Excercise 5 - Nucleic Acids

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1 Excercise 5 - Nucleic Acids Student s name: Date: Points: Assistant s signature: Index numer: Isolation and purification of DNA is a key step in most of the procedures used in molecular biology, diagnostics and biochemistry. The main goal of this stage is to obtain high quality and purity of the biological material, irrespective of the source of its origin. Choosing the right insulation method depends on: the type of nucleic acid we want to obtain (genomic, mitochondrial, plasmid DNA, RNA,etc.) origin (plant, animal, bacterial, viral, etc.), type of material from which we carry out isolation (from tissues, organ, cell culture, etc.), expected results (cleanliness, quality, isolation time, etc.) destination (PCR, cloning, etc.). There are many DNA isolation procedures that result in biologically active DNA, chemically stable and free of RNA and proteins. However, due to the size and sensitivity of the chromosomal DNA it is virtually impossible to isolate its unaltered form. Part of the DNA undergoes mechanical damage. During DNA isolation from a cell the following parameters should be taken into account, conditioning the behavior of the native DNA structure: 1. ph: hydrogen bonds between complementary chains are stable in the environment of ph = 4 10, phosphodiester bonds in the DNA skeleton are stable in the range of ph = 3 12, N-glycosidic bonds with purine bases (adenine and guanine) are hydrolyzed at ph <3. 2. Temperature: there are significant differences in the thermal stability of hydrogen bonds in the double helix, but most of the DNA becomes unraveled at a temperature of C, N-glycoside and phosphodiester bonds are stable up to 100 C. 3. Ionic strength: DNA is more stable and soluble in salt solutions, in the salt concentration lower than 0.1M hydrogen bonds between complementary chains are weakened. 4. Cellular conditions: Before the release of DNA it is necessary to break down the cell wall (fungal cells, bacterial cells and others) and lysis of cellular membranes (animal cells, etc.); the ease of breaking the wall depends on the type of organism, in some cases it is necessary to intense grinding or use ultrasound (cells yeast, tobacco), whereas in others (E. coli) enzymatic hydrolysis of the cell wall is possible. There are several enzymes in the cell, that hydrolyse DNA; the most important of these are deoxyribonucleases that hydrolyze phosphodiester bonds. In addition, native DNA occurs in cells in complex with proteins (histones, helicases, polymerases etc.), these proteins must be separated during extraction. 1

2 5. DNA mechanical resistance: Mild manipulation is not always possible during DNA isolation; grinding, shaking, mixing and other mechanical activities can cause DNA cleavage, usually it does not cause destruction of secondary DNA structure, but reduces the length of the molecule (fragmentation). Evaluation of isolation performance The evaluation of the obtained DNA rely on the spectrophotometric measurement of the amount of acid obtained. The sample should contain about 1 mg of DNA in 1 ml. The measurement is carried out at wavelength λ= 260 nm and λ= 280 nm as reference using PCR water, and then the A260nm / A280nm ratio is determined. Values are normal in the range of 1.6 to 1.8. The values of the ratio A260nm / A280nm <1.6 indicate the contamination of the preparation with proteins, values> on contamination with the reagents used. The DNA content in the solution is determined by the formula: DNA content = A260nm x 50 x R where: A260nm - absorbance at wavelength = 260 nm 50-1 absorbance unit = 50 μg double stranded DNA R - dilution. Excercise 1 Aim: Isolation and DNA composition testing. Principle of the method: The most commonly used material for isolation of DNA is whole peripheral blood. 1 to 5 ml of venous blood is taken To prevent clotting, blood should be collected on EDTA (disodium salt of edetic acid), which chelates both Ca2 + ions (prevents blood clotting) and hemoglobin (DNA polymerase inhibitor) and prevents degradation of DNA caused by the presence of DNAses. The main stages of DNA isolation from peripheral blood are: lysis of cells that do not have nuclei such as erythrocytes and their rejection with serum as unnecessary pollution. collection of leukocytes of interest to us, their lysis, RNAase purification to remove single-stranded nucleic acids, mainly RNA, and deproteinization to remove histone and nonhistone proteins. DNA precipitation with isopropyl alcohol. drying of DNA in the air. hydration of the DNA, i.e. preparation of the sample for analysis or storage. Reagents: solution for lysis of red blood cells white cell lysis solution RNAse A solution 2

3 protein precipitating solution 100 % isopropanol 70% ethanol solution for hydration of DNA Procedure : 1.1. DNA isolation from whole venous blood For DNA isolation, whole venous blood is used taken to EDTA containing tubes to prevent its clumping. Blood should be stored at about 4 o C to prevent haemolysis. DNA is isolated from lymphocyte mass, obtained as a result of blood centrifugation after previous lysis of erythrocytes and removing excess hemolysate. Cell lysis 1. To a Falcon centrifuge tube (15 ml capacity), add 4.5 ml of the Red Blood Cell Lysis Solution, and then add 1.5 ml of blood. 2. The tube should be inverted to mix the contents. 3. The sample should be incubated for 3 minutes at room temperature. 4. After incubation, the sample should be stirred again by inversion. 5. The sample should be centrifuged at 2000 x g for 10 min. 6. After centrifugation, the supernatant should be removed, leaving approximately 100 ul of the supernatant above the white blood cell pellet, and then shake the sample well to disperse the cells in the liquid. 7. Add 1.5 ml of the white cell lysis solution to the tube to disperse the cells and pipet up-down to induce cell lysis. 8. The sample should be incubated at 37 C for 5 min. RNAase purification 1. Add 5 ml of RNAse A Solution to the previously obtained sample and mix the contents for 25 times, rotating the tube. 2. The sample should be incubated at 37 C for 15 minutes. Protein precipitation 1. The sample after incubation should be cooled to room temperature, and then 0.5 ml protein precipitating solution should be added, next shake the sample at high speed for 20 seconds. 2. Then centrifuge the sample at 2000 x g for 10 min. The precipitated proteins form a brown pellet. If it is not clear, shake it again, then incubate on ice for 5 minutes and then again centrifuge the sample. 3

4 DNA precipitation 1. The supernatant containing DNA should be transferred to a clean 15 ml Falcon centrifuge tube containing 1.5 ml of 100% isopropanol and mix by turning the tube 50 times. 2. The sample should be centrifuged at 2000 x g for 3 minutes. The DNA can be seen in the form of a small white pellet. 3. After centrifugation, remove the supernatant and quickly dry the edge of the tube by contact with clean tissue paper filter. 4. Add 1.5 ml of 70% ethanol to the test tube and mix the DNA several times by shaking the test tube. 5. The sample should be centrifuged at 2000 x g for 1 minute and the ethanol removed (Note: the pellet may be loose, so ethanol should be removed slowly). 6. After centrifuging, invert and dry the test tube on clean filter paper and then dry it in the open Falcons, in a heating block at 37 C, for about 10 minutes. DNA hydration 1. Add 250 ul of the Hydration Solution to the dry residue. 2. The sample should be incubated at 65 C for 20 minutes, periodically tapping the tube for faster DNA scattering Conclusions: 1.2. Qualitative determination of deoxyribose Principle of the method: DNA preparation in Dische reagent (H2SO4 concentration, glacial CH3COOH and diphenylamine) at 100 C undergoes hydrolysis and then deoxyribose forms a condensation product with diphenylamine of bluish purple color. 4

5 diphenylamine deoxyribose Hydroxylevuline aldehyde Bluish purple complex compound Procedure: Prepare 2 tubes and proceed as shown in a table Reagents Sample number 1 2 H2O 0.1 ml --- DNA --- 0,1 ml Dische reagent 0,5 ml 0,5 ml Incubation in 100 o C for 5 min Excercise 2 Aim: Detection of RNA components. A ready, appropriately diluted, RNA hydrolyzate is available. The hydrolysis was carried out in 1 M HCl for 1 h 3.1. Detection of purines Principle of the method: Purines are detected in the RNA hydrolyzate adjusted to ph 4, because in this environment sodium hydrogen sulfate (IV) (NaHSO3) (acidic sodium sulfite) reduces Cu2 + to Cu + ions. Newly formed copper ions (I) form insoluble purine salts. This gives an iridescent, yellowish precipitate in the test tube containing the purine. Reagents: RNA hydrolyzate at ph M CuSO4 solution saturated NaHSO3 solution 5

6 Procedure: Prepare 2 test tubes and follow the diagram in the table (the order of reagents addition is important): Reagents Tube number 1 2 H2O 0,5 ml --- RNA hydrolyzate ph ,5 ml 0.25 M CuSO4 solution 0,1 ml 0,1 ml saturated NaHSO3 solution 0,1 ml 0,1 ml Conclusions: 3.2. Detection of pentoses Aim: Detection of ribose according to the Bial method. Principle of the method: Pentoses under the influence of concentrated hydrochloric acid are dehydrogenated into furfural, which in the presence of iron (III) ions give the complex with orcine of a green or green-blue color. Ribose bound with purine bases is involved in this reaction, and bound to pyrimidines - no. 6

7 aldopentose ketopentose Orcine Reagents: RNA hydrolyzate 10% orcinol solution Color complex FeCl3 solution in HCl Procedure: Prepare 2 test tubes, sign as 1 and 2 respectively. Then prepare samples according to a table: Reagents Sample number 1 2 H2O 0,5 ml --- RNA hydrolyzate --- 0,5 ml 10% orcinol solution 0,2 ml 0,2 ml FeCl3 solution in HCl 1 ml 1 ml Mix and incubate in 100 o C for 5 minutes Conclusions: 7

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