Isolation of Bacterial DNA
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1 N( EXPERIMENT 19 Isolation of Bacterial DNA Theory To isolate a functional macromolecular component from bacterial cells, you must accomplish three things. First, you must efficiently disrupt the bacterial cell wall and cell-membrane system to facilitate extraction of desired components. Second, you must work under conditions that either inhibit or destroy the many degradative enzymes (nucleases, proteases) released during cell disruption. Finally, you must employ a fractionation procedure that separates the desired macromolecule from other cellular components in satisfactory yield and purity. The isolation of bacterial DNA described in this experiment, patterned after the work of Marmur (1961), accomplishes these objectives. Bacterial cells are disrupted by initial treatment with the enzyme, egg-white lysozyme, which hydrolyzes the peptidoglycan that makes up the structural skeleton of the bacterial cell wall. The resultant cell walls are unable to withstand osmotic shock. Thus, the bacteria lyse in the hypotonic environment. The detergent, sodium dodecyl sulfate, (SDS, sodium dodecyl sulfate) then completes lysis by disrupting residual bacterial membranes. SDS also reduces harmful enzymatic activities (nucleases) by its ability to denature proteins. The chelating agents, citrate and EDTA (ethylenediamine tetraacetic acid), also inhibit nucleases by removing divalent cations required for nuclease activity. This experiment employs a variety of fractionation methods to purify the bacterial DNA. Perchlorate ion is used to dissociate proteins from DNA. Chloroform isoamyl alcohol is used to denature and precipitate proteins by lowering the dielectric constant of the aqueous medium. The precipitated material is removed by centrifugation. DNA is then isolated from the solution by ethanol precipitation, which allows spooling of the long, fibrous DNA strands onto a glass rod. Although the residual RNA and proteins in the solution also coagulate, they do not form fibers and are left in the solution as a granular precipitate. The resultant crude DNA is dissolved and precipitated again with isopropanol under conditions that favor specific DNA precipitation. These steps form a general procedure for the isolation of chromosomal DNA from virtually any bacterium. If you intend subsequently to assay the biological activity of the isolated DNA in a transformation assay (as in Experiment 20), it is necessary to start this isolation with the appropriate species and strain of bacteria possessing the genotype of interest. This experiment will also provide you with experience in characterizing DNA samples by determining their ultraviolet absorption spectra and observing their thermal denaturation by means of the hyperchromic effect. These topics were discussed in the introduction to Section V. Supplies and Reagents For growth of wild-type (Trp ) Bacillus subtilis (if this experiment is to be done together with Experiment 20. Other kinds of frozen bacterial cells can be used if Experiment 20 will not be performed.) Stock culture of wild-type B. subtilis (Bacillus Genetic Stock Center strain 1A2) Inoculating needle 333
2 334 SECTION V Nucleic Acids Luria broth (see Appendix) 37 C Incubator-shaker Fermenter Centrifuges, continuous and laboratory Sterile saline solution (8.5 g NaCl/liter) 0.15 M NaCl, 0.l M Na 2 EDTA Lysozyme solution, 10 mg/ml 25% (wt/vol) SDS solution 5.0 M NaClO 4 Chloroform isoamyl alcohol (24:1, vol/vol) 95% Ethanol Dilute (1/10X) saline-citrate (0.015 M NaCl, M Na 3 -citrate) Standard (1X) saline-citrate (0.15 M NaCl, M Na 3 - citrate) Concentrated (10X) saline-citrate (1.5 M NaCl, 0.15 M Na 3 -citrate) 3.0 M Sodium acetate, 1 mm EDTA, ph 7.0 Isopropanol Chloroform Spectrophotometer and quartz cuvettes Protocol Growth of Wild-Type Bacillus subtilis for DNA Isolation This portion of the experiment may be performed by an instructor in advance. 1. Inoculate (sterile technique) 100 ml of sterile Luria broth (200- to 400-ml flask) with a sample of wild-type Bacillus subtilis obtained from a stock culture slant. Incubate this solution on a shaker at 37 C for 15 to 24 hr (until the culture is densely turbid and is entering the stationary phase of growth). 2. Transfer the starter culture (sterile technique) into a fermenter containing 10 to 12 liters of sterile Luria broth and continue the bacterial growth at 37 C while stirring ( 400 rpm) and aerating at 1 liter of air per liter of fluid per minute. Monitor the growth periodically by determining the turbidity of the culture (absorbance at 660 nm) during the 3- to 5-hr growth until this culture just enters the stationary phase of growth. 3. Stop the growth by cutting off both air and agitation. Use a continuous centrifuge (e.g., Sharples) to harvest the cells as quickly as possible. 4. Suspend the cell paste in an equal volume of cold (1 to 4 C) sterile saline solution. Complete the cell-washing procedure by harvesting the B. subtilis cells (10 min, 20,000 g, 1 to 3 C) with a high-speed centrifuge. 5. Decant the supernatant fluid from the cell pellet after centrifugation and store the washed cells in Parafilm or waxed-paper-wrapped lots at 20 C until use (2-g lots are convenient). The expected yield is 35 to 45 g of cells per 10 liters. Isolation of Bacterial DNA 1. Suspend 2 g of bacterial cell paste in 25 ml of 0.15 M NaCl, 0.1 M Na 2 EDTA solution. The bacterial paste is most easily suspended by first adding a small amount of the NaCl EDTA solution, making a slurry, and then slowly adding the rest of the NaCl EDTA solution. After suspension, add 1 ml of a 10-mg/ml lysozyme solution and incubate the suspension at 37 C for 30 min, gently agitating occasionally. 2. After the 30-min incubation, complete the lysis of the bacteria by adding 2 ml of a 25% SDS solution and heating this preparation for 10 min in a 60 C water bath. Cool the solution to room temperature in a bath of tap water. 3. Add 7.5 ml of 5.0 M NaClO 4 to the 25 ml of lysed bacteria cells and mix gently. Then add 32.5 ml of CHCl 3 isoamyl alcohol (24:1) (i.e., a volume equal to the volume of lysed cell preparation containing 1.0 M perchlorate). Slowly shake the solution (30 60 oscillations per minute, by hand or on a low-speed shaker) in a tightly stoppered flask for 30 min at room temperature. 4. Transfer the suspension to a 250-ml polypropylene centrifuge bottle, and separate the resulting emulsion by centrifuging for 5 min at 10,000 g at room temperature. 5. During the centrifugation step, remove any residual enzymes from a glass rod by heating one end of the rod in a flame and allowing the sterile end to air cool in a thoroughly washed 250-ml beaker. 6. After the centrifugation step, carefully pipette the clear aqueous phase (top layer) away from the coagulated protein emulsion at the interface between the aqueous and organic phases of the centrifuged solution. Do not attempt to
3 EXPERIMENT 19 Isolation of Bacterial DNA 335 remove all of the aqueous phase; rather, avoid contaminating the aqueous phase with material from the other layers. Place the aqueous phase, which contains the extracted nucleic acids, in the 250-ml beaker. Dispose of the CHCl 3 -containing waste in the special container provided by your instructor. 7. Gently stir the nucleic acid solution with the sterilized rod while slowly and gently adding 2 volumes (about 60 ml) of 95% ethanol. Pour the ethanol gently down the side of the beaker so that it is layered over the viscous aqueous phase. Continue to stir this preparation gently with the sterilized glass rod so that the ethanol is very gradually mixed throughout the entire aqueous phase. Avoid stirring vigorously, which would shear the DNA strands and make the DNA more difficult to isolate. The DNA will form a white fibrous film at the water ethanol interface, and you should be able to spool all of the gelatinous, threadlike, DNA-rich precipitate onto the glass rod. Continue gentle stirring until the aqueous and ethanol phases are completely mixed and no more DNA precipitate can be collected on the glass rod. Drain off excess fluid from the spooled crude DNA by pressing the rod against the walls of the beaker until no further fluid can be squeezed from the spooled preparation. 8. Dissolve the crude DNA by stirring the glass rod with its spool of material in 9 ml of dilute (1/10X) saline-citrate in a test tube. Difficulty in dissolving the sample indicates failure to remove sufficient alcohol from the sample during step 7. If such difficulty is encountered, continue working the sample within the solution until you obtain as uniform a suspension as possible. 9. To the DNA solution add 1 ml of 3.0 M sodium acetate, 1 mm EDTA, ph 7.0 solution and transfer the preparation to a 100-ml beaker. Gently swirl the sample while slowly dripping in 6.0 ml of isopropanol. If fibrous DNA is readily apparent, collect the DNA threads by stirring and spooling with a sterilized glass rod as described in step 7. If a gel-like preparation develops, add an extra 1.0 ml of isopropanol and stir to spool the DNA onto the sterilized glass rod. Remove excess fluid from the spooled DNA by pressing the sample against the walls of the beaker. 10. Wash the sample (by submersion and brief swirling of the DNA on the rod) in test tubes containing, in order, 10 ml of 70% ethanol (made by adding 2.6 ml H 2 O to 7.4 ml of 95% ethanol), and then 10 ml of 95% ethanol. 11. If you plan to isolate the DNA only for use in the genetic transformation outlined in Experiment 20, or if a day or longer will elapse before you will characterize the DNA as described below, store the DNA in a stoppered tube (2 C) as a spool submerged on the rod in 95% ethanol. Dissolve the DNA as described in step 1 of the following section when you are ready to use it. Characterization of DNA 1. Remove the rod and the spooled DNA from the 95% ethanol, blot away all obvious residual fluid with a clean piece of filter paper, and then dissolve the DNA by stirring the glass rod, with its spooled DNA, in a test tube containing 10 ml of dilute (1/10X) saline-citrate (sterile solution if the DNA is to be used in Experiment 20). This solution can be stored at 2 C. Spectral Characterization of DNA 2. Dilute a 0.5 ml sample of the DNA solution with 4.5 ml of standard (1X) saline-citrate and determine the absorbance of this diluted sample at 260 nm against a dilute saline-citrate blank containing no DNA. If the absorbance at 260 nm is greater than 1.0, dilute the sample with a precisely known volume of standard (1X) saline-citrate until you obtain an absorbance of between 0.5 and Determine the absorbance of this solution at 5- nm intervals between 240 and 300 nm, so as to obtain an absorption spectrum for the isolated DNA. (Remember that quartz cuvettes must be used in this wavelength range.) Blank the spectrophotometer to read zero absorbance with 1X saline-citrate at each wavelength. (If equipment for the automated collection of this absorbance spectrum is available, follow the instructions for that instrument.) Include this absorption spectrum in your report. 4. Calculate the A 260 A 280 ratio for your sample, and compare it with the value expected for pure DNA (2.0).
4 336 SECTION V Nucleic Acids 5. Assuming that a 1-mg/ml solution of native DNA has an absorbance at 260 nm of 20 and that all of your absorbance at 260 nm is due to native DNA, calculate the concentration (in milligrams per milliliter) of the DNA in your undiluted DNA solution and the total yield (in micrograms or milligrams) of DNA you obtained. 6. Assuming 100% recovery of the cellular DNA and assuming the wet bacterial cell paste was 80% water, calculate the percent of the dry weight in the bacterial cells that is DNA. How do your results agree with the DNA content of a typical bacterium, which is about 3% of the cell s dry weight? If your results do not agree with this value, discuss possible reasons for the discrepancy. Melting of DNA The Hyperchromic Effect 1. If your laboratory is equipped with a spectrophotometer with a thermoprogrammer designed for determination of melting curves of nucleic acid samples (Gilford, Beckman Instruments), determine a melting curve of a sample of your DNA dissolved in the required volume (usually 1 ml or less) of standard (1X) salinecitrate so as to give an initial absorbance of about 1 at 260 nm. Use the results from the preceding section to decide how much you need to dilute the stock DNA solution. Follow the instructions provided by the instrument s manufacturer in programming the rate of temperature increase, protecting the sample against evaporation, etc. 2. If your laboratory does not have access to a spectrophotometer with a thermoprogrammer, use the quick cool method described in steps 3 through 6 below to obtain a melting curve for your DNA. This method does not observe a true equilibrium between native and denatured ( melted ) DNA and is appreciably less accurate than the method described in step 1, but it will suffice to illustrate the principles underlying the thermal separation of DNA strands. 3. Using your stock DNA solution from step 11, prepare 10 ml of a diluted DNA solution in the dilute saline-citrate (1/10X) that has a final absorbance at 260 nm of about 0.5. Use the results from the preceding section to decide how much you need to dilute the stock DNA solution. Determine the absorbance at 260 nm of this dilute solution at room temperature (about 20 C). 4. Transfer 1.0 ml of the same dilute DNA solution into each of six microcentrifuge tubes and cap them tightly. Incubate one tube at each of the following temperatures for 20 min: 50 C, 65 C, 75 C, and 85 C. In addition, incubate two of the tubes at 100 C (i.e., a boiling water bath). 5. After 20 min of incubating the tubes at the indicated temperatures, quickly cool all of them by placing them in an ice-water bath and gently agitating them. Allow one of the tubes heated at 100 C to cool slowly to room temperature by placing it in a rack at your bench (30 to 40 min will be required). 6. As quickly as possible after they have cooled, determine the absorbance at 260 nm of the solution in each tube. For a blank, use a sample of dilute saline-citrate (1/10X) that contains no DNA. 7. During heating, the double-stranded structure of DNA melts ; that is, the strands separate. The nucleotide bases in the separated strands are no longer stacked in a regular fashion, which results in an increase in their absorbance of UV light. This is called the hyperchromic effect or hyperchromicity. When melted DNA is quickly cooled in solutions with very low salt concentrations, the strands are very slow to reassociate into their original base-paired, double-stranded form. Thus, you can estimate the degree of melting at each temperature even though you are measuring the absorbance at 260 nm at room temperature. The DNA sample that was heated to 100 C and slowly cooled to room temperature, on the other hand, is expected to form normal double-stranded DNA again (reanneal) and to exhibit little hyperchromic effect. Do your data support this expectation? 8. Prepare a plot of the absorbance at 260 nm at each temperature for the unheated sample and each of the rapidly cooled samples. Estimate the melting temperature (T m ) for your DNA. (T m is the temperature at which the DNA sample is half melted. Assume that the DNA is
5 EXPERIMENT 19 Isolation of Bacterial DNA 337 completely melted at 100 C. Calculate the hyperchromic effect for your DNA as the percent increase in absorbance at 260 nm on complete melting. Calculate the percent guanine plus cytosine (% GC) base pairs in your DNA using the following formula % GC 2.44 (T m 81.5 C 16.6 log 10 [Na ]) For dilute (1/10X) saline-citrate, this becomes % GC 2.44 (T m 53.9 C ). B. subtilis DNA is known to contain about 43% GC. How does this agree with your results? Discuss the results of this experiment. Exercises 1. The DNA of cells containing nuclei (eukaryotic cells) is tightly associated with proteins called histones, most of which are rather basic proteins (i.e., they have high isoelectric points). Considering the basic character of most histones and the amino acids that contribute to this basic character, what enzymes would probably be most effective in disrupting histones from eukaryotic cellular DNA? Would you expect the procedure used in this experiment for the isolation of bacterial DNA to be useful for the isolation of DNA from eukaryotic cells? 2. This isolation of bacterial DNA utilizes reagents in the neutral ph range. What effects, if any, would you expect extremes in acid or alkali to have on the fibrous character of the DNA isolated in this procedure? Why? 3. Examine the procedure described in Experiment 21 for the isolation of plasmid DNA from bacteria. It is very different from the procedure used to isolate chromosomal DNA in this experiment. Could it be used to isolate chromosomal DNA from bacteria? Why or why not? Explain why such different procedures are used for isolating these two types of DNA. 4. What other physical and chemical treatments besides heating would be expected to allow you to demonstrate the hyperchromicity of singlestranded DNA? Explain how these treatments bring about strand separation. 5. Most naturally occurring RNA molecules are singled-, not double-stranded duplexes, yet RNA also shows a hyperchromic effect on melting. Explain. 6. The equation used for calculating % GC bases pairs in this experiment predicts that increasing % GC and increasing Na concentration increases the T m of DNA. Explain the physical basis for these effects. REFERENCES Mandel, M., and Marmur, J. (1968). Use of ultraviolet absorbance-temperature profiles for determining the guanine plus cytosine content of DNA. Methods Enzymol 12B:195. Marmur, J. (1961). A procedure for the isolation of desoxyribonucleic acid from microorganisms. J Mol Biol 3:208. Puglisi, J. D., and Tinoco, I. (1989). Absorbance melting curves of RNA. Methods Enzymol 180:304.
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