2DNA Purification Requirements Many applications require purified DNA.Purity and amount of DNA required (and process used) depends on intended application.Example applications:Tissue typing for organ transplantDetection of pathogensHuman identity testingGenetic researchPurified DNA is an essential requirement for molecular biology, molecular diagnostic and research applications.Different applications have different requirements for purity. For example, contaminants in purified DNA can inhibit the polymerase chain reaction or inhibit cutting of DNA by restriction enzymes. Although PCR can be performed on crude extracts, sensitive assays such as quantitative PCR and most diagnostic applications require a high degree of purity.
3DNA Purification Challenges Separating DNA from other cellular components such as proteins, lipids, RNA, etc.Avoiding fragmentation of the long DNA molecules by mechanical shearing or the action of endogenous nucleases. Effectively inactivating endogenous nucleases (DNase enzymes) and preventing them from digesting the genomic DNA is a key early step in the purification process. DNases can usually be inactivated by use of heat or chelating agents.Challenges in DNA purification include:1. Separation of DNA from the rest of the cellular components. Ideally, the final DNA preparation should be as pure as possible.2. Preservation of DNA molecules during the purification process.Inactivation of nucleases is key to successful DNA purification. Endogenous DNase enzymes will quickly degrade DNA and must be inactivated effectively. Most DNA purification methods use chelating agents or heat to inactivate endogenous nucleases. Chelating agents sequester magnesium ions, which are essential for nuclease function. Heat denatures proteins, and therefore inactivates nuclease function.
4Quality is ImportantBest yields are obtained from fresh or frozen materials.Blood/Tissues must be processed correctly to minimize destruction of DNA by endogenous nucleases.DNA yield will be reduced if endogenous nucleases are active.Prompt freezing, immediate processing or treatment with chelating agents (such as EDTA) minimizes nuclease effects.It is important to process starting materials quickly to minimize DNA degradation by endogenous nuclease activity. If blood or tissues are not processed promptly they should be frozen until use. Some tissues require preprocessing such as mechanical grinding prior to use. Tissue can be ground up and frozen prior to processing.
5Nucleic Acid Purification There are many DNA purification methods. All must:Effectively disrupt cells or tissues (usually using detergent)Denature proteins and nucleoprotein complexes (a protease/denaturant)Inactivate endogenous nucleases (chelating agents)Purify nucleic acid target away from other nucleic acids and protein (could involve RNases, proteases, selective matrix and alcohol precipitations)All DNA purification methods accomplish the steps listed here but use different methods to perform each step. The cells and nuclei must be lysed to release DNA, proteins must be denatured and nucleases inactivated. Finally, the DNA must be effectively separated from the other cellular components.
6Disruption of Cells/Tissues Most purification methods disrupt cells using lysis buffer containing:Detergent to disrupt the lipid bilayer of the cell membraneDenaturants to release chromosomal DNA and denature proteinsAdditional enzymes are required for lysis of some cell types:Gram-positive bacteria require lysozyme to disrupt the bacterial cell wall.Yeasts require addition of lyticase to disrupt the cell wall.Plant cells may require cellulase pre-treatment.Most mammalian cells and cultured cells can be lysed easily using detergents. Organisms that have cell walls in addition to a cell membrane usually require treatment with additional enzymes to effectively disrupt the cell wall. For example, lysozyme is used to digest the thick peptidoglycan cell wall of Gram-positive bacteria and Lyticase (or zymolase) enzyme is used to disrupt the polysaccharide cell wall of yeasts. For some plant species, mechanical disruption of tissues in addition to detergent treatment is sufficient. For other plant species, use of cellulase enzyme is required to disrupt the cell wall structure and maximize DNA yield.
7Disruption of Cells: Membrane Disruption Detergents are used to disrupt the lipid:lipid and lipid:protein interactions in the cell membrane, causing solubilization of the membrane.Ionic detergents (such as sodium dodecyl sulfate; SDS) also denature proteins by binding to charged residues, leading to local changes in conformation.When the lipid membrane is disrupted by the action of detergent, the contents of the cell are released. Ionic detergents can denature proteins by binding to charged side chains, causing local changes in protein conformation and consequent loss of function.
8Protein DenaturationDenaturation = Modification of conformation to unfold protein, disrupting secondary structure but not breaking the peptide bonds between amino acid residues.Denaturation results in:Decreased protein solubilityLoss of biological activityImproved digestion by proteasesRelease of chromosomal DNA from nucleoprotein complexes (“unwinding” of DNA and release from associated histones)In the mammalian cell, DNA is typically present in a nucleoprotein complex (chromatin), where the DNA is wound around proteins called histones. This supercoiling of DNA allows the 1.8m of genomic DNA in a human cell to fit into the nucleus. More about chromatin structure is available in Alberts et al, Molecular Biology of the Cell, Garland Publishing, 1994, pages 342–46.
9Protein Denaturing Agents Ionic detergents, such as SDS, disrupt hydrophobic interactions and hydrogen bonds.Chaotropic agents such as urea and guanidine disrupt hydrogen bonds.Reducing agents break disulfide bonds.Salts associate with charged groups and at low or moderate concentrations increase protein solubility.Heat disrupts hydrogen bonds and nonpolar interactions.Some DNA purification methods incorporate proteases such as proteinase K to digest proteins.More information on protein denaturation is available at:
10Inactivation of Nucleases Chelating agents, such as EDTA, sequester Mg2+ required for nuclease activity.Proteinase K digests and destroys all proteins, including nucleases.Some commercial purification systems provide a single solution for cell lysis, protein digestion/denaturation and nuclease inactivation.
11Removal of RNASome procedures incorporate RNase digestion during cell lysate preparation.In other procedures, RNase digestion is incorporated during wash steps.
12Separation of DNA from Crude Lysate DNA must be separated from proteins and cellular debris.Separation MethodsOrganic extractionSalting outSelective DNA binding to a solid support
13Separation by Organic Extraction DNA is polar and therefore insoluble in organic solvents.Traditionally, phenol:chloroform is used to extract DNA.When phenol is mixed with the cell lysate, two phases form. DNA partitions to the (upper) aqueous phase, denatured proteins partition to the (lower) organic phase.DNA is a polar molecule because of the negatively charged phosphate backbone.This polarity makes it more soluble in the polar aqueous phase.More about how phenol extraction works at: bitesizebio.com/2008/02/12/
14Separation by Salting Out Salts associate with charged groups.At high salt concentration, proteins are dehydrated, lose solubility and precipitate. Usually sodium chloride, potassium acetate or ammonium acetate are used.Precipitated proteins are removed by centrifugation.DNA remains in the supernatant.Salting out is thought to dehydrate the environment around the protein, causing the surface of the protein to become less hydrophilic and forcing the protein to come out of solution. More information is available at:
15Ethanol Precipitation of DNA Methods using organic extraction or salting-out techniques result in an aqueous solution containing DNA.The DNA is precipitated out of this solution using salt and isopropanol or ethanol.Salt neutralizes the charges on the phosphate groups in the DNA backbone.The alcohol (having a lower dielectric constant than water) allows the sodium ions from the salt to interact with the negatively charged phosphate groups closely enough to neutralize them and let the DNA fall out of solution.Nucleic acids are hydrophilic due to the negatively charged phosphate groups in the phosphate-sugar backbone of the molecule. The positively charged sodium or ammonium ions in the salt neutralize the negatively charged phosphate groups on the DNA backbone. In the presence of alcohol, the positively charged ion is brought into close enough proximity to the phosphate groups to neutralize the charge and cause the DNA molecule to become less polar (more hydrophobic) and come out of solution.
16Separation by Binding to a Solid Support Most modern DNA purification methods are based on purification of DNA from crude cell lysates by selective binding to a support material.Support MaterialsSilicaAnion-exchange resinAdvantagesSpeed and convenienceNo organic solventsAmenable to automation/miniaturizationDNA purification column containing a silica membrane
17SilicaDNA binds selectively to silica in the presence of high concentrations of chaotropic salts (e.g., guanidinium HCl).Protein does not bind under these conditions.Silica membranes or columns are washed with an alcohol-based solution to remove the salts.DNA is eluted from the membrane with a low-ionic-strength solution, such as a low-salt buffer or water.AdvantagesFast purificationAmenable to automationNo centrifugation required (can use vacuum)No organic solvents or precipitation steps
18Magnetic Separation: Silica or Charge-Based Several commercial systems are based on capture of DNA from solution using magnetic particles.Magnetic Particle TypesSilica-based (bind/release DNA depending on salt concentration)Charge-based (particle charge changes based on pH of solution, binding/releasing negatively charged DNA).AdvantagesFastNo organic extraction or precipitationsAmenable to automationClose-up view of the silica-coated surface of a magnetic bead
19Anion-Exchange Columns Based on interaction between negatively charged phosphates in DNA and positively charged particles.DNA binds under low-salt conditions.Protein and RNA are washed away using higher salt buffers.DNA is eluted with high salt (neutralizes negative charge on DNA).Eluted DNA is recovered by ethanol precipitation.AdvantagesNo organic solventsFast, but more hands-on than silica (requires ethanol)
20Example: Solution-Based Protocol This example uses an easy, solution-based approach to cell lysis, protein denaturation and DNA purification. Centrifugation is used to remove precipitated materials from solution.View Animation
21Example: Silica Membrane-Based Protocol Here is an example protocol using a silica membrane to capture DNA. Centrifugation or vacuum pressure is used to pull materials through the membrane.View Animation
22DNA QuantitationOnce DNA is purified, it is usually quantified. Typical quantities are in the milligram-picogram rangegram (g)milligram (mg) = 10-3 g; 0.001gmicrogram (µg) = 10-3 mg; gnanogram (ng) = 10-3 µg; 10-6 mg; gpicogram (pg) = 10-3 ng; 10-6 µg; 10-9 mg; g
23Quantification Methods Spectrophotometry: Use of light absorbance to measure concentration. Many biological substances absorb light. The spectrophometer measures absorbance of light at specific wavelengthsMost commonly used methodDNA concentration can be calculated based on absorbance at 260 nm: A = e × c × l (Beer-Lambert Law) A = absorbance e = extinction coefficient c = concentration l = path length
25DNA QuantificationHigh-sensitivity fluorescence methods are also availablePicoGreen® method. Mix sample with reagent, wait 5 minutes and read with a fluorimeter.Lower sensitivity methodsEstimation of concentration based on comparison to a known concentration standard on an agarose gel.StandardDNA Sample
26Summary DNA purification methods all do the following: Disrupt cells and denature/digest of proteinsSeparate DNA from proteins, RNA and other cellular componentsPrepare a purified DNA solutionOlder methods relied on laborious organic extraction and precipitation procedures.Newer methods are faster, using selective binding of DNA to silica or magnetic beads, and are amenable to automation and miniaturization.