Table 4. Summary of wavelength scan of untreated and alkaine-treated samples from 200-300 nim ‘DNA Sample ‘ena(tim) Aime Untreated I 7238 029 Treated 227 | 0.44 Describe and Interpret. ‘The table shows an increase in absorbance in the treated sample of DNA. This Phenomenon is called hyperchromicity. The purine and pyrimidine bases of nucleotides are what strongly absorb UV light. In @ double helical DNA, the base-base interaction and hydrogen bonds limit the amount of light that can be absorbed by the bases. When an alkaline component is added to the DNA sample, disruption of the hydrogen bonding between base pairs of the DNA double helix occurs and this will in tum cause separation of the two strands of DNA. When DNA is separated into single strands, the purine and pyrimidine bases are free to absorb more UV light because of the absence of the limiting hydrogen bonds present in the double stranded form, It should be noted, however, that in hyperchromicity, the max wavelength of the samples should be the same. ‘Table 2. Absorbace readings of DNA at 250 nm and 280 nm concentration of DNA and it spurity in ROSE, Wang and ‘pooling methods. DNA Sample ‘A280 Gone. DNA(ugimL) | A2607A280 ROSE 0.035 1.05 06 Wang 0.068 375 7.08 Spooling 0.034 | 3.45 202 Describe and interpret. The table shows the absorbance of DNA samples measured at 260 and 280 nanometers. The absorbance at 260 nanometers was done in order to determine the concentration of nucleic acids, since nucleic acids only absorb light at a wavelength of 260 nm. Absorbance at 280 nm was also oblained since the ratio ‘A260:A280 is used to assess the purity of nucleic acids. For pure DNA, the A260:A280 ratio should be approximately 1.8 and for pure RNA it should be close to 2. Based on the data gathered, it can be concluded that the Spooling method of extracting DNA gives the best yield among the three methods, The 260.280 ratio has a low sensitivity for protein contamination in nucleic acids. ‘The concentration of the DNA in micrograms per milliter was calculated via the Beer-Lambert's Law. Given the absorbance at 260 nanometers, divide it with 0.020 (ugimL)'cm", for double-stranded DNA, which ‘corresponds as the average extinction coefficient at that wavelength. For stranded DNA, the value increases to 0.027 (ugimL) "em". The resulting value from the said calculation would now be the concentration in ug/ml. Explain rationale of A260:A280 to approximate DNA purity. When: © 260:280<<<<<1.8 © When the ratio gives a value that is much lesser than 1.8, it indicates a sample of nucleic ‘acids significantly contaminated by orotein. The discrepancy between the ratios must be very large sinca the A260:A280 ratio lacks sensitivity for protein contamination in nucleic acids. Other contaminants, such as phenol, may lower the ratio. = 1,8<260:280<2.0 ‘© When the 260:280 ratio falls between 1.8 and 2.0, it indicates a pure sample of nucleic acids. ratio that falls between these values possesses a great degree of purity. © 2,0<<<<<<260:280 © A ration greater than 2.0 indicates the presence of impurities other than proteins, such as ‘organic compounds with carboxylic groups, which absorb strongly at 260 nanometers RMA ws DNA? - 2DNA Extraction from different Animal Tissues ‘Table3. Functions ofthe lferent reagents used in the Wang Method of DNA extraction Reagent Function Enzyme reaction solution + 1%wv SDS + SDS (Sodium dodecyl sulfate) acts as anionic surfactant and denatures the proteins that may act as contaminants in the DNA sample. Denaturation occurs because itis amphiphilic and disrupts the + SmMEDTA non-covalent bonds of proteins. +10 mM Tris-Cl, pH 8 ‘+ EDTA chelates divalent cations and disrupts cell integrity Tris-Cl buffers the solution Ethanol, 70% aqueous solution Tis a volatile compound used to solublise DNA. When air-dried, ethanol is easily evaporated leaving pure DNA in the solution. Tsopropanol, absolute, and 40% viv aqueous solution Selectively precipitates DNA out of the solution and further removes alcohol-soluble proteins to ensure high purty and DNA yield. Proteinase K stock solution, 20 mgiml (Store at -20°C) itis a protease with broad specificity, has a high activity and can digest proteins at short times. This reagent ensures complete protein digestion eliminating impurties in the sample. Proteinase K does not need divalent ‘ations, such as Magnesium and Calcium. This allows EDTA to chelate these ions while Proteinase K performs its work ‘Sodium iodide (Nal) solution = 7.6MNal © 20mMEDTA = 40 mM Tris-Cl, pH 8 Solublizes proteins and polypeptides in the solution at high Concentrations. DNA is insoluble to this solution. Sodium iodide is a crystalline salt that must be stored in the dark at room temperature because it can readily decompose. Furthermore, low temperature can cause it to recrystalize Tris EDTA (TE) buffer = mMEDTA © 10 mM Tris, pH 8 1 Disruption and lysis of cell + EDTA (ethylenetriaminetetraacetic acid) is a known chelating agent. It binds divalent cations, specifically Magnesium and Calcium which maintain | the cell membrane’s integrity. Uponbinding with the cations, the membrane is disrupted and lysed. + Tris (Tris(hydroxymethyljaminomethane) works as_a biological buffer. A buffer is of tremendous importance because DNA is highly plt-sensitive. The pKa of Tris is equal to 8.1, which means that it can prevent drastic pH shifts and maintain the pH at a range of 7.0 to 9.0. The second role of Tris is to interact with the lipopolysaccharidemembrane and make it more permeable in order for disruption and then lysis to occur. Wang's lysis solution 1% wiv Triton X-100 0.32M sucrose 5 mM MgC, 410 mM Tris-Cl, pH7.5 Elimination of erythrocytes since mammalian red blood calls or erythrocytes do not contain nuclei. Hence, genomic DNA cannot be extracted from these. Another fact is that in whole blood, there are ‘approximately 1000x more erythrocytes than leukocytes which contain rhuclei. Thus, eliminating erythrocytes is needed in order to recover DNA that is high in both yield and purity. * Triton X-100 functions as 2 non-ionic detergent. Mainly, it solubilises the proteins of the cell membranes, rendering these easier to disrupt. + Sucrose selectively lyses erythrocytes by hypotonic shack, since these are more susceptible than leukocytes. ‘= MgCl, is a salt and it functions to selectively precipitate nuclel- containing cells out of the solution. Also, the Magnesium cations counteract the Phosphate anions of the nucleic acids and protect DNA from degradation. + The buffer protects the solution from pH shifts since the cell debris, generated can drastically alter itGel electrophoresis separates DNA according to its different attributes, namely: net charges and molecular weights. SDS-Page, type of electrophoresis used in this experiment, separates DNA according to the differences in molecular weights. Samples used are treated with sodium dodecy! sulfate disrupt intermolecular forces of attraction between the subunits and can also be a helpful in determining the DNA sequence. Generally, the substances are sieve through the gel and they move according to their molecular weights. The cone farthest the base of the gel has the lightest molecular weight while the one nearest has the heaviest. ‘Tables. Interpretation of results observed from gol electrophoresis of different DNA Samples. ‘Sample. Interpretation Tanet A Hinalll ladder | The Hindlll digest of lambda DNA (cl857indi Sam 7) yields 8 fragments suitable for use as molecular weight standards for agarose gel electrophoresis. The approximate mass of DNA in each of the bands is provided (assuming a 1.0 Ug load) for approximating the mass of DNA in comparably intense samples of similar size. This serves as a standard measurement for the different DNA ie sample: a Sai eae) Lane2: Plant DNA Four bands are seen in this lane. These bands represent different DNA fragmenis Lane3: Plant DNA with different molecular weight. The one nearest the base of the gel is the heaviest while the one farthest Is the lightest. The restriction enzyme applied during the extraction process cuts the DNA strand obtained from the plant sample into four fragments, attributing to the four bands seen on the gel. Also since, these bands correspond to the bands on the standard. The different molecular weight of these fragments can be approximated using the values indicated by the standard. Tane4:E, caliDNA___| An E. coli DNA is typically circuler, called plasmids. Uncut plasmids migrate more TaneS: E. coli DNA rapidly than the same plasmid when linearized. Preparations of uncut plasmid contain at least two topologically.different forms of DNA, corresponding to supercoiled forms and — nicked ~—aifcles. «(Retrieved —_ from http://arbl.cumbs.colostate.edu/hbooks/genetics/biotech/gels/agardna.html) This can be seen in the two streaks found in both lane 4 and 5. The farther streak is the lighter DNA. Also, it can be seen that the nearer streak coresponds to a streak found in the standard. With ths, its molecular weight can be approximated Using the values indicated by the A Hindlll ladder. Replication of sample testing ‘was mainly done to ensure proper approximation of the molecular weight of the DNA.Tane6: DNA from heparin tube Heparin is an anticoagulant. it inhibits the activity of thrombin consequently inhibiting the conversion of fibrinogen to fibrin. Heparin interferes with the downstream applications of recovered DNA. The effect of heparin can be seen in the gel since no DNA streaks can be found on the lane, The lane is very much ‘comparable to the negative control indicating the absence of DNA. Lane7: DNA from EDTA tube EDTA-Disodium is the usual anticoagulant used for DNA extraction. It works by chelating the calcium ions, which render these inactive to participate in coagulation. Also, EDTA-Disodium does not interfere with the downstream applications of recovered DNA ensuring efficient recovery of DNA in the sample. Anticoagulants were used since sample DNA was retrieved from blood. Blood Clotting Would stop us from obtaining the sample of interest, which is whole blood containing DNA. It can be seen in the streak that two distinct bands are formed. It can be inferred that the restriction enzyme applied during extraction cuts the DNA into two strands. Also the nearer band, DNA strand with the heavier molecular weight, corresponds to a band in the standard. With this, one can approximate its molecular weight. Lane8: Negative Control “This lane serves as the negative control and bares no DNA. It can be clearly sean that no streaks are imprinted on the gel. This is mainly used as @ standard for samples that contains no DNA.