(Updated) Nucleic Acid Extraction Bulleted.pdf

Nucleic Acid Extraction the process of isolating DNA from other cellular materials like proteins and membranes. It's a crucial initial step in molecular techniques as downstream analysis relies heavily on the quality of the extracted DNA. Sources of DNA Plasmid DNA Viral...

Nucleic Acid Extraction the process of isolating DNA from other cellular materials like proteins and membranes. It's a crucial initial step in molecular techniques as downstream analysis relies heavily on the quality of the extracted DNA. Sources of DNA Plasmid DNA Viral Nucleic Acids Genomic DNA from Blood and Biological Fluids Genomic DNA from Tissue and Cells Genomic DNA from Forensic Samples Genomic DNA from Plant and Fungi Genomic DNA from Food and Feed Ancient DNA DNA Isolation/Extraction: A critical part of molecular techniques due to the reliance of downstream procedures and analysis on the quality of the DNA used. It involves separating DNA from other cellular components like proteins and membranes. DNA was first isolated in 1869 by Frederich Miescher and is now routinely performed for various molecular methods, including clinical diagnostic applications. The process aims to remove potential inhibitors to PCR amplification and produce a stable, high-quality DNA solution suitable for long-term storage. Principles of DNA Isolation: Key steps include: ○ Breaking cells open (cell disruption or lysis) to expose the DNA. ○ Removing membrane lipids using a detergent. ○ Removing proteins with a protease (optional but commonly done). ○ Precipitating the DNA with an alcohol (usually ice-cold ethanol or isopropanol). ○ Resolubilizing the DNA in a slightly alkaline buffer or ultrapure water (optional). Additional points: ○ Adding a chelating agent binds divalent cations (Mg2+ and Ca2+) to inhibit DNase activity. ○ Cellular and histone proteins bound to DNA can be removed by protease, precipitation with sodium/ammonium acetate, or phenol-chloroform extraction. DNA Isolation/Extraction Process: Generally involves three basic steps: ○ Lysis of cells to release DNA.  ○ Separation of DNA from other cell components. ○ Isolation of the purified DNA. Basic Steps in Isolating DNA: The typical workflow includes: ○ Separating white blood cells (WBCs) from red blood cells (RBCs) if necessary. ○ Lysing WBCs or other nucleated cells. ○ Denaturing/digesting proteins. ○ Separating contaminants (e.g., proteins, heme) from DNA. ○ Precipitating DNA if needed. ○ Resuspending DNA in the final buffer. Lysis of Cells: The first step is to release DNA from cells, nuclei, or organisms. Lysis techniques include: ○ Chemical lysis: Using chaotropic agents, detergents, salts, or strong bases. ○ Enzymatic lysis: Employing enzymes like proteinase K and lysozyme to target cell proteins. ○ Physical lysis: Utilizing external forces like milling, sonication, boiling, or freeze-thaw cycling. The choice of lysis method depends on the complexity of the cells being lysed. Separation of DNA from Other Cell Components: Protein contamination is common due to the presence of histones and other proteins. Protein removal is crucial as proteins can hinder DNA analysis. Methods for protein removal include: ○ Detergents: SDS and Triton X-100 solubilize proteins. ○ Chaotropic acids: Guanidine hydrochloride and guanidinium thiocyanate denature proteins. ○ Protease digestion: Proteinase K breaks down proteins into smaller molecules. Isolation of the DNA: Two primary separation methods: ○ Liquid-liquid extraction ○ Solid-phase extraction Liquid-Liquid Extraction: Involves liquid phase separation and precipitation based on the differential solubility of DNA in immiscible liquids.  Phenol, often mixed with chloroform and isoamyl alcohol, is commonly used. Chloroform/isoamyl alcohol helps in phase separation and prevents foaming. DNA is precipitated from the aqueous phase using alcohol. This method is effective but labor-intensive and involves hazardous chemicals. DNA precipitation: ○ Alcohols (ethanol, isopropanol) or salts (sodium chloride, ammonium chloride, ammonium acetate) are used to precipitate DNA, yielding a pure and concentrated product. ○ Other precipitating agents include acetone and lithium chloride. ○ The DNA pellet is obtained by centrifugation, dried, and resuspended in buffer or ultrapure water. Solid-Phase Extraction: Separates DNA based on size or affinity. It's favored for being less hazardous, easier, adaptable to automation, and suitable for high-volume sample preparation. Basic steps: lysis, binding, washing, and eluting. Solid-Phase Extraction Techniques: Gel filtration Ion-exchange chromatography Affinity chromatography Gel Filtration:  Separates DNA by size exclusion using a gel matrix (e.g., Sephadex (most common)) in a spin column Larger molecules pass through while smaller ones are retained Ion-Exchange Chromatography: Based on the selective binding of negatively charged DNA to positively charged surfaces Commonly uses diethylaminoethyl cellulose (DEAE-C) resin DNA is released by displacement with free ions Affinity Chromatography: The most common method, utilizes reversible adsorption of DNA to silica surfaces under specific conditions (chaotropic salts) Binding occurs due to adsorption and hydrogen bonding DNA is released by removing salt/alcohol and hydrating the surfaces Silica-Gel-Membrane Technology: A procedure based on binding nucleic acids to a silica-gel membrane in the presence of chaotropic salts Contaminants are washed away, and pure nucleic acids are eluted under low- or no-salt conditions Advantages: fast, convenient, economical, avoids toxic reagents and alcohol precipitations DNA Extraction from Different Sources The ease of DNA extraction varies depending on the cell type: ○ Prokaryotic cells (bacteria) are simpler, with a lipid bilayer and cytoplasm containing a circular chromosome. Lysis releases the DNA for extraction ○ Eukaryotic cells (humans, animals, plants) have a lipid bilayer, cytoplasm, and membrane-bound organelles, including the nucleus with chromosomes Specific Considerations ○ Bacteria: Cell wall differences between Gram-positive and Gram-negative bacteria influence extraction methods ○ Plants: Cell walls and high polysaccharide/polyphenol content complicate extraction ○ Humans/Animals: Lack of cell walls makes lysis and extraction easier DNA Sources and Yields ○ Typical DNA amounts vary based on the sample type (e.g., oral swab: 100-1500 ng, tissue: 50-500 ng per gram) ○ Viral DNA: Extracted from tissue cultures or directly from virus particles ○ Plasmid DNA: Isolated from bacterial cultures, often using the alkaline lysis method DNA Extraction from Whole Blood Ficoll-Directed Density Gradient Centrifugation: ○ Separates blood components based on density using Ficoll-Hypaque medium ○ WBCs, containing DNA, are collected from a specific layer Quick Extraction Methods: ○ Proteinase K Protocol: Overnight digestion of blood with proteinase K and other reagents at 37°C ○ Phenol Method: Mixing blood with Tris-HCl-saturated phenol and water, followed by shaking and centrifugation Proteinase K: A broad-spectrum serine protease used in molecular biology Digests protein and removes contamination from nucleic acid preparations Inactivates nucleases that can degrade DNA/RNA during purification Active in the presence of denaturants (SDS, urea) and protease inhibitors Stable over a wide pH range (4-12) Phenol-Chloroform Extraction: A liquid-liquid extraction technique for isolating DNA, RNA, and protein Relies on phase separation by centrifugation of an aqueous sample and a phenol:chloroform:guanidinium thiocyanate solution  RNA is in the aqueous phase, DNA at the interphase, and protein in the organic phase Can be used for DNA purification alone in the absence of guanidinium thiocyanate DNA Extraction from Dry Blood Spots: This method involves extracting DNA from dried blood spots on filter paper It's useful for various applications, including newborn screening and forensic analysis Noninvasive Human DNA Isolation: Hair: ○ Hair with root is incubated in NaOH buffer or digested with dithiothreitol (DTT) and proteinase K ○ Genomic DNA is then extracted from the solution Saliva: ○ Buccal swabs or mouthwash samples are collected ○ Samples are lysed with a buffer containing proteinase K ○ DNA is extracted from the solution Urine: ○ Urine is centrifuged to collect cells ○ Cells are lysed with a buffer containing proteinase K ○ DNA is extracted from the solution Comparisons: DNA from blood and hair samples is generally of higher quality for restriction analysis compared to buccal swab and urine samples DNA Preparation from Microorganisms: Chemical Method Combines enzymes and chemical reagents for DNA extraction  Detergent cell lysis is a milder alternative to physical disruption, often used in conjunction with homogenization and grinding Detergents disrupt the lipid barrier of cells by solubilizing proteins and disrupting molecular interactions Detergent types: ○ Nonionic and zwitterionic detergents (e.g., CHAPS, Triton-X series) are milder and less denaturing, used when protein function needs to be preserved ○ Ionic detergents (e.g., SDS) are strong solubilizing agents that tend to denature proteins Lysis requirements vary depending on the cell type (animal, bacteria, yeast) due to the presence or absence of a cell wall Animal tissues require both detergent and mechanical lysis due to their dense and complex nature Bacteria: ○ Lysozyme is effective for Gram-positive bacteria due to their high peptidoglycan content ○ Lysozyme and EDTA combination is used for Gram-negative bacteria ○ Mechanical disruption can be used for Gram-positive bacteria and spores Fungal cells are difficult to disrupt due to capsules or spores ○ Lyticase (zymolyase) digests fungal cell walls ○ Alkaline chemicals, detergents, and xanthogenates can also break cells ○ CTAB method is commonly used for fungal genomic DNA extraction DNA Preparation from Microorganisms: Chelex-100 Method Chelex-100, a chelating resin, isolates fungal DNA quickly and efficiently Releases DNA from cells by boiling while protecting it with resin beads Conidial suspension is mixed with Chelex-100 resin, incubated, and centrifuged Supernatant is collected after further incubation and centrifugation Advantages: ○ Quick DNA extraction from spores ○ DNA is ready for direct use in molecular analyses ○ Cost-effective, especially for large-scale sample processing DNA Purification Involves removing proteins, carbohydrates, lipids, and cell debris from the crude cell extract Techniques include organic, inorganic, and spin column methods Organic Purification Phenol and chloroform/isoamyl alcohol are mixed with the sample (25:24:1 ratio) Phenol denatures proteins but doesn't fully inhibit RNase activity The mixture inhibits RNase activity  Proteinase K and RNase can be added to remove lipids and degrade RNA Centrifugation creates a biphasic emulsion: ○ Organic layer (bottom): Contains precipitated proteins ○ Aqueous layer (top): Contains DNA The aqueous layer is mixed with chloroform to remove residual phenol DNA is precipitated from the pure aqueous layer using high salt concentration and ethanol or isopropanol DNA precipitate is collected by centrifugation, washed with ethanol, and dissolved in Tris-EDTA or sterile distilled water Multiple phenol extractions or protease treatment may be needed for samples with high protein content Inorganic Purification Involves incubating nuclei with proteinase K at 65°C for auto-inactivation After incubation, the extracted DNA can be used directly for analysis without further purification Salting out is another method using saturated NaCl to precipitate protein DNA is purified from the supernatant by adding ethanol These approaches yield pure DNA using non-toxic substances and are fast and inexpensive Solid Phase Nucleic Acid Extraction A solid-phase system absorbs nucleic acid based on pH and salt content of the buffer Absorption principles: ○ Hydrogen-bonding with a hydrophilic matrix under chaotropic conditions ○ Ionic exchange with an anion exchanger under aqueous conditions ○ Affinity and size exclusion mechanisms Commonly performed using a spin column and centrifugation for faster purification  Solid supports include silica matrices, glass particles, diatomaceous earth, and anion-exchange carriers Solid Phase Nucleic Acid Extraction Steps Key steps: cell lysis, nucleic acid adsorption, washing, and elution Column conditioning: Prepares the column for sample adsorption using a buffer Cell extract application: Lysate is applied to the column Nucleic acid adsorption: Desired nucleic acid binds to the column under high pH and salt conditions Washing: Removes contaminants using a washing buffer Elution: TE buffer or water releases the purified nucleic acid from the column Diatomaceous Earth Extraction Diatomaceous earth (kieselguhr or diatomite) has high silica content Used for purifying plasmid and other DNA by immobilizing DNA onto its particles in the presence of a chaotropic agent Diatomaceous earth-bound DNA is washed with an alcohol-containing buffer DNA is eluted in a low-salt buffer or distilled water Affinity Extraction Magnetic separation is a simple and efficient method for nucleic acid purification Uses magnetic carriers with immobilized affinity ligands or biopolymers with affinity to the target nucleic acid Magnetic beads are preferred due to their larger binding capacity Nucleic acid binding may involve "wrapping around" the support Magnetic Bead Isolation Magnetic beads are positively charged and bind to the negatively charged DNA backbone at low pH At higher pH, beads lose their charge and DNA binding ability After lysis, genomic DNA is isolated by: ○ Binding to magnetic beads in a low pH buffer ○ Immobilizing beads with a magnet ○ Washing ○ Eluting in a higher pH buffer Anion-Exchange Extraction Uses anion-exchange resin based on the interaction between positively charged diethylaminoethyl cellulose (DEAE) groups and negatively charged DNA phosphates Resin consists of silica beads with large pores, hydrophilic surface, and high charge density Works over a wide range of pH (6-9) and salt concentrations (0.1-1.6M) Allows separation of DNA from RNA and other impurities Anion-Exchange Resin Purification is based on the interaction between negatively charged DNA phosphates and positively charged DEAE groups on the resin Salt concentration and pH control DNA binding and elution DNA binds tightly to DEAE over a wide range of salt concentrations Impurities are washed away with medium-salt buffers Genomic DNA is eluted with a high-salt buffer Comparison of Nucleic Acid Purification Technologies Anion-Exchange Resin: ○ Principle: Solid-phase, anion-exchange chromatography ○ Procedure: Binding and elution controlled by salt and pH; alcohol precipitation ○ Advantages: Ultrapure DNA, versatile formats, optimal for sensitive applications Silica-Gel-Membrane Technology: ○ Principle: Selective adsorption to silica-gel membranes ○ Procedure: Binding under high salt, elution under low salt ○ Advantages: High-purity nucleic acids, fast, inexpensive, no silica slurry or alcohol precipitation Magnetic-Particle Technology: ○ Principle: Binding to magnetic silica particles ○ Procedure: Binding under high salt, elution under low salt  ○ Advantages: High-purity nucleic acids, fast, inexpensive, versatile formats, easy automation DNA Preparation from Formalin-Fixed, Paraffin-Embedded (FFPE) Tissues Routine methods involve deparaffinization, washing, protein digestion, and DNA purification Deparaffinization: Incubation with xylene followed by centrifugation Washing: Pellets are washed with ethanol Lysis and homogenization: Followed by incubation at 65°C and 95°C Proteinase K digestion: Increases DNA yield Purification: Supernatant is purified using phenol/chloroform, spin column, or inorganic methods Another method uses glycine in an alkaline environment to reverse cross-linking These methods yield DNA suitable for PCR amplification Automated DNA Isolations Advantages: ○ Rapid, fully automated isolation of high-quality DNA and RNA ○ Reliable and reproducible results ○ Flexibility in sample type, volume, elution volume, and post-elution processes ○ Automatic pipetting for PCR setup ○ Available from various companies Automated DNA Isolations Using Magnetic Particles Samples are lysed using a buffer with chaotropic salts and proteinase K Magnetic Glass Particles (MGPs) bind DNA Unbound substances are removed by washing Purified DNA is eluted Automated DNA Isolations Using Vacuum Devices Uses silica-based resin and columns, automatically loaded Flow-through is enhanced by a vacuum Allows 96 parallel preparations Storage of Extracted DNA Refrigerator: Months Frozen (-20°C to -80°C): Years (long-term) Prevent nuclease activity EDTA chelates magnesium to inhibit nucleases Storage buffers are slightly basic (e.g., Tris buffer) to prevent DNA hydrolysis Nucleic Acid Storage Requirements Not recommended at 2-25°C (less than or equal 4 months) and 2-8°C (1-3 years) Less than or equal 7 years (-20°C) Recommended at -70°C for >7 years for long term storage in ethanol Quantity from UV Spectrophotometry DNA and RNA absorb maximally at 260 nm Proteins absorb at 280 nm Chemicals absorb at 230 nm Background scatter absorbs at 320 nm Concentration is calculated using specific formulas for DNA and RNA Purity from UV Spectrophotometry A260/A280 ratio indicates purity 1.8-2.0: Good DNA or RNA 2.0: Reagent contamination Using Spectrophotometer to Quantitate DNA and RNA Measure the absorbance at 260 nm and 280 nm Calculate concentration using the appropriate formula Assess purity using the A260/A280 ratio Yield Calculated based on dsDNA concentration and total sample volume Agarose Gel Electrophoresis Separates DNA or RNA molecules by size using an electric field and agarose matrix Smaller molecules move faster and farther Agarose Gel Electrophoresis Applications Estimating DNA molecule size after restriction enzyme digestion Analyzing PCR products Separating DNA or RNA before transfer techniques (Southern, Northern) Separating plasmid backbone Agarose Gel Electrophoresis A method used to separate DNA or RNA molecules based on their size Achieved by moving negatively charged nucleic acid molecules through an agarose matrix using an electric field Shorter molecules migrate faster and farther than longer ones Agarose Gel Electrophoresis: Factors Affecting Migration Size of DNA molecule: Smaller molecules move faster Agarose concentration: Higher concentration impedes larger molecules more DNA conformation: Supercoiled DNA moves fastest, followed by linear and open circular Applied voltage: Higher voltage increases migration speed but can cause overheating and band distortion Electrophoresis buffer: Composition and ionic strength affect DNA mobility Intercalating dyes: Can alter DNA migration Agarose Gel Electrophoresis: Visualization Ethidium bromide (EtBr): ○ Classic dye, fluoresces under UV light when bound to DNA ○ Detects bands with >20 ng DNA ○ Mutagenic  SYBR Green: ○ More expensive but 25 times more sensitive than EtBr DNA Degradation in Agarose Gel Electrophoresis DNA can degrade during electrophoresis and storage Proper storage conditions are crucial for maintaining DNA integrity Fluorometry Uses fluorescent dyes that bind specifically to DNA or RNA Requires a negative control and a standard of known concentration Fluorometer measures emitted fluorescent light after excitation More sensitive than spectrophotometry and less susceptible to contamination Avoid using glass cuvettes Fluorometry: Advantages About 1,000x more sensitive than spectrophotometric absorbance Less affected by protein and RNA contamination