Presentation on theme: "Isolation and Quantification of Nucleic Acids in Plants Chris Yuen, Ph.D. June 13, 2006 Advances in Bioscience Education Leeward Community College."— Presentation transcript:
Isolation and Quantification of Nucleic Acids in Plants Chris Yuen, Ph.D. June 13, 2006 Advances in Bioscience Education Leeward Community College
The Central Dogma of Genetics Replication Transcription Translation mRNA non-coding RNA (rRNA, tRNA, siRNA, etc.)
Plants cells contain three distinct sets of DNA: nuclear, plastidic, mitochondrial
The cell interior is separated from its surrounding environment by a phospholipid bilayer: the plasma membrane Phospholipids of the plasma membrane are amphipathic, containing both a polar (hydrophilic) head and a nonpolar (hydrophobic) tail.
Plant cells are enclosed within a rigid extracellular polysaccharide matrix: the cell wall Cellulose microfibrils, the main constituent of plant cell walls, as viewed through an electron microscope
Nucleic Acid Extraction Requirements 1. Disruption of cell wall and membranes to liberate cellular components. 2. Inactivation of DNA- and RNA-degrading enzymes (DNases, RNases). 3. Separation of nucleic acids from other cellular components. Extraction/Precipitation method Adsorption Chromatography method
Getting Prepared: Creating a Nuclease-Free Environment Living organisms produce several enzymes designed to degrade DNA and RNA molecules. There are several things you can do to minimize the risk of exposing your samples to external DNases and RNases. Autoclave solutions. This is usually sufficient for getting rid of DNases, and most RNases as well. Treat solutions with 0.1% DEPC. DEPC inactivates nucleases by covalently modifying the His residues in proteins. Generally considered unnecessary for DNA extraction. Not compatible with solutions containing Tris or HEPES. Have a dedicated set of pipettors or use aerosol barrier tips. Wear gloves. You should be doing this anyway for safety reasons, but skin cells also produce RNase7, a potent RNA-degrading enzyme. Bake glass, metal, or ceramic equipment at high temp.
Overview of the Extraction/Precipitation Method
Step 1: Disruption of cell walls by grinding Step 2: Lysis of cells in extraction buffer Step 1+2: mechanical disruption and homogenization in extraction buffer Extraction/Precipitation Method Grind sample into a fine powder to shear cell walls and membranes Mix thoroughly with extraction buffer to dissolve cell membranes and inhibit nuclease activity A homogenizer allows cells to be mechanically disrupted within the extraction buffer Crude lysate
Purposes of the Extraction Buffer 1. Dissolve cellular membranes 2. Inactivation of DNase and RNase 3. Assist in the removal of contaminants Detergents Chaotropic salts Metal chelators Salts Reducing agents CTAB PVP Extraction/Precipitation Method + Plasma membrane (phospholipid bilayer) Detergent molecules Use of Detergents to Lyse Cells: Like Dissolves Like Mixed micelle SDS
Crude lysate containing nucleic acids and other cell constituents Mix thoroughly with an equal volume of organic solvent e.g. phenol, chloroform, or phenol:chloroform Centrifuge The aqueous phase contains water- soluble molecules, including nucleic 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 Perform additional extractions for increased purity Collect aqueous phase Extraction/Precipitation Method Step 3: Organic extraction Organic Aqueous Interphase
Pellet down nucleic acids. Wash pellet with 70% ethanol to remove residual salts and other contaminants. Pellet down nucleic acids. Wash pellet with 70% ethanol to remove residual salts and other contaminants. Discard ethanol and allow pellet to dry. After Add alcohol and salt to precipitate nucleic acids from the aqueous fraction Supernatant Pellet 70% EtOH Dissolve pellet (H 2 O, TE, etc.) Step 4: Nucleic Acid Precipitation Extraction/Precipitation Method BeforeAfter CentrifugeWashCentrifuge
Overview of the Adsorption Chromatography Method Adsorption: the binding of molecules or particles to a surface
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.
Step 1: Prepare crude lysate Silica-gel membrane Apply to column Step 2: Adsorb to silica surface Adsorption Chromatography Method Centrifuge Flow through (discard) Nucleic acids Surface silanol groups are weakly acidic, and will repel nucleic acids at near neutral or high pH due to their negative charge Extraction Buffer composition favors DNA and RNA adsorption to silica: Low pH High ionic strength Chaotropic salt Nucleic acids bind to the membrane, while contaminants pass through the column.
Using Nucleases to Remove Unwanted DNA or RNA Add DNase Add RNase + DNase (protein) + RNase (protein) Depending on when nuclease treatment is performed, it may be necessary to repeat purification steps for protein removal (e.g. phenol/chloroform extraction).
Assessing the Quality and Yield of Nucleic Acids
Nucleic Acid Analysis via UV Spectrophotometry 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] ≈ A 260 x (50 µg/mL) [ssDNA] ≈ A 260 x (33 µg/mL) [ssRNA] ≈ A 260 x (40 µg/mL) DNA Absorption Spectra
How pure is your sample? The A 260 /A 280 ratio is ~1.8 for dsDNA, and ~2.0 for ssRNA. Ratios lower than 1.7 usually indicate significant protein contamination. The A 260 /A 230 ratio of DNA and RNA should be roughly equal to its A 260 /A 280 ratio (and therefore ≥ 1.8). Lower ratios may indicate contamination by organic compounds (e.g. phenol, alcohol, or carbohydrates). Turbidity can lead to erroneous readings due to light interference. Nucleic acids do not absorb light at the 320 nm wavelength. Thus, one can correct for the effects of turbidity by subtracting the A 320 from readings at A 230, A 260 and A 280.
Running your sample through an agarose gel is a common method for examining the extent of DNA degradation. Good quality DNA should migrate as a high molecular weight band, with little or no evidence of smearing. genomic DNA RNA (degraded) Checking for Degradation: DNA
Checking for Degradation: RNA Ribosomal RNA (rRNA) makes up more than 80% of total RNA samples. Total RNA preps should display two prominent bands after gel electrophoresis. These correspond to the 25S and 18S rRNAs, which are 3.4 kb and 1.9 kb in Arabidopsis (respectively). Good quality RNA will have: No evidence of smearing 25S/18S ratio between S 18S
Today’s Lab Objectives: Use the RNeasy Extraction Kit to isolate total RNA from Arabidopsis thaliana. Determine RNA yield