Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein opyright◎2001 ISBNS:0-471-37972-7( Paper);0-47 (Electronic) 7 DNA Purification Sibylle He What Criteria Could You Consider When Selecting Purification Strategy ...68 How Much Purity Does Your Application uire How Much nucleic Acid Can be produced from a given Amount of Starting Material? Do You Require High Molecular Weight Material?........ 168 How Important ls Speed to Your Situation? How Important Is Cost? 169 How Important Is Reproducibility(Robustness)of the Procedure? What Interferes with nucleic acid Purification? What Practices Will Maximize the Quality of DNA Purification? How Can You Maximize the Storage Life of Purified DNA Isolating DNA from Cells and Tissue What Are the Fundamental Steps of dna Purification? 72 What Are the Strengths and Limitations of ContemporarY O Purification Methods? What Are the Steps of Plasmid Purification? What Are the Options for Purification after In Vitro Reactions? Spun Column Chromatography through Gel Filtration Resins 167
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 Silica Resin-Based Strategies Isolation from Electrophoresis Gels What Are Your Options for Monitoring the Quality of Your dNA Preparation Bibliography 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 ucleic acids within 30 minutes, but speed usually comes at the price of reduced yield and/or purity, especially when working with complex samples. Herzer
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 published 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? 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 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 37C 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 moses, 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
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 nucleases to prevent nuclease degradation. Most lysis buffers contain protein-denaturing and enzymeinhibiting 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 potential 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 contamination is minor, consider repurifying the sample with a procedure that removes protein. Shearing Large DNA molecules (genomic DNA, bacterial artificial chromomoses, yeast artificial chromosomes) can be easily sheared during purification. Avoid vortexing, repeated pipetting (especially 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- tream 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 c The reagents of a DNA purification method can also contribute ontaminants 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 phenolPhenol 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 hibit 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 ) and Poly a polymerase from E coli (Sippel, 1973). Ag can also be a problem but some enzyme activity can be recovered by adding BSa to 500 ug/ml final concentration(Ausubel et al 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 H
Chemical Contaminants Materials that interfere with nucleic acid isolation or downstream 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 solubilize samples, such as guanidinium isothiocyanate, can inhibit some enzymes when present in trace amounts. Ethanol precipitation 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 recommended 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 interphase. 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 transformation due to the increased salt concentration (Woods, 1994). Phosphate buffers may also inhibit some enzymes, namely T4 Polynucleotide kinase (Sambrook et al., 1989), alkaline phosphatase (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 500mg/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 isolated 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, ce death sets in after active growth has ceased, which can produc 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 hase to avoid co-purification of nicked plasmid If old samples can't be avoided, scaling up the purification can ompensate 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 cant 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 -80oC. 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
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 preparations 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 literature to determine if your sample requires any special physical or mechanical means to generate the lysate. DNA Purification 171