Nucleic Acid Extraction Methods & Techniques: A Comprehensive Guide PDF

Nucleic acid extraction & gel electrophoresis Piya Wongyanin, Ph.D.  Outline Purpose of nucleic acid extraction Review the main steps in the nucleic acid extraction protocol and the chemistry involved in each step – Genomic DNA extraction – Plasmid DNA...

Nucleic acid extraction & gel electrophoresis Piya Wongyanin, Ph.D.  Outline Purpose of nucleic acid extraction Review the main steps in the nucleic acid extraction protocol and the chemistry involved in each step – Genomic DNA extraction – Plasmid DNA extraction – RNA extraction Agarose gel eletrophoresis  What is nucleic acid ? Nucleic acid are molecules that store information for cellular growth and reproduction There are two types of nucleic acids: – Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) These are polymers consisting of long chains of monomers called nucleotides A nucleotide consists of a nitrogenous base, pentose sugar and phosphate group.  What is DNA ? DNA-Deoxyribo Nucleic Acid DNA - The complex chemical compound found in chromosomes that contains the genetic code. DNA is a very large molecule, made up of smaller units called nucleotide Each nucleotide has three parts: a sugar (deoxyribose), a phosphate molecule and a nitrogenous base. The nitrogenous base is the part of the nucleotide that carries genetic information. The base found in DNA are four: adenine, cytosine, guanine and thymine (ATP, CTP, GTP and TTP)  What is RNA ? RNA-Ribo Nucleic Acid RNA – Nucleic acid involved in putting genetic code into action. –Remember DNA=blueprints for life but “someone” Has to be able to read those blueprints and get that into outside of the nucleus RNA is assembled as a chain of nucleotide Each nucleotide has three parts: a sugar (ribose), a phosphate molecule and a nitrogenous base. The base found in RNA are four: adenine, cytosine, guanine and uracil (ATP, CTP, GTP and UTP) Nucleic acid  Purpose of nucleic acid Extraction To obtain DNA or RNA in a relatively purified form which can be used for further investigations, i.e. PCR, sequencing, etc What do we need DNA for? Detect genetic disorders and diseases Detect, enumerate species Detect/sequence specific DNA regions Create new DNA “constructs” (recombinant DNA) What do we need RNA for? When/where are genes being transcribed? Measuring gene expression Detect RNA viruses  Nucleic acid extraction method Conventional method – Phenol/ Chloroform extraction Commercial kit – Silica based – Magnetic based Automate nucleic acid extraction  Basic Protocol for DNA extraction (Conventional method) Most DNA extraction protocols consist of four parts 1. A technique to lyse the cells gently and solubilize the DNA 2. Enzymatic or chemical methods to remove contaminating proteins, RNA, or macromolecules 3. DNA precipitation by ethanol 4. DNA dilution in water or buffer  Nucleic acids extraction  Cell lyses  Homogenization (mechanical disruption/mixing)  Physical lysis (sonication, vortex in the presence of glass beads)  Temperature-based lysis (freeze/thaw)  Enzymatic (Lysozyme)  Chemical lysis buffers (Tris HCl, EDTA and SDS)  Extraction/Precipitation Method Detergents Purposes of the Extraction Buffer Chaotropic salts 1. Dissolve cellular membranes Metal chelators 2. Inactivation of DNase and RNase Salts 3. Assist in the removal of contaminants Reducing agents CTAB PVP Use of Detergents to Lyse Cells: Like Dissolves Like Mixed micelle Plasma membrane (phospholipid bilayer) Detergent molecules + SDS  Sodium dodecyl sulphate (SDS) is anionic detergent which will lyse cells and denature proteins Proteinase K digest protein (non-specific) Chelator Ethylene diamine tetra-acetic acid (EDTA) bind divalent cation (Mg2+/Ca2+/Mn2+) required as co-factors for degradative DNase Chaotropic salt is a salt that disrupts stabilizing intra-molecular forces such as hydrogen bonding. It will make hydrophobic proteins more soluable in water: guanidinium salt. Salt Neutralization of the negative charges on DNA allows it to precipitate in alcohol: NaCl, C2H3NaO2. Reducing agents Often included in extraction buffers designed for plant DNA extraction, because it is a strong reducing agent which can remove tannins and other polyphenols often present in the crude plant extract: b-Mercaptoethanol. CTAB (cetrimonium bromide) is supposed to be the separation of polysaccharides from nucleic acid PVP (polyvinylpyrrolidone) is added to remove phenolic compounds from plant DNA extracts. DNA extraction from bacteria  DNA extraction from bacteria: overview cell harvest cell growth and lysis DNA concentration DNA purification Bacterial genomic DNA prep: cell extract Lysis: Detergents Organic solvent Proteases (lysozyme) Heat “cell extract”  Genomic DNA prep: removing proteins and RNA chloroform Need to mix gently! (to avoid shearing breakage of the genomic DNA) Add the enzyme RNase to degrade RNA in the aqueous layer DNA purification: phenol/chloroform extraction 1:1 phenol : chloroform or 25:24:1 phenol : chloroform : isoamyl alcohol Phenol: denatures proteins, precipitates form at interface between aqueous and organic layer Chloroform: increases density of organic layer Isoamyl alcohol: prevents foaming  2 ways to concentrate the genomic DNA (Absolute) “spooling” Ethanol precipitation  Nucleic acid precipitation  Ice-cold 100 % isopropanol or ethanol Plasmids: vehicles of recombinant DNA Bacterial cell genomic DNA plasmids Non-chromosomal DNA Replication: independent of the chromosome Many copies per cell Easy to isolate Easy to manipulate  Preparation of Plasmid DNA  Minipreps of plasmid DNA (Alkaline lysis miniprep)  NaOH denatures chromosomal and plasmid DNA  The mixture is neutralized with potassium acetate (pH 5.5)  preferential recovery of circular plasmid > linear chromosomal DNA  Chromosomal DNA and proteins precipitate  Plasmid DNA and RNA are precipitated by ethanol  RNA is destroyed by DNase-free RNase Plasmid purification: alkaline lysis Alkaline conditions denature DNA Neutralize: genomic DNA can’t renature (plasmids CAN because they never fully separate) Genomic DNA prep in plants -- how get rid of carbohydrates? CTAB: Cationic detergent CH3 CH3 (low ionic N+ Br- conditions) CH3 C16H33 (MC 6.61-6.62)  Extraction/Precipitation Method Step 1: Disruption of cell walls by grinding Step 1+2: mechanical disruption and homogenization in extraction buffer Grind sample into a fine powder to shear cell walls and membranes Step 2: Lysis of cells in extraction buffer A homogenizer allows cells to be mechanically disrupted within the extraction buffer Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit nuclease activity Crude lysate  Extraction/Precipitation Method Step 3: Organic extraction Mix thoroughly with Aqueous an equal volume of organic solvent Centrifuge Collect aqueous phase e.g. phenol, chloroform, or phenol:chloroform Interphase Organic Perform additional extractions for increased purity Crude lysate containing The aqueous phase contains water- nucleic acids and other soluble molecules, including nucleic cell constituents acids. Proteins and lipids become trapped in the organic phase, and are thus separated away. Insoluble plant debris become trapped in the interphase between the two layers  Extraction/Precipitation Method Step 4: Nucleic Acid Precipitation Before After Supernatant 70% EtOH Centrifuge Wash Centrifuge Pellet Dissolve pellet (H2O, TE, etc.) Add alcohol and salt to Pellet down nucleic acids. precipitate nucleic acids from the aqueous fraction Wash pellet with 70% ethanol to remove residual salts and other contaminants. Discard ethanol and allow pellet to dry.  DNA extraction by commercial kit Adsorption Chromatography Method (Silica based) Basic Principle Nucleic acids within a crude lysate are bound to a silica surface The silica surface is washed with a solution that keeps nucleic acids bound, but removes all other substances The silica surface is washed with a solution unfavorable to nucleic acid binding. The solution, containing purified DNA and/or RNA, is recovered.  Adsorption Chromatography Method Step 1: Prepare crude lysate Step 2: Adsorb to silica surface Apply to column Centrifuge Nucleic acids Silica-gel membrane Extraction Buffer composition favors Flow through DNA and RNA adsorption to silica: (discard) Low pH High ionic strength Chaotropic salt Nucleic acids bind to the membrane, while contaminants pass through the column. Surface silanol groups are weakly acidic, and will repel nucleic acids at near neutral or high pH due to their negative charge  Adsorption Chromatography Method Step 3: Wash away residual contaminants Centrifuge Wash buffer Nucleic acids Nucleic acids Flow through (discard) Step 4: Elute nucleic acids Centrifuge Elution buffer Nucleic acids Elution Buffer composition is unfavorable to surface binding: High pH Low ionic strength Nucleic acids DNA extraction by commercial kit (magnetic based) RNA extraction  RNA in a typical eukaryotic cell: 80-85% is ribosomal RNA 15-20% is small RNA (tRNA, small nuclear RNAs) About 1-5% is mRNA -- variable in size -- but usually containing 3’ polyadenylation  RNA Purification Total RNA from biological samples – Organic extraction – Affinity purification mRNA from total RNA – Oligo(dT) resins mRNA from biological samples – Oligo(dT) resins  Total RNA Purification Goal: Isolate RNA from other cellular components – Cells or tissue must be rapidly and efficiently disrupted – Inactivate RNases – Denature nucleic acid-protein complexes – RNA selectively partitioned from DNA and protein Isolation from different tissues/sources raises different issues  Ribonucleases (RNases) RNases are naturally occurring enzymes that degrade RNA Common laboratory contaminant (from bacterial and human sources) Also released from cellular compartments during isolation of RNA from biological samples Can be difficult to inactivate  Protecting against RNase Wear gloves at all times Use RNase-free tubes and pipet tips Use dedicated, RNase-free, chemicals Pre-treat materials with extended heat (180oC for several hours), wash with DEPC-treated water, NaOH or H2O2 Supplement reactions with RNase inhibitors Include a chaotropic agent (guanidine) in the procedure – Chaotropic agents such as guanidine inactivate and precipitate RNases and other proteins  Organic Extraction of total RNA Purifying RNA: the key is speed Break the cells/solubilize components/inactivate RNases by the addition of guanidinium thiocyanate (very powerful denaturant) Extract RNA using phenol/chloroform (at low pH) Precipitate the RNA using ethanol/LiCl Store RNA: in DEPC-treated H20 (-80°C) in formamide (deionized) at -20°C Organic Extraction of total RNA  Organic Extraction of total RNA (Kit)  TRIzol reagent (mono-phasic solution of phenol and guanidine isothiocyanate ) and chloroform Aqueous (upper) phase : RNA Interphase : Denatured proteins and larger fragments of DNA Organic (lower) phase: Denatured proteins and small fragments of DNA Organic Extraction of total RNA Affinity purification of total RNA Affinity purification of total RNA  Messenger RNA Isolation mRNA molecules have a tail of A’s at the 3’ end (polyA tail) Oligo(dT) probes can be used to purify mRNA from other RNAs mRNA can be eluted from oligo(dT) matrix using water or low-salt buffer Messenger RNA Isolation  Messenger RNA Isolation Isolating mRNA from total RNA – Purifying total RNA first enables larger sample sizes to be processed; this results in higher mRNA yield – Two purifications; takes longer than isolating mRNA directly Isolating mRNA directly from a biological sample – Quicker than doing an initial total RNA isolation followed by mRNA selection – However, sample size is limited Assessing the Quality and Yield of Nucleic Acids  Nucleic Acid Analysis via UV Spectrophotometry DNA Absorption Spectra By measuring the amount of light absorbed by your sample at specific wavelengths, it is possible to estimate the concentration of DNA and RNA. Nucleic acids have an absorption peak at ~260nm. [dsDNA] ≈ A260 x (50 µg/mL) [ssDNA] ≈ A260 x (33 µg/mL) [ssRNA] ≈ A260 x (40 µg/mL)  How pure is your sample? The A260/A280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination. The A260/A230 ratio of DNA and RNA should be roughly equal to its A260/A280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates). Gel Electrophoresis  Agarose Gel Electrophoresis Gel electrophoresis is a widely used technique for the analysis of nucleic acids and proteins. Agarose gel electrophoresis is routinely used for the preparation and analysis of DNA. Gel electrophoresis is a procedure that separates molecules on the basis of their rate of movement through a gel under the influence of an electrical field.  DNA is negatively charged. When placed in an electrical field, DNA will migrate toward the positive pole (anode). An agarose gel is used to slow the movement of DNA and separate by size. H O2   DNA - + Power Polymerized agarose is porous, allowing for the movement of DNA Scanning Electron Micrograph of Agarose Gel (1×1 µm)  How fast will the DNA migrate? strength of the electrical field, buffer, density of agarose gel… Size of the DNA! *Small DNA move faster than large DNA …gel electrophoresis separates DNA according to size DNA small large - + Power Within an agarose gel, linear DNA migrate inversely proportional to the log10 of their molecular weight.  Agarose D-galactose 3,6-anhydro L-galactose Sweetened agarose gels have been eaten in the Far East since the 17th century. Agarose was first used in biology when Robert Koch* used it as a *Lina Hesse, technician and illustrator for culture medium for Tuberculosis a colleague of Koch was the first to bacteria in 1882 suggest agar for use in culturing bacteria Agarose is a linear polymer extracted from seaweed. An agarose gel is prepared by combining agarose powder and a buffer Buffer solution. Flask for boiling  Agarose  Electrophoresis Equipment Power supply Gel tank Cover Electrical leads  Casting tray Gel combs Gel casting tray & combs  Preparing the Casting Tray Seal the edges of the casting tray and put in the combs. Place the casting tray on a level surface. None of the gel combs should be touching the surface of the casting tray.  Agarose Buffer Solution Combine the agarose powder and buffer solution. Use a flask that is several times larger than the volume of buffer.  Melting the Agarose Agarose is insoluble at room temperature (left). The agarose solution is boiled until clear (right). Gently swirl the solution periodically when heating to allow all the grains of agarose to dissolve. ***Be careful when boiling - the agarose solution may become superheated and may boil violently if it has been heated too long in a microwave oven.  Pouring the gel Allow the agarose solution to cool slightly (~60ºC) and then carefully pour the melted agarose solution into the casting tray. Avoid air bubbles. Each of the gel combs should be submerged in the melted agarose solution. When cooled, the agarose polymerizes, forming a flexible gel. It should appear lighter in color when completely cooled (30-45 minutes). Carefully remove the combs and tape. Place the gel in the electrophoresis chamber.  DNA buffer      wells Anode Cathode (positive) (negative) Add enough electrophoresis buffer to cover the gel to a depth of at least 1 mm. Make sure each well is filled with buffer.  Sample Preparation Mix the samples of DNA with the 6X sample loading buffer (w/ tracking dye). This allows the samples to be seen when loading onto the gel, and increases the density of the samples, causing them to sink into the gel wells. 6X Loading Buffer:  Bromophenol Blue (for color) Glycerol (for weight)  Loading the Gel Carefully place the pipette tip over a well and gently expel the sample. The sample should sink into the well. Be careful not to puncture the gel with the pipette tip.  Running the Gel Place the cover on the electrophoresis chamber, connecting the electrical leads. Connect the electrical leads to the power supply. Be sure the leads are attached correctly - DNA migrates toward the anode (red). When the power is turned on, bubbles should form on the electrodes in the electrophoresis chamber.  Cathode (-)  wells DNA  Bromophenol Blue (-)  Gel Anode (+) After the current is applied, make sure the Gel is running in the correct direction. Bromophenol blue will run in the same direction as the DNA.  DNA Ladder Standard -  12,000 bp  5,000 DNA migration  2,000  1,650 Note: bromophenol blue migrates at  1,000 approximately the  850 same rate as a 300  650 bp DNA molecule  500  400 bromophenol blue  300  200 +  100 Inclusion of a DNA ladder (DNAs of know sizes) on the gel makes it easy to determine the sizes of unknown DNAs.  Staining the Gel Ethidium bromide binds to DNA and fluoresces under UV light, allowing the visualization of DNA on a Gel. Ethidium bromide can be added to the gel and/or running buffer before the gel is run or the gel can be stained after it has run. ***CAUTION! Ethidium bromide is a powerful mutagen and is moderately toxic. Gloves should be worn at all times. Safer alternatives to Ethidium Bromide Methylene Blue BioRAD - Bio-Safe DNA Stain Ward’s - QUIKView DNA Stain Carolina BLU Stain …others advantages disadvantages Inexpensive Less sensitive Less toxic More DNA needed on gel No UV light required Longer staining/destaining time No hazardous waste disposal  Staining the Gel Place the gel in the staining tray containing warm diluted stain. Allow the gel to stain for 25-30 minutes. To remove excess stain, allow the gel to destain in water. Replace water several times for efficient destain. Ethidium Bromide requires an ultraviolet light source to visualize  Visualizing the DNA (ethidium bromide) DNA ladder DNA ladder  1 2 3 4 5 6 7 8  wells  5,000 bp  2,000  1,650  1,000  850  650  500 PCR Product  400  300  200 Primer dimers  100 + - - + - + + - Samples # 1, 4, 6 & 7 were positive