武汉大学生命科学学院：《分子生物学》（英文版）7 DNA Purification

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 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? 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 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- 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 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 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 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, 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 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

4. Load the appropriate amount of sample. Nothing will impair the quality and yield of a purification strategy more than overloading the system. Too much sample can cause an increase in the viscosity of the dna preparation and lead to shearing of genomic DNA. If you do not know the exact amount of start ing material, use 60 to 70% of your estimate How Can You Maximize the Storage Life of Purified DNA? The integrity of purified DNA in solution could be c mised by nuclease, pH below 6.0 and above 9.0, heavy metals, UV light, and oxidation by free radicals. EDTA is often added to chelate divalent cations required for nuclease activity and to prevent heavy metal oxidative damage. Tris-based buffers will provide a safe ph of 7 to 8 and will not generate free radicals, as Jane accur with PBS(Miller, Thomas, and Frazier, 1991; Muller and 1993). Free-radical oxidation seems to be a key player in breakdown and ethanol is the best means to control this process (Evans et al., 2000) Low temperatures are also important for long-term stability Storage at 4C is only recommended for short periods( days) (Krajden et al., 1999). Even though some studies have shown that storage under ethanol is safe even at elevated temperatures Sharova, 1977), better stability is obtained at -80C Storage at -20C can lead to degradation, but this breakdown is prevented by the addition of carrier DNA. RNa stored in serum has also been shown to degrade at -20oC(Halfon et aL., 1996) Another approach for intermediate storage is freeze drying DNA-containing samples intact(Takahashi et al., 1995). The DNA within freeze-dried tissue was stable for 6 months, but RNA began degrading after 10 weeks of storage. The control of moisture and temperature had a significant effect on shelf life of samples. The long term stability of DNA-containing samples is still being inves- tigated(Visvikis, Schlenck, and Maurice, 1998), but some compa- nies offer specialized solutions(e.g, RNA Later from Ambion Inc )allowing storage at room temperature. ISOLATING DNA FROM CELLS AND TISSUES What Are the Fundamental Steps of DNAPurification? The fundamental processes of DNa purification from cells and tissues are sample lysis and the segregation of the nucleic acid away from contaminants. While dna is more or less universal to all species, the contaminants and their relative amounts will differ H

4. Load the appropriate amount of sample. Nothing will impair the quality and yield of a purification strategy more than overloading the system.Too much sample can cause an increase in the viscosity of the DNA preparation and lead to shearing of genomic DNA. If you do not know the exact amount of start￾ing material, use 60 to 70% of your estimate. How Can You Maximize the Storage Life of Purified DNA? The integrity of purified DNA in solution could be compro￾mised by nuclease, pH below 6.0 and above 9.0, heavy metals, UV light, and oxidation by free radicals. EDTA is often added to chelate divalent cations required for nuclease activity and to prevent heavy metal oxidative damage. Tris-based buffers will provide a safe pH of 7 to 8 and will not generate free radicals, as can occur with PBS (Miller,Thomas, and Frazier, 1991; Muller and Janz, 1993). Free-radical oxidation seems to be a key player in breakdown and ethanol is the best means to control this process (Evans et al., 2000). Low temperatures are also important for long-term stability. Storage at 4°C is only recommended for short periods (days) (Krajden et al., 1999). Even though some studies have shown that storage under ethanol is safe even at elevated temperatures (Sharova, 1977), better stability is obtained at -80°C. Storage at -20°C can lead to degradation, but this breakdown is prevented by the addition of carrier DNA. RNA stored in serum has also been shown to degrade at -20°C (Halfon et al., 1996). Another approach for intermediate storage is freeze drying DNA-containing samples intact (Takahashi et al., 1995).The DNA within freeze-dried tissue was stable for 6 months, but RNA began degrading after 10 weeks of storage. The control of moisture and temperature had a significant effect on shelf life of samples. The long term stability of DNA-containing samples is still being inves￾tigated (Visvikis, Schlenck, and Maurice, 1998), but some compa￾nies offer specialized solutions (e.g., RNA LaterTM from Ambion, Inc.) allowing storage at room temperature. ISOLATING DNA FROM CELLS AND TISSUES What Are the Fundamental Steps of DNA Purification? The fundamental processes of DNA purification from cells and tissues are sample lysis and the segregation of the nucleic acid away from contaminants. While DNA is more or less universal to all species, the contaminants and their relative amounts will differ 172 Herzer

considerably. The composition of fat cells differs significantly from muscle cells. Plants have to sustain high pressure, contain chloro- plasts packed with chromophores, and often have a very rigid outer cell wall. Bacteria contain lipopolysaccharides that can interfere with purification and cause toxicity problems when present in downstream applications. Fibrous tissues such as heart and skeletal muscle are tough to homogenize. These variations have to be taken into consideration when developing or selecting a lysis method Lysis Detergents are used to solubilize the cell membranes. Popular choices are SDS, Triton X-100, and CTAB(hexadecyltrimethyl ammonium bromide). CTAB can precipitate genomic DNA, and it is also popular because of its ability to remove polysaccharides from bacterial and plant preparations(Ausubel et al., 1998 Enzymes attacking cell surface components and/or components of the cytosol are often added to detergent-based lysis buffers ysozyme digests cell wall components of gram-positive bacteria ymolase, and murienase aid in protoplast production from east cells. Proteinase K cleaves glycoproteins and inactivates(to some extent) RNase/DNase in 0.5 to 1% SDS solutions. Heat is also applied to enhance lysis. Denaturants such as urea, guani dinium salts, and other chaotropes are applied to lyse cells and inactivate enzymes, but extended use beyond what is recom mended in a procedure can lead to a reduction in quality and Sonication, grinding in liquid nitrogen, shredding devices such as rigid spheres or beads, and mechanical stress such as filtration have been used to lyse difficult samples prior to or in conjunc- tion with lysis solutions. Disruption methods are discussed at http://www.thescientist.com/yr199%/nov/profile2_981109.html Segregation of DNa from Contaminants The separation of nucleic acid from contaminants are discussed below within the question, What Are The Strengths and Limita tions of Contemporary Purification Methods? DNA Precipitation To concentrate nucleic acids for resuspension in a more suitable buffer, solvents such as ethanol(75-80%)or isopropanol(final concentration of 40-50%)are commonly used in the presence of salt to precipitate nucleic acids(Sambrook, Fritsch, and Maniatis, DNA Purification 173

considerably.The composition of fat cells differs significantly from muscle cells. Plants have to sustain high pressure, contain chloro￾plasts packed with chromophores, and often have a very rigid outer cell wall. Bacteria contain lipopolysaccharides that can interfere with purification and cause toxicity problems when present in downstream applications. Fibrous tissues such as heart and skeletal muscle are tough to homogenize. These variations have to be taken into consideration when developing or selecting a lysis method. Lysis Detergents are used to solubilize the cell membranes. Popular choices are SDS, Triton X-100, and CTAB(hexadecyltrimethyl ammonium bromide). CTAB can precipitate genomic DNA, and it is also popular because of its ability to remove polysaccharides from bacterial and plant preparations (Ausubel et al., 1998). Enzymes attacking cell surface components and/or components of the cytosol are often added to detergent-based lysis buffers. Lysozyme digests cell wall components of gram-positive bacteria. Zymolase, and murienase aid in protoplast production from yeast cells. Proteinase K cleaves glycoproteins and inactivates (to some extent) RNase/DNase in 0.5 to 1% SDS solutions. Heat is also applied to enhance lysis. Denaturants such as urea, guani￾dinium salts, and other chaotropes are applied to lyse cells and inactivate enzymes, but extended use beyond what is recom￾mended in a procedure can lead to a reduction in quality and yield. Sonication, grinding in liquid nitrogen, shredding devices such as rigid spheres or beads, and mechanical stress such as filtration have been used to lyse difficult samples prior to or in conjunc￾tion with lysis solutions. Disruption methods are discussed at http://www.thescientist.com/yr1998/nov/profile2_981109.html. Segregation of DNA from Contaminants The separation of nucleic acid from contaminants are discussed below within the question, What Are The Strengths and Limita￾tions of Contemporary Purification Methods? DNA Precipitation To concentrate nucleic acids for resuspension in a more suitable buffer, solvents such as ethanol (75–80%) or isopropanol (final concentration of 40–50%) are commonly used in the presence of salt to precipitate nucleic acids (Sambrook, Fritsch, and Maniatis, DNA Purification 173

1989; Ausubel et al, 1998). If volume is not an issue, ethanol preferred because less salt will coprecipitate and the pellet is more easily dried. Polyethylene glycol(PEG) selectively precipi- tates high molecular weight DNA, but it is also more difficult to dry and can interfere with downstream applications(Hillen Klein, and Wells, 1981). Trichloroacetic acid (TCA) precipitates evenlowMwpolymersdownto(5kda)(http://biotech- server biotech.ubc. ca/biotech/bisc437/lecture/e-na-isoIn/ na-isoin3. html), but nucleic acids cannot be recovered in a fund tional form after precipitation Salt is essential for DNA precipitation because its cations counteract the repulsion caused by the negative charges of the phosphate backbone. Ammonium acetate is useful because it is volatile and easily removed, and at high concentration it selec- tively precipitates high molecular weight molecules. Lithium chlo- ride is often used for RNa because Li* does not precipitate double-stranded DNA, proteins, or carbohydrates, although the single-stranded nucleic acids must be above 300 nucleotides. To efficiently precipitate nucleic acids, incubation at low tem peratures(preferably <-20oC)for at least 10 minutes is required followed by centrifugation at 12,000 xg for at least five minutes. Temperature and time are crucial for nucleic acids at low con- centrations, but above 0. 25 mg/ml, precipitation may be carried out at room temperature. Additional washing steps with 70% ethanol will remove residual salt from pelleted DNA. Pellets are dried in a speed vac or on the bench and are resuspended in water or TE(10mM Tris, I mM EDTA) Do not attempt to precipitate nucleic acids below a concentration of 20ng/ml unless carrier such as RNA, DNA, or a high molecular weight co-precipitant like glycogen is added. In the range from 20ng/ml to 10/ml, either add carrier or extend precipitation time, and add more ethanol. Polyethylene glycol (PEG) precipitation is even more concentra- tion dependent and will only work at DNA concentrations above 10 ug/ml (Lis and Schleif, 1975). Pellets will dissolve better in low salt buffers (water or TE)and at concentrations below 1 mg/ml Gentle heating can also help to redissolve nucleic acids What Are the Strengths and Limitations of Contemporary Purification methods? Salting out and DNA Precipitation Mechanism Some of the first dna isolation methods were based on the use of chaotropes and cosmotropes to separate cellular components Herzer

1989; Ausubel et al., 1998). If volume is not an issue, ethanol is preferred because less salt will coprecipitate and the pellet is more easily dried. Polyethylene glycol (PEG) selectively precipi￾tates high molecular weight DNA, but it is also more difficult to dry and can interfere with downstream applications (Hillen, Klein, and Wells, 1981). Trichloroacetic acid (TCA) precipitates even low MW polymers down to (5 kDa) (http://biotech￾server.biotech.ubc.ca/biotech/bisc437/lecture/e-na-isoln/ na-isoln3.html), but nucleic acids cannot be recovered in a func￾tional form after precipitation. Salt is essential for DNA precipitation because its cations counteract the repulsion caused by the negative charges of the phosphate backbone. Ammonium acetate is useful because it is volatile and easily removed, and at high concentration it selec￾tively precipitates high molecular weight molecules. Lithium chlo￾ride is often used for RNA because Li+ does not precipitate double-stranded DNA, proteins, or carbohydrates, although the single-stranded nucleic acids must be above 300 nucleotides. To efficiently precipitate nucleic acids, incubation at low tem￾peratures (preferably £-20°C) for at least 10 minutes is required, followed by centrifugation at 12,000 ¥ g for at least five minutes. Temperature and time are crucial for nucleic acids at low con￾centrations, but above 0.25 mg/ml, precipitation may be carried out at room temperature. Additional washing steps with 70% ethanol will remove residual salt from pelleted DNA. Pellets are dried in a speed vac or on the bench and are resuspended in water or TE (10mM Tris, 1mM EDTA). Do not attempt to precipitate nucleic acids below a concentration of 20 ng/ml unless carrier such as RNA, DNA, or a high molecular weight co-precipitant like glycogen is added. In the range from 20ng/ml to 10mg/ml, either add carrier or extend precipitation time, and add more ethanol. Polyethylene glycol (PEG) precipitation is even more concentra￾tion dependent and will only work at DNA concentrations above 10mg/ml (Lis and Schleif, 1975). Pellets will dissolve better in low￾salt buffers (water or TE) and at concentrations below 1 mg/ml. Gentle heating can also help to redissolve nucleic acids What Are the Strengths and Limitations of Contemporary Purification Methods? Salting out and DNA Precipitation Mechanism Some of the first DNA isolation methods were based on the use of chaotropes and cosmotropes to separate cellular components 174 Herzer

based on solubility differences(Harrison, 1971; Lang, 1969).A chaotrope increases the solubility of molecules("salting-in")by changing the structure of water, and as the name suggests, the driving force is an increase in entropy. A cosmotrope is a structure-maker; it will decrease the solubility of a molecule (salting-out"). Guanidium salts are common chaotropes applied in DNA purification. Guanidinium isothiocyanate is the most potent because both cation and anion components are chaotropic Typical lyotropes used for salting out proteins are ammonium and potassium sulfate or acetate. An all solution based nucleic acid purification can be performed by differentially precipitating con taminants and nucleic acids Cells are lysed with a gentle enzyme-or detergent-based buffe (often SDS/proteinase K). A cosmotrope such as potassium acetate is added to salt out protein, SDS, and lipids but not the bulk of nucleic acids. The white precipitate is then removed by centrifugation. The remaining nucleic acid solution is too dilute and in a buffer incompatible with most downstream applications, o the dna is next precipitated as described above Features Protocols and commercial products differ mainly in lysis buffe composition. Yields are generally good, provided that sample lysis was complete and dNa precipitation was thorough. These proce- dures apply little mechanical stress, so shearing is generally not a problem Limitations If phenolic contaminants (i.e, from plants) are a problem adding 1% polyvinylpyrrolidone to your extraction buffer can absorb them (John, 1992; Pich and Schubert, 1993; Kim et al 1997). Alternatively, add a CTAB precipitation step to remove polysaccharides(Ausubel et al., 1998) Extraction with Organic Solvents, Chaotropes, and DNA Precipitation Mechanism Chaotropic guanidinium salts lyse cells and denature proteins, and reducing agents(B-mercaptoethanol, dithiothreitol) prevent oxidative damage of nucleic acids. Phenol, which solubilizes and extracts proteins and lipids to the organic phase, sequestering them away from nucleic acids, can be added directly to the lysis buffer, or a phenol step could be included after lysis with either DNA Purification l75

based on solubility differences (Harrison, 1971; Lang, 1969). A chaotrope increases the solubility of molecules (“salting-in”) by changing the structure of water, and as the name suggests, the driving force is an increase in entropy. A cosmotrope is a structure-maker; it will decrease the solubility of a molecule (“salting-out”). Guanidium salts are common chaotropes applied in DNA purification. Guanidinium isothiocyanate is the most potent because both cation and anion components are chaotropic. Typical lyotropes used for salting out proteins are ammonium and potassium sulfate or acetate. An all solution based nucleic acid purification can be performed by differentially precipitating con￾taminants and nucleic acids. Cells are lysed with a gentle enzyme- or detergent-based buffer (often SDS/proteinase K). A cosmotrope such as potassium acetate is added to salt out protein, SDS, and lipids but not the bulk of nucleic acids. The white precipitate is then removed by centrifugation. The remaining nucleic acid solution is too dilute and in a buffer incompatible with most downstream applications, so the DNA is next precipitated as described above. Features Protocols and commercial products differ mainly in lysis buffer composition. Yields are generally good, provided that sample lysis was complete and DNA precipitation was thorough. These proce￾dures apply little mechanical stress, so shearing is generally not a problem. Limitations If phenolic contaminants (i.e., from plants) are a problem, adding 1% polyvinylpyrrolidine to your extraction buffer can absorb them (John, 1992; Pich and Schubert, 1993; Kim et al., 1997). Alternatively, add a CTAB precipitation step to remove polysaccharides (Ausubel et al., 1998). Extraction with Organic Solvents, Chaotropes, and DNA Precipitation Mechanism Chaotropic guanidinium salts lyse cells and denature proteins, and reducing agents (b-mercaptoethanol, dithiothreitol) prevent oxidative damage of nucleic acids. Phenol, which solubilizes and extracts proteins and lipids to the organic phase, sequestering them away from nucleic acids, can be added directly to the lysis buffer, or a phenol step could be included after lysis with either DNA Purification 175

GTC- or SDs-based buffers as above. GTC/phenol buffers often require vortexing or vigorous mixing The affinity of nucleic acids for this two-phase extraction system is pH dependent. Acidic phenol is applied in RNA extractions because DNA is more soluble in acidic phenol; smaller DNA mol ecules(50kb)will be found in the organic phase and larger DNA molecules(>50kb)in the interphase. When purifying RNA via this procedure, it is essential to shear the dNa to ensure a light interphase Phenol titrated to a ph of 8 is used to separate dNA from pro- teins and lipids, since DNA is insoluble in basic phenol. Whether protocols call for a GtC/phenol, a GTC, or an SDs based step followed by phenol, it is best to follow a phenol extraction with chloroform in order to extract residual phenol from the aqueous phase. Phenol is highly soluble in chloroform, and chloroform is not water soluble Remaining lipids may also be removed by this step Phenol extractions are followed by nucleic acid precipitation steps as described above. Features Though caustic and toxic, this strategy still has wide use because vield, purity, and speed are good, and convenient for working with all numbers of samples. Limitations If lysis is incomplete, the interphase between organic and aqueous layers becomes very heavy and difficult to manipulate, and may trap DNA Phenol is not completely insoluble in water, so if chloroform steps are skipped, residual phenol can remain and am applications. High salt concentrations can also lead to phase inversion, where the aqueous phase is no longer on top(problematic if colorless phenol is used ). Diluting he aqueous phase and increasing the amount of phenol will correct this inversion. When working with GTC/phenol-based extraction buffers. cross-contamination of rna with dna. and vice versa, is frequent. Glass Milk/Silica Resin-Based Strategies Mechanism Nucleic acids bind to glass milk and silica resin under denatur ing conditions in the presence of salts( Vogelstein and Gillespie, 1979). Recent findings indicate that binding of some nucleic 176 Herzer

GTC- or SDS-based buffers as above. GTC/phenol buffers often require vortexing or vigorous mixing. The affinity of nucleic acids for this two-phase extraction system is pH dependent. Acidic phenol is applied in RNA extractions because DNA is more soluble in acidic phenol; smaller DNA mol￾ecules (50 kb) in the interphase. When purifying RNA via this procedure, it is essential to shear the DNA to ensure a light interphase. Phenol titrated to a pH of 8 is used to separate DNA from pro￾teins and lipids, since DNA is insoluble in basic phenol. Whether protocols call for a GTC/phenol, a GTC, or an SDS based step followed by phenol, it is best to follow a phenol extraction with chloroform in order to extract residual phenol from the aqueous phase. Phenol is highly soluble in chloroform, and chloroform is not water soluble. Remaining lipids may also be removed by this step. Phenol extractions are followed by nucleic acid precipitation steps as described above. Features Though caustic and toxic, this strategy still has wide use because yield, purity, and speed are good, and convenient for working with small numbers of samples. Limitations If lysis is incomplete, the interphase between organic and aqueous layers becomes very heavy and difficult to manipulate, and may trap DNA. Phenol is not completely insoluble in water, so if chloroform steps are skipped, residual phenol can remain and interfere with downstream applications. High salt concentrations can also lead to phase inversion, where the aqueous phase is no longer on top (problematic if colorless phenol is used). Diluting the aqueous phase and increasing the amount of phenol will correct this inversion. When working with GTC/phenol-based extraction buffers, cross-contamination of RNA with DNA, and vice versa, is frequent. Glass Milk/Silica Resin-Based Strategies Mechanism Nucleic acids bind to glass milk and silica resin under denatur￾ing conditions in the presence of salts (Vogelstein and Gillespie, 1979). Recent findings indicate that binding of some nucleic 176 Herzer