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 starting 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 compromised 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 investigated (Visvikis, Schlenck, and Maurice, 1998), but some companies 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 chloroplasts 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, guanidinium salts, and other chaotropes are applied to lyse cells and inactivate enzymes, but extended use beyond what is recommended 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 conjunction 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 Limitations 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 precipitates 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://biotechserver.biotech.ubc.ca/biotech/bisc437/lecture/e-na-isoln/ na-isoln3.html), but nucleic acids cannot be recovered in a functional 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 selectively precipitates high molecular weight molecules. Lithium chloride 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 temperatures (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 concentrations, 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 concentration dependent and will only work at DNA concentrations above 10mg/ml (Lis and Schleif, 1975). Pellets will dissolve better in lowsalt 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 contaminants 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 procedures 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 molecules (<50 kb) will be found in the organic phase and larger DNA molecules (>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 proteins 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 denaturing conditions in the presence of salts (Vogelstein and Gillespie, 1979). Recent findings indicate that binding of some nucleic 176 Herzer