What Can You Do to Achieve Minimum Radioactive Dose? Attitude Consider the benefits of an attitude whereby everyone working with radioactivity continuously ponders if they are working in the safest, most efficient manner. Is a particular radioactive experi- ment necessary? Can the amount of radioactivity in an experiment be reduced? Is there a faster, safer way to carry out the work? Questions like these will reduce cost and radioactive exposure. An institution’s RSO is also required to implement a continuing education program regarding the principles of keeping personnel exposure dose low. If you find yourself becoming stressed while handling radioac- tivity, or if that “incessant clicking sound” of the count-rate meter is causing a heightened sense of alarm, you can always step away from the bench to put things into perspective. Estimate how much dose you are receiving from your activities and relate those back to your annual allowable dose. Time Work quickly and neatly. In the example above, a finger lingering for 1 minute over an open 1mCi vial of 32 P will receive 17 mrem, whereas a 10 second exposure receives a sixfold lower dose. Practicing the manipulations of your experiments with non- radioactive materials will identify problem areas and ultimately enable you to work faster and safer. Working with radioactivity while feeling panicked or rushed will slow you down or cause an accident. If you can’t smoothly do the motion in 10 seconds, take 20. You’ll improve through time and all along will be well aware of your estimated dose. You’ll automatically be striving to lower your dose. Distance Dosage decreases with distance.Why? A radiation source is like a light bulb. As the rays radiate outward in a sphere, they cover a wider area but become less potent at any single point. Use the inverse square law to your advantage. Can you pipette with a longer pipettor? Can you place the reaction vial even a few inches farther from you and others in the lab? Can you place a film cassette containing a radioactive membrane farther away from your work area? Small steps such as these can go a long way in reducing dose to you and your colleagues. 162 Volny Jr. Shielding A good shielding strategy will effectively reduce dose rate without preventing you from working smoothly and safely. It will not force you to get closer or stay longer in high radiation areas. If the use of lead-lined gloves makes you feel like you’re working in a vat of honey and increase the likelihood of a spill, you might want to consider alternative shielding. Shielding for Beta Emitters Acrylic plastic (Plexiglas TM ) is used for the pure beta emitters, like 32 P, 33 P, and 35 S. A half inch thick piece of acrylic will stop essentially 100% of all betas, even for strong emitters, such as 32 P. Shielding for Gamma Emitters Lead will attenuate rather than completely obstruct gamma or X radiation. You may see in some literature that for a particular gamma-emitting isotope, a certain thickness of lead is required to “reduce the dose rate by a factor of 10.”This means that if a source is giving off a field of 100 mrem/h without shielding, the dose rate with that particular thickness of lead will be brought down to 10 mrem/h. For example, 125 I needs to have 0.25mm of lead shield- ing in order to reduce the dose rate by a factor of 10. Each suc- cessive layer of 0.25 mm will continue to decrease the dose rate by a factor of 10. Lead is best used as shielding for an isotope giving off both gamma radiation and beta particles, rather than a combination of acrylic and lead. Volatile Nuclides The three isotopes you are likely to encounter with volatile properties are 3 H, 35 S, and 125 I. Their chemical properties and the incredibly complex reactions involved with radiolytic decay cause these two isotopes to form gaseous by-products. If you work with any of these isotopes, your RSO and institution may have approved fume hoods for their use. Isotopes That Do Not Require Shielding Tritium, being a very weak beta emitter, travels only a few microns in air. Acrylic shielding would be of no use. What you do not want to do is to ingest tritium. Tritium in an aqueous form is 25,000 times more radiotoxic than tritium in a gaseous form. Working Safely with Radioactive Materials 163 How Can You Organize Your Work Area to Minimize Your Exposure to Radioactivity? If feasible, select bench space at the corner of the room, rather than in a central location, to reduce unnecessary traffic. Clearly delineate this work area as radioactive. Although it is not always possible due to space restrictions, it is recommended that if your lab is working with different radioisotopes that there be separate work areas for each radioisotope. Check with your RSO about any additional requirements listed on the institution’s license. Of main importance will be arranging your work space. Begin with absorbent material, perhaps a double thick section, taped onto the bench.A waste container that shields against the radioac- tivity should be placed in a location that makes it easy, quick, and safe to dispose of pipette tips, hot gloves, and the like.A box made of acrylic with a lid is sufficient for 32 P, 33 P, and 35 S, while for 125 I, lead-impregnated acrylic will help attenuate the gamma rays. Each radioisotope may need its own separate container for waste, depending on your institution’s disposal protocols. If you are using 32 P, acrylic shielding between you and the source is strongly recommended. There are many commercially available shields that will meet your needs. Once you establish your radioac- tive area, do a couple of practice runs to make sure that your work area is properly organized. Bring the RSO in so that she/he can approve your radioactive area and perhaps make further suggestions. You’ll want to examine closely any areas or actions that have the potential for high doses.An open vial of 32 P, an Eppendorf tube with 50 ml of 32 P, and a tray containing your blot with hybridiza- tion solution mixed with radioactive probe will all be obvious areas where you’ll need to pay close attention. The open vial may simply need an acrylic pipette guard on your pipettor in order to bring the dose down 10,000 fold. The Eppendorf incubation tube can be kept in an acrylic box, or behind acrylic shielding while the labeling reaction is going on. You may devise a way of not picking the reaction tube up with your fingers while you remove the reaction mix with a pipettor. The blotting container may present a potential spill. Finding a safe, out-of-the-way place, preferably in a fume hood and behind some acrylic shielding will go a long way toward reducing dose. How Can You Concentrate a Radioactive Solution? Three convenient approaches are lyophilization, a spinning vacuum chamber, and drying with a gentle stream of nitrogen gas. 164 Volny Jr. There is significant risk of contamination when using a lyophilizer or spinning vacuum chamber, so most facilities dedicate specific equipment for radioactive work. Blowing a very gentle stream of nitrogen gas over the solution works efficiently, but practice is required to avoid blowing the radioactive solution out of its container. The nitrogen stream method is straightforward (Figure 6.3). Attach a small glass pipette/dropper tip to tubing that is attached to the gas regulator of a tank of dry nitrogen gas, being careful not to break the top of the pipette into your hand. Turn on the gas flow, keeping the gas flow as gentle as possible. This procedure requires very little nitrogen flow. Before im- pinging upon the surface of the radioactive material, test the gas flow on a vial containing a like amount of water. Adjust the flow so that there is no splashing of the liquid but only a noticeable indentation of the liquid’s surface. Once you are satisfied that it is safe, gently direct the stream of gas onto the surface of the radioactive liquid, ensuring no splashing. Do all of this in a hood and in a location that is safe and will be able to contain acciden- tal spills. Continue blowing off the solution until dryness. Overdrying can sometimes be of concern, so it is best not to leave the area and come back to it after an extended period. It is also best to bring the solution to complete dryness so that when you bring it up into a known amount of solution, you will have an accurate idea of the concentration. Working Safely with Radioactive Materials 165 Figure 6.3 Removal of solvent from a non-volatile radiochemical using dry nitrogen. From Guide to Working Safely with Radio- labelled Compounds, Amerh- sam International, plc, 1974, Buckinghamshire, U.K. Reprinted by permission of Amersham Pharmacia Biotech. BIBLIOGRAPHY Feinberg, A. P., and Vogelstein, B. 1983. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132:6–13. U.S. Nuclear Regulatory Commission Regulatory Guide, Office of Standards Development. Regulatory Guide 10.5, Applications for Type A Licenses of Broad Scope. Revision 1, December 1980. U.S. Nuclear Regulatory Commission Regulatory Guide, Office of Standards Development. Regulatory Guide 10.7, Guide for the Preparation of Applica- tions for Licenses for Laboratory and Industrial Use of Small Quantities of Byproduct Material, Revision 1, August, 1979. U.S. Nuclear Regulatory Commission Regulatory Guide, Office of Standards Development. Regulatory Guide 10.2, Guidance to Academic Institutions Applying for Specific Byproduct Material Licenses of Limited Scope. Revision 1, December 1976. Guide to the Self-decomposition of Radiochemicals. Amersham International, plc, Buckinghamshire, U.K., 1992. Guide to Working Safely with Radiolabelled Compounds. Amersham Interna- tional, plc, Buckinghamshire, U.K., 1974. International Air Transport Association (IATA) Dangerous Goods Regulations, 6.2, Packing Instructions. Code of Federal Regulations (CFR) 173.421, 173.422, 173.424, and 173.427. Addi- tional Requirements for Excepted Packages Containing Class 7 (Radioactive) Materials. 166 Volny Jr. Appendix A. Physical Properties of Common Radionuclides Beta Energy, Specific Activity, Radionuclide Halflife max(MeV) max Tritium (hydrogen-3) 12.4 years 0.0186 28.8 Ci/matom a 1.06 TBq/matom a Carbon-14 5730 years 0.156 62.4 mCi/matom 2.31 GBq/matom Sulfur-35 87.4 days 0.167 1.49 kCi/matom 55.3 TBq/matom Phosphorus-32 14.3 days 1.709 9.13 kCi/matom 338 TBq/matom Phosphorous-33 25.3 days 0.249 5140 Ci/matom Iodine-125 59.6 days Electron 2.18 Ci/matom capture b 80.5 TBq/matom a Source: Data reproduced from Guide to Working Safely with Radiolabelled Compounds (Amerhsam International, 1974). a A milliatom is the atomic weight of the element in milligrams. b Electron capture is a radioactive transformation in which the nucleus absorbs an electron from an inner orbital. The remaining orbital electrons re-arrange to fill the empty electron shell and in so doing energy is released as electromagnetic radiation at X-ray wavelengths and/or electrons. 167 7 DNA Purification Sibylle Herzer What Criteria Could You Consider When Selecting a Purification Strategy? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 168 How Much Purity Does Your Application Require?. . . . . . . . 168 How Much Nucleic Acid Can Be Produced from a Given Amount of Starting Material? . . . . . . . . . . . . . . . . . . . . . . . . 168 Do You Require High Molecular Weight Material? . . . . . . . . 168 How Important Is Speed to Your Situation? . . . . . . . . . . . . . 168 How Important Is Cost? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 How Important Is Reproducibility (Robustness) of the Procedure? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169 What Interferes with Nucleic Acid Purification? . . . . . . . . . . 169 What Practices Will Maximize the Quality of DNA Purification?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171 How Can You Maximize the Storage Life of Purified DNA? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 Isolating DNA from Cells and Tissue . . . . . . . . . . . . . . . . . . . . . 172 What Are the Fundamental Steps of DNA Purification?. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172 What Are the Strengths and Limitations of Contemporary Purification Methods? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 174 What Are the Steps of Plasmid Purification? . . . . . . . . . . . . . 180 What Are the Options for Purification after In Vitro Reactions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Spun Column Chromatography through Gel Filtration Resins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic) Filter Cartridges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185 Silica Resin-Based Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 Isolation from Electrophoresis Gels . . . . . . . . . . . . . . . . . . . . 187 What Are Your Options for Monitoring the Quality of Your DNA Preparation? . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191 WHAT CRITERIA COULD YOU CONSIDER WHEN SELECTING A PURIFICATION STRATEGY? How Much Purity Does Your Application Require? What contaminants will affect your immediate and downstream application(s)? As discussed below and in Chapter 1, “Planning for Success in the Laboratory,” time and money can be saved by determining which contaminants need not be removed. For example, some PCR applications might not require extensively purified DNA. Cells can be lysed, diluted, and amplified without any further steps. Another reason to accurately determine purity requirements is that yields tend to decrease as purity requirements increase. How Much Nucleic Acid Can Be Produced from a Given Amount of Starting Material? While it is feasible to mathematically calculate the total amount of nucleic acid in a given sample, and values are provided in the research literature (Sambrook et al., 1989; Studier and Moffat, 1986; Bolivar et al., 1977; Kahn et al., 1979; Stoker et al., 1982), the yields from commercial purification products and noncommercial purification strategies are usually significantly less than these maxima, sometimes less than 50%. Since recoveries will vary with sample origin, consider making your plans based on yields pub- lished for samples similar if not identical to your own. Do You Require High Molecular Weight Material? The average size of genomic DNA prepared will vary between commercial products and between published procedures. How Important Is Speed to Your Situation? Some purification protocols are very fast and allow isolation of nucleic acids within 30 minutes, but speed usually comes at the price of reduced yield and/or purity, especially when working with complex samples. 168 Herzer How Important Is Cost? Reagents obviously figure into the cost of a procedure, but the labor required to produce and apply the reagents of purification should also be considered. How Important Is Reproducibility (Robustness) of the Procedure? Some methods will not give consistent quality and quantity. When planning long-term or high-throughput extractions, validate your methods for consistency and robustness. What Interferes with Nucleic Acid Purification? Nuclease One of the major concerns of nucleic acid purification is the ubiquity of nucleases. The minute a cell dies, the isolation of DNA turns into a race against internal degradation. Samples must be lysed fast and completely and lysis buffers must inactivate nucle- ases to prevent nuclease degradation. Most lysis buffers contain protein-denaturing and enzyme- inhibiting components. DNases are much easier to inactivate than RNases, but care should be taken not to reintroduce them during or after purification. All materials should be autoclaved or baked four hours at 300°F to inactivate DNases and RNases, or you should use disposable materials. Use only enzymes and materials guaranteed to be free of contaminating nucleases. Where appropriate, work on ice or in the cold to slow down poten- tial nuclease activity. Smears and lack of signal, or smeared signal alone, and failure to amplify by PCR are indicative of nuclease contamination. The presence of nuclease can be verified by incubating a small aliquot of your sample at 37°C for a few hours or overnight, followed by evaluation by electrophoresis or hybridization. If nuclease conta- mination is minor, consider repurifying the sample with a proce- dure that removes protein. Shearing Large DNA molecules (genomic DNA, bacterial artificial chro- momoses, yeast artificial chromosomes) can be easily sheared during purification. Avoid vortexing, repeated pipetting (espe- cially through low-volume pipette tips), and any other form of mechanical stress when the isolate is destined for applications that require high molecuar weight DNA. DNA Purification 169 Chemical Contaminants Materials that interfere with nucleic acid isolation or down- stream applications involving the purified DNA can originate from the sample. Plants, molds, and fungi can present a challenge because of their rigid cell wall and the presence of polyphenolic components, which can react irreversibly with nucleic acids to create an unusable final product. The reagents of a DNA purification method can also contribute contaminants to the isolated DNA. Reagents that lyse and solu- bilize samples, such as guanidinium isothiocyanate, can inhibit some enzymes when present in trace amounts. Ethanol precipita- tion of the DNA and subsequent ethanol washes eliminate such a contaminant. Phenol can also be problematic. If you experience problems with DNA purified by a phenol-based strategy, apply chloroform to extract away the phenol. Phenol oxidation products may also damage nucleic acids; hence re-distilled phenol is rec- ommended for purification procedures. A mixture of chloroform and phenol is often employed to maximize the yield of isolated DNA; the chloroform reduces the amount of the DNA-containing aqueous layer at the phenol inter- phase. Similar to phenol, residual chloroform can be problematic, and should be removed by thorough drying. Drying is also employed to remove residual ethanol. Overdried DNA can be difficult to dissolve, so drying should be stopped shortly after the liquid can no longer be observed. Detailed procedures for the above extraction, precipitation and washing steps can be found in Sambrook, Fritsch, and Maniatis (1989) and Ausubel et al. (1998). Ammonium ions inhibit T4 polynucleotide kinase, and chloride can poison translation reactions (Ausubel et al., 1998). The common electrophoresis buffer, TBE (Tris, borate, EDTA) can inhibit enzymes (Ausubel et al., 1998) and interfere with trans- formation due to the increased salt concentration (Woods, 1994). Phosphate buffers may also inhibit some enzymes, namely T4 Polynucleotide kinase (Sambrook et al., 1989), alkaline phos- phatase (Fernley, 1971), Taq DNA polymerase (Johnson et al., 1995), and Poly A polymerase from E. coli (Sippel, 1973). Agarose can also be a problem but some enzyme activity can be recovered by adding BSA to 500 mg/ml final concentration (Ausubel et al., 1998). EDTA can protect against nuclease and heavy metal damage, but could interfere with a downstream application. The anticoagulant heparin can contaminate nucleic acids iso- lated from blood, and should be avoided if possible (Grimberg et al., 1989). Taq DNA polymerase is inhibited by heparin, which 170 Herzer can be resolved by the addition of heparinase (Farnert et al., 1999). Heparin also interacts with chromatin leading to release of denatured/nicked DNA molecules (Strzelecka, Spitkovsky, and Paponov, 1983). Narayanan (1996) reviews the effects of anticoagulants. What Practices Will Maximize the Quality of DNA Purification? The success of DNA purification is dependent on the initial quality of the sample and its preparation. It would be nice to have a simple, straightforward formula that applies to all samples, but some specimens have inherent limitations. The list below will help guide your selection and provide remedies to nonideal situations: 1. Ideally start with fresh sample. Old and necrotic samples complicate purification. In the case of plasmid preparations, cell death sets in after active growth has ceased, which can produce an increase in unwanted by-products such as endotoxins that interfere with purification or downstream application. The best growth phase of bacterial cultures for plasmid pre- parations may be strain dependent. During the log phase of bacterial culture, actively replicating plasmids are present that are “nicked” during replication rather than being supercoiled. Still some researchers prefer mid to late log phase due to the high ratio of DNA to protein and low numbers of dead cells. Others only work with plasmids that have grown just out of log phase to avoid co-purification of nicked plasmid. If old samples can’t be avoided, scaling up the purification can compensate for losses due to degradation. PCR or dot blotting is strongly recommended to document the integrity of the DNA. 2. Process your sample as quickly as possible. There are few exceptions to this rule, one being virus purification. When samples can’t be immediately purified, snap freeze the intact sample in liquid nitrogen or hexane on dry ice (Franken and Luyten, 1976; Narang and Seawright, 1990) or store the lysed extract at -80°C. Commercial products, such as those from Ambion, Inc., can also protect samples from degradation prior to nucleic acid purification. Samples can also be freeze-dried, as discussed below in the question, How Can You Maximize the Storage Life of Purified DNA?. 3. Thorough, rapid homogenization is crucial. Review the lit- erature to determine if your sample requires any special phys- ical or mechanical means to generate the lysate. DNA Purification 171 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss,. . . . . . . 185 Silica Resin-Based Strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . 186 Isolation from Electrophoresis Gels . . . . . . . . . . . . . . . . . . . . 187 What Are. . . . . . . . . . 180 What Are the Options for Purification after In Vitro Reactions? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184 Spun Column Chromatography