Isolation and Quantification of
Nucleic Acids in Plants
The Central Dogma of Genetics
non-coding RNA (rRNA, tRNA, siRNA, etc.)
Transcription
Replication
mRNA
Translation
Deoxyribonucleotide
Ribonucleotide
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
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
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
Extraction/Precipitation Method
Detergents
Chaotropic salts
Metal chelators
Salts
Reducing agents
CTAB
PVP
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
Plasma membrane
(phospholipid bilayer)
Mixed micelle
Detergent molecules
+
SDS
Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly with
an equal volume of
organic solvent
e.g. phenol, chloroform,
or phenol:chloroform
Aqueous
Centrifuge
Collect aqueous phase
Interphase
Organic
Perform additional extractions for increased purity
Crude lysate containing
nucleic acids and other
cell constituents
The aqueous phase contains watersoluble 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
Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation
Before
After
Supernatant
Centrifuge
70% EtOH
Wash
Centrifuge
Pellet
Add alcohol and salt to
precipitate nucleic acids
from the aqueous fraction
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
Dissolve pellet
(H2O, TE, etc.)
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.
Adsorption Chromatography Method
Step 1: Prepare crude lysate
Apply to column
Step 2: Adsorb to silica surface
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
Adsorption Chromatography Method
Step 3: Wash away residual contaminants
Wash buffer
Centrifuge
Nucleic acids
Nucleic acids
Flow through
(discard)
Step 4: Elute nucleic acids
Elution buffer
Centrifuge
Nucleic acids
Elution Buffer composition is
unfavorable to surface binding:
High pH
Low ionic strength
Nucleic acids
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).
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).
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.
Checking for Degradation: DNA
genomic
DNA
RNA
(degraded)
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.
Checking for Degradation: RNA
25S
18S
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
Today’s Lab Objectives:
Use the RNeasy Extraction Kit to isolate total
RNA from Arabidopsis thaliana.
Determine RNA yield