform method is the gold standard for DNA extraction. It provides high yield and is relatively inexpensive. Perhaps the only drawback is the toxicity of the reagents (phenol and chloroform) and, accordingly, the need to work in a exhaust cabinet.
Any approach to isolation includes three main points - destruction of cell and nuclear membranes, protein degradation and separation of nucleic acids from the rest of the cellular contents. The gradual emergence and development of PCR, sequencing and other methods of working with nucleic acids led to a natural evolution of approaches to isolation. One way or another, they were aimed at reducing costs and labor costs for isolation, using non-toxic reagents and increasing the quality and quantity of nucleic acid output. The emergence of new methods is a matter of time. Yes, the extraction of nucleic acids is routine for any genetic laboratory. Some methods are already so automated that they practically do not require human participation. But the diversity of biological materials, life forms and research tasks will allow creativity to always remain part of the process. After all, each organism, tissue and organ may require its own unique approach.
In conclusion, the choice of the DNA isolation methodology depends on many factors are based on certain criteria: the task; • primarily selected material; • the amount of time allotted for analysis; • the expected result (the required degree of purity and the concentration of nucleic ki- slot); • the cost of the procedure; • laboratory infrastructure, the presence of trained personnel; further use of the resulting DNA (PCR, synthesis, cloning).
The choice of a specific method for isolating nucleic acids is still a difficult task, on the solution of which it depends on obtaining the correct and reliable results that. The wrong choice of the DNA isolation method or its incorrect implementation can lead either to obtain a contaminated DNA unsuitable for research, or its loss.
Nucleic acids are large biomolecules. They consist of nucleotides, which are monomeric components: 5-carbon sugar, phosphate group and nitrogen base. There are two main classes of nucleic acids - deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). Nucleic acids are chemical compounds that found in nature. They carry information in cells and make up genetic material. These acids are very common in all living organisms on Earth. Nucleic acids are creating, encode and store genetic information in every living cell.
At the birth and throughout life, deoxyribonucleic acid (DNA) can accumulate mutations or changes in its sequence. Mutations can be neutral or pathogenic, that is, to lead to the development of the diseases. Changes in the sequence of DNA can be congenital and acquired. Congenital disorders are transmitted from parents to offspring and lead to the emergence of pathology at birth or at a young age. And acquired mutations, for example, in the case of cancer, occurs in the process of life, lead to a disease at a late age. Detection of DNA disorders is extremely important for making a diagnosis, treatment and prognosis. In the case of a tumor, DNA is released from the tumor cells. In case of congenital pathology, DNA extracted from leukocytes of peripheral blood. There are differences in methods and their modifications of DNA extraction. The fate of the patient may depend on the methods used to extract DNA. To study the causes of the diseases or the search for mutations, it is necessary to destroy cells and extract DNA. Therefore, it is very important to find right method to extract DNA. There are a number of techniques allowing extract DNA (liquid -phase and solid phase methods). The classic method is the release of DNA by phenol-chloroform extraction. This method is considered outdated and is practically not used in practice. With obsolete methods, new ones, such as the absorption method on the columns. In practice and when performing experiments, methods that are not toxic or do not require much time can be used, but there is an opinion that this leads to a loss of quality of DNA. In this paper, we used both methods to compare them. The phenol-chloroform extraction method is used for comparison. Next, the quantity and quality of DNA using the method of spectrophotometry was evaluated. We extracted DNA with two different methods and studied it in a comparative aspect. In our study was used the Nanodrop 2000C spectrophotometer (Thermo Phisher Scientific, USA) to assess the quantity and quality of DNA. The method is based on the property of nucleic acids to absorb light at 260 nm, while proteins absorb light at 280 nm. The peak intensity of 260 nm estimated the concentration of nucleic acid, in accordance with the A260/280 ratio, the presence of proteins was estimated. The presence of helates (ethylenediaminetetraacetic acid) was evaluated in relation to A260/230. We showed that both methods are comparable and can be used in the current work of the different laboratory.

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