Isolation and Quantification of Nucleic Acids in Plants

1. Isolation and Quantification of Nucleic Acids in Plants

2. The Central Dogma of Genetics non-coding RNA (rRNA, tRNA, siRNA, etc.) Transcription Replication mRNA Translation

3. Deoxyribonucleotide Ribonucleotide

4. Plants cells contain three distinct sets of DNA: nuclear, plastidic, mitochondrial

5. 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.

6. 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

7. 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

8. 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.

9. Overview of the Extraction/Precipitation Method

10. 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

11. Detergents Chaotropic salts Metal chelators Salts Reducing agents CTAB PVP Extraction/Precipitation Method Purposes of the Extraction Buffer 1. Dissolve cellular membranes 2. Inactivation of DNase and RNase 3. Assist in the removal of contaminants Use of Detergents to Lyse Cells: Like Dissolves Like Mixed micelle Detergent molecules Plasma membrane (phospholipid bilayer) + SDS

12. Extraction/Precipitation Method Step 3:Organic extraction Mix thoroughly with an equal volume of organic solvent Aqueous Centrifuge Collect aqueous phase e.g. phenol, chloroform, or phenol:chloroform Interphase Organic Perform additional extractions for increased purity Crude lysate containing nucleic acids and other cell constituents 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

13. Extraction/Precipitation Method Step 4:Nucleic Acid Precipitation Before After After Supernatant 70% EtOH Centrifuge Wash Centrifuge Pellet Dissolve pellet (H2O, TE, etc.) • Pellet down nucleic acids. • 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. Add alcohol and salt to precipitate nucleic acids from the aqueous fraction

14. Overview of the Adsorption Chromatography Method Adsorption: the binding of molecules or particles to a surface

15. 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.

16. 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 DNA and RNA adsorption to silica: • Low pH • High ionic strength • Chaotropic salt Flow through (discard) 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

17. 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

18. Using Nucleases to Remove Unwanted DNA or RNA Add DNase + DNase (protein) Add RNase + 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).

19. Assessing the Quality and Yield of Nucleic Acids

20. 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)

21. 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). 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 A320 from readings at A230, A260 and A280.

22. Checking for Degradation: DNA genomic DNA 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. RNA (degraded)

23. 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 1.8 - 2.3 25S 18S

24. Today’s Lab Objectives: Use the RNeasy Extraction Kit to isolate total RNA from Arabidopsis thaliana. Determine RNA yield