Isolation and Quantification of Nucleic Acids Catherine Unabia, Ph.D. June 12, 2007 Advances in Bioscience Education Leeward Community College

The DNA double helix Molecule contains all of the information of life as well as to serve as a template for its own replication

Flow of information DNA to mRNA to protein In Eukaryotes, the mRNA transcript is exported through nuclear pores to the cytoplasm Nucleic Acids are key to this essential process of life

Nucleic Acid structure

Nucleic Acid Polymers

Purines Pyrimidines Complementary Base pairs

DNA RNA polymerase transcribes gene into mRNA RNA Protein mRNA rRNA tRNA translation Amino acids specified by code DNA template is used to build complementary RNA strand

Biotechnology uses nucleic acids in many ways Creating recombinant DNA –inserting new traits like disease resistance –repairing genetic defects Identifying particular genes and their functions –PDI –Which genes are involved in development of cancer? Mapping genes on chromosomes Comparing gene sequences between organisms

Plants cells contain three distinct sets of DNA: nuclear, plastidic, mitochondrial Many types of RNA are also present in the cell

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

The cell interior is separated from its surrounding environment by a phospholipid bilayer: the plasma membrane Phospholipids of the plasma membrane and the nuclear membrane are amphipathic, containing both a polar (hydrophilic) head and a nonpolar (hydrophobic) tail.

Nucleic Acid Extraction Objectives 1.Disruption of cell wall and membranes to liberate cellular components. - Grinding or digestion of cellulose with enzyme 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 Special Problems with some Plant Tissues: Polysaccharides may be abundant and may be separated together with the nucleic acids Pigments and Secondary Products such as polyphenols may interfere with separation, or co-separate with nucleic acids Cells may contain active RNA or DNases that quickly cut the material you are trying to isolate!

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.

Protocols for Isolating Nucleic Acids Extraction/Precipitation Method Adsorption Chromatography Method Adsorption: binding of molecules or particles to a surface Chromatography: separation based on binding

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 (EDTA) Salts Reducing agents CTAB (cetyltrimethylammonium bromide) PVP (polyvinylpyrollidine) 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

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 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. Chaotropic salts disrupt molecular structure based on hydrogen bonds; hydrophilic interactions

Centrifuge Nucleic acids Step 3: Wash away residual contaminants Adsorption Chromatography Method Wash buffer Nucleic acids Flow through (discard) Nucleic acids Elution buffer Elution Buffer composition is unfavorable to surface binding: High pH Low ionic strength Step 4: Elute nucleic acids Centrifuge Nucleic acids

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 Degraded DNA may be unsuitable for most uses

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

How can we use isolated DNA? Restriction Digest Hybridization with specific probes using Southern Blot PCR amplification of particular genes Recombination, cloning Sequencing