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Extraction of DNA from Calf or Hog Thymus/Isolation of Yeast RNA
Nucleic acids may be divided into two groups RNA and DNA. DNA contains almost all the genetic information while RNA serves as the bridge between the DNA and proteins.
Study of both DNA and RNA initially involves proper extraction/isolation. The storehouse of eukaryotic DNA is the nucleus (and in the mitochondria), so experimentally, DNA is extracted from tissues that have a high nuclear to cytoplasmic mass ratio, such as the tissues of the thymus gland and spleen. The thymus gland is a particularly good site for DNA extraction because it functions as the primary site for T lymphocyte differentiation. The T lymphocytes it acts upon have a round nucleus that occupies a greater proportion of the cellÐŽÂ¦s volume (with only a thin layer of cytoplasm surrounding the nucleus). The high lymphocyte content of the thymus gland, makes DNA extraction much more efficient, convenient and productive. In contrast, RNA extraction is done for cells that have a high cytoplasmic to nuclear mass ratio, such as the fungus Saccharomyces cerevisiae (commonly known as bakerÐŽÂ¦s yeast) used in this experiment.
The extraction procedures for both DNA and RNA basically outline the same steps: release of cell contents (through cell membrane lysis), separation of contaminants (lipids, proteins, etc.) from the desired nucleic acid (DNA, RNA), then precipitation of the separated nucleic acids while making sure that the nucleic acids are not digested or damaged. The samples obtained are then further purified and stored for testing and future use.
The weight of the crude DNA fibers was taken. The extracted DNA was analyzed spectrophotometrically at A260 and A280. The A280/A260 ratio was used to calculate and estimate %DNA purity. The % DNA purity is 29.
The weight of the RNA isolate was taken, and from that value % recovery was calculated. The % recovery was 5.33.
II. Results and Discussion
A. Extraction of DNA
Mass of hog thymus = 8.58g
Mass of crude DNA fibers: 0.28g
A260/A280 = 0.1588 ~ 0.16
A pure DNA solution would have a ratio of 1.8-2.0.
%DNA purity= -8.57%
A calibration plot was constructed, based on the table of values given in page 28 of the Chemistry 40.1 laboratory manual. The inverse of the values on the left column (A280/A260) were calculated to yield the A260/A280 values shown in Table 1 below. The values for % nucleic acid were then plotted against the A260/A280 ratios to come up with Figure 2. A trendline equation was obtained as well:
y = 15.402x ÐŽV 11.038
Table 1 ÐŽV Calibration Plot
A260/A280 % Nucleic Acid
By plugging the computed A260/A280 ratios into the trendline equation, the % purity of the DNA extract can then obtained.
y = 15.402x ÐŽV 11.038
% nucleic acid = 15.402 (A260/A280) ÐŽV 11.038
= 15.402 (0.16) ÐŽV 11.038
= -8.57% purity
In calculating for the concentration of the DNA extract, it was assumed that a DNA solution with a concentration of 50 Ñ“Ðg/mL has an A260/A280 ratio of 1. The principle of ratio and proportion was then used.
1.00 = 0.16
50 Ñ“Ðg/mL DNA concentration
DNA concentration = (0.16)(50 Ñ“Ðg/mL)
= 8.0 Ñ“Ðg/mL
B. Isolation of RNA
Mass of dry yeast: 3.0 g
Mass of RNA extract: 0.16 g
% recovery: 5.33%
Description of Product: yellowish
% recovery = mass of RNA extract, g x 100
mass of dry yeast, g
= 0.16 g x 100
The first step in the experiment involving the extraction of DNA from hog thymus is cell lysis. The hog thymus was first thoroughly cleaned by removing residual fat, blood vessels, and other extraneous tissues as this precaution reduces the chance of contamination from unwanted lipids and proteins that may be present. Cell lysis through fine mincing and homogenizing the hog thymus is accompanied by the addition of citrate buffer---done in order to keep the acidity at near-physiological pH---approx. 7.4 (at this pH, stability of crucial hydrogen bonds and other linkages in the DNA double helix are maintained). The addition of sodium citrate buffer has two other significances. First, it chelates Mg2+ and Mn2+, cations that serve as cofactors for DNAses. This chelating action of sodium citrate prevents the degradative enzymes to take effect in performing their function. Secondly, sodium citrate chelates other divalent cations (e.g. Ca2+) that could form salts with the anionic phosphate groups in the DNA backbone.
Centrifugation was done next in order to separate the nuclei from other cellular organelles as well as cellular debris. The denser nuclei sank down to the bottom after centrifugation, forming the residue, whilst the ÐŽÂ§lighterÐŽÐ cellular organelles such as the mitochondria, etc. remained suspended in the supernatant. After discarding off the supernatant, the residue was suspended in 24 ml of 2.0 M NaCl. This was done because the salt increases the ionic strength of the solvent that in turn produces a ÐŽÂ§salting inÐŽÐ effect. Salting in then allowed the proteins of the nucleus to again, dissolve for easier separation later through a second centrifugation. Another effect of salt addition is that it could, with strong possibility, weaken the interactions between the negative charges carried by DNA and the positive charge of basic proteins. SDS solution was then added and the resulting solution incubated for 10 minutes in a warm water bath. The denaturant SDS was added to release the DNA by breaking the nuclear membrane (SDSÐŽÂ¦ polar heads and non-polar tails disintegrated the lipid bi-layer membrane of the nucleus). The warm water bath helped in the denaturation of the unwanted proteins embedded in the nucleus/nuclear membrane.
As was mentioned, a second centrifugation was done (at 7000 rpm for 5 minutes) to separate the DNA from the lysed nuclear membrane, denatured proteins, etc. DNA was then precipitated out of the supernatant through the addition of cold 95% ethanol, with the resulting DNA ÐŽÂ§fibersÐŽÐ coiled around the J-tube. Ethanol decreases the solubility of the DNA through lowering the dielectric constant of the solution. Ethanol precipitation ensures that the isolated DNA is relatively pure and free of unwanted RNA and protein contaminants---solution constituents that do not precipitate as fibers upon the addition of ethanol. After collecting the fibers and washing away residual lipids with cold 70% ethanol, the DNA was suspended in sodium citrate buffer for storage while keeping a mildly alkaline pH of 7.4 in order to keep the DNA dissolved.
Upon analyzing the results from spectrophotometric analysis, we can make several observations. First of all, we can see that from 8.58 g of hog thymus, 0.28 g of DNA fibers was obtained, giving us a recovery of around 3.26%. Theoretically, only 5 ÐŽV 15% of the dry weight of cells is composed of DNA, but we must remember that the recovered 3.26% was still unpurified DNA, and thus may still have contained many contaminants, especially bound nucleoproteins. If the DNA fibers were weighed again after purification, it would have probably weighed less than 0.28 g.
In measuring absorbance, the spectrophotometer had to be set to 260 and 280 nm. As mentioned in the, proteins absorb strongly at 280 nm, while DNA absorbs best at 260 nm. Using the A260/A280 ratio as a measure of DNA purity not only takes into account the amount of DNA in the sample, but also the amount of proteins, which are the most likely contaminants in this experiment. A high A260/A280 ratio would mean that the DNA extract has a high purity, while a low ratio would indicate that the extract contains a lot of protein contaminants compared to DNA molecules.
Supposedly, an A260/A280 ratio within the range of 1.80 ÐŽV 2.00 indicates a high purity of the DNA extract. Since we obtained a ratio of 0.16, we were already expecting that we will be getting a very low % purity. True enough, we obtained a very low value for % purity and it is even a negative value of -8.57%. This discrepancy can be explained by the trendlineÐŽÂ¦s poor approximation of the actual data in Table 1, as supported by the relatively low R2 value of 0.89. Also, our calculated ratios fell outside the range of values given in Table 1. It would have been better if they fell within the range of values, so that there would be no need for extrapolation, which may lead to erroneous results. Even the validity of the table of values given in the laboratory manual (which was used to derive Table 1) is questionable, since it seems to give an exponential graph instead of a linear one, as shown in Figure 2. The discrepancy in the value we have computed can also be attributed to the fact that we didnÐŽÂ¦t perform the purification part of the extraction experiment.
The calculated DNA concentration of 8.0 Ñ“Ðg/mL is relatively low, which further supports the idea the reason why we obtained a negative value for % purity. Possible sources of error are involved in the discrepancies of the values obtained. First of all, handling the thymus tissue with bare hands (without rubber gloves) may have introduced DNAses to the sample, thus leading to some degradation. Also, mechanical stress during washing and mincing may have caused denaturation and damage to the DNA. Furthermore, the temperature could not be kept low at all times, especially during centrifugation and incubation periods. This then allowed DNAses to resume normal activity.
Contaminants were also a major problem. We were not sure exactly what the thymus gland was supposed to look like, so it was difficult to distinguish between thymus and extraneous tissue. It is highly probable that we left a lot of residual fat and blood vessels without even knowing it, which could then have led to contamination of our DNA extract. Also, RNAse solution was supposed to be added during the purification procedure to degrade contaminating RNA. But due to the lack of RNAse solution, this step was substituted instead with the addition of more SDS solution. Obviously, the substitution does not yield the same results, and the RNA may have interfered with absorption of the DNA extract at 260 nm, since it is a nucleic acid like DNA.
The first thing done in the isolation of yeast RNA was the addition of 1%NaOH and water to the yeast sample and heating it in a boiling water bath. After the boiling period it was then strained through cheesecloth, and the residue discarded. The supernatant liquid was then centrifuged and the resulting pellet also discarded. These first few steps were basically done to lyse yeast cells and separate the cell debris from the rest of the cellular components. Heat was applied to promote the lysis of the cells and the extraction of nucleic acids and water-soluble proteins. Heat also inactivated nucleases that could have degraded our RNA sample.
The supernatant we obtained was then acidified. The acidified supernatant was then centrifuged and filtered through cheesecloth several times until the supernatant was clear. This step was for the removal of proteins from the solution. The slightly acidic environment also prevents the alkali hydrolysis of the RNA in the sample.
The resulting supernatant was then evaporated to approximately 5 mL. This was done to concentrate the RNA in the solution for easier isolation and evaporate solutes and other unwanted substances. The solution was then cooled to around 40 degrees Celsius before the addition of acidified ethanol. The addition of ethanol was not done until the solution reached 40 degrees Celsius because ethanol is highly flammable and also easily evaporates. The addition of ethanol lowers the dielectric constant of water and lessens the solubility of the RNA, and thus making it easier to precipitate. The added ethanol was acidified so that the protons contributed by the acid would protonate the negatively charged phosphate in the RNA and prevent electrostatic repulsion. Without repulsion, the RNA would precipitate better. The protonation reaction also occurs faster for RNA than for DNA and for this reason the precipitate can more or less be assumed to be made up of RNA molecules. To further lower the solubility of RNA in the solution, the mixture was then placed in an ice bath.
After the ice bath, the samples were again centrifuged to separate the remaining solute along with other unwanted biomolecules from the precipitated RNA. Several washings of ethanol and ether were then done. This was done for the removal of any remaining lipids and non-polar contaminants from the sample. The sample was then air dried to allow for the evaporation of the added ethanol and ether.
Horton, Robert H., et al. Principles of Biochemistry. 3rd ed. Prentice-Hall Publishers. New Jersey. 2002.
Biochemistry Laboratory Manual, UP Diliman, 2002
Campbell M. K. Biochemistry 3rd edition. 1998. Harcourt Brace College Publishers. USA
Zubay, Geoffrey L. et al., Principles of Biochemistry, Wm. C. Brown Publishers, Iowa, 1995.
IV. Answers to Questions
EXTRACTION OF DNA FROM HOG THYMUS
1. Why should the extraction steps in the experiment be carried out at low temperature and almost neutral pH?
The extraction should be carried out at low temperatures in order to slow down the action of degradative enzymes such as deoxyribonucleases or DNAses. Also, higher temperatures would cause unwinding of the DNA double helix (80 ÐŽV 90 â€žaC) and destabilization of the phosphodiester and N-glycosidic bonds (greater than 100 â€žaC). The pH level should also be kept near neutrality, in order to keep hydrogen bonds, phosphodiester linkages, and purine N-glycosidic bonds stable.
2. Give the purpose of each step in the extraction of crude nucleoprotein.
The first step was to clean the thymus gland by removing residual fat and blood vessels. This was done in order to reduce contamination from unwanted lipids and proteins that may be present in the extraneous tissues. Mincing the thymus tissue made it easier to homogenize in citrate buffer, which was done to mechanically lyse the thymus cells and release the DNA-containing nuclei from within.
The added sodium citrate buffer actually has three important roles. First of all, sodium citrate chelates Mg2+ and Mn2+, cations that serve as cofactors for DNAses. In effect, the chelating action of sodium citrate renders the degradative enzymes incapable of performing their function. Secondly, sodium citrate chelates other divalent cations as well, such as Ca2+, which could form salts with the anionic phosphate groups in the DNA backbone. And finally, the solutionÐŽÂ¦s buffering action helps maintain a nearly neutral pH of 7.4, which as mentioned in #1 above, stabilizes crucial hydrogen bonds and other linkages in the DNA double helix.
Through centrifugation, the denser nuclei and unbroken cells were made to sink down to the bottom of the spacers, forming the residue. The supernatant, which contained mitochondria, lysosomes, and other cellular organelles, was discarded. The residue was then resuspended in 2.0 M NaCl solution. The NaCl increases the ionic strength of the solvent, resulting in a salting in effect that allows the nucleoprotein to redissolve. The high concentration of salt will also help later in weakening interactions between the negatively charged DNA (due to its phosphate groups) and positively charged or basic proteins. Sodium dodecyl sulfate or SDS, a strong anionic detergent, was then added to the solution to lyse the nuclear membrane and release the DNA. Through the action of its polar (hydrophilic) heads and non-polar (hydrophobic) tails, SDS effectively disintegrated the lipid bilayer membrane of the nucleus. The temperature was also raised to 40 â€žaC to help denature the proteins imbedded in the nuclear membrane.
The solution was centrifuged once again, but this time, the sediment (containing the lysed nuclear membranes, denatured proteins, and other cellular contaminants) was the one discarded. The addition of ice-cold 95% ethanol to the supernatant caused the precipitation of the DNA as fibers. Ethanol lowers the dielectric constant of the solution, consequently decreasing the solubility of the DNA. This ethanol precipitation step ensures that the isolated DNA is relatively pure and free of unwanted RNA and protein contaminants, which do not precipitate as fibers upon the addition of ethanol. The cold temperature of the ethanol also helps lower the solubility of DNA, as well as inhibiting the action of DNAses. After collecting the fibers and washing away residual lipids with cold 70% ethanol, the DNA was resuspended in sodium citrate buffer for storage, with a mildly alkaline pH of 7.4 that keeps the DNA dissolved.
3. Which step ensures the isolation of pure DNA solution? Why?
The ethanol precipitation step ensures that the isolated DNA is relatively pure and free of unwanted RNA and protein contaminants, which do not precipitate as fibers upon the addition of ethanol. The cold temperature of the ethanol also helps lower the solubility of DNA, as well as inhibits the action of DNAses.
4. Why should the absorbance be taken at 260 and 280 nm?
Proteins absorb strongly at 280 nm, while DNA absorbs best at 260 nm. Using the A260/A280 ratio as a measure of DNA purity not only takes into account the amount of DNA in the sample, but also the amount of proteins, which are the most likely contaminants in this experiment. A high A260/A280 ratio would mean that the DNA extract has a high purity, while a low ratio would indicate that the extract contains a lot of protein contaminants compared to DNA molecules.
ISOLATION OF YEAST RNA
What is/are the purpose/s of each step and each reagent in the isolation process?
The first step was the addition of NaOH, which served to disrupt the cell membrane and lyse the cell, consequently releasing the RNA from within. The NaOH also increases the pH of the solution, resulting in the denaturation of contaminant proteins as well as the deactivation of degradative enzymes such as ribonucleases or RNAses. The heating also helped loosen the cell membrane by increasing the kinetic energy of the lipid molecules, thus aiding in the release of more RNA. The mixture was then filtered and centrifuged to remove the denatured proteins, lysed lipid membranes, and other insoluble contaminants.
The addition of glacial acetic acid reduces the pH and once again contributes to the denaturation of more proteins. It also helps prevent alkali RNA hydrolysis, thus ensuring that the desired RNA is not degraded. The mixture goes through a few more rounds of filtration and centrifugation to eliminate the precipitated proteins. The mixture was then evaporated to a volume of only 5 mL. This was done to increase the concentration of RNA in the solution, thus making it easier to isolate it later. The mixture was allowed to cool to 40 â€žaC first before the addition of acidified ethanol, since ethanol has a relatively low boiling point and evaporates quickly at higher temperatures.
As in DNA extraction, the ethanol lowers the dielectric constant of the solution and reduces the solubility of RNA, causing it to precipitate from solution. The HCl protonates the phosphate groups in nucleic acid backbones, thus minimizing the charge repulsions between molecules and helping them to aggregate and precipitate. This protonation reaction occurs much faster for RNA than DNA, which then guarantees that the precipitate is mostly made up of RNA rather than DNA.
The mixture was placed in an ice bath for 30 minutes to allow the RNA to precipitate. The low temperature helped to lower the solubility of RNA even more, hence maximizing the yield of RNA extract. The last round of centrifugation separated the RNA precipitate from the unneeded supernatant. And finally, the washings with ethanol and ether removed any residual lipids and other nonpolar contaminants.