1. Isolation and Quantification of Nucleic Acids in Plants
Chris Yuen, Ph.D.
June 13, 2006
Advances in Bioscience Education
Leeward Community College

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. (phospholipid bilayer)
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.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Pellet down nucleic acids.
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. Nucleic acids within a crude lysate are bound to a silica 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.

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
25S
18S

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