Nucleic Acid Isolation

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Isolation and Quantification of Nucleic Acids in
Plants Chris Yuen, Ph.D. June 13,
2006 Advances in Bioscience Education Leeward
Community College
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The Central Dogma of Genetics
non-coding RNA (rRNA, tRNA, siRNA, etc.)
Transcription
Replication
mRNA
Translation
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Plants cells contain three distinct sets of DNA
nuclear, plastidic, mitochondrial
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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.
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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
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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
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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.
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Overview of the Extraction/Precipitation Method
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Extraction/Precipitation Method
Step 1 Disruption of cell walls by grinding
Step 12 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
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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
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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 phenolchloroform
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
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Extraction/Precipitation Method
Step 4 Nucleic Acid Precipitation
After
Before
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
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Overview of the Adsorption Chromatography Method
Adsorption the binding of molecules or particles
to a surface
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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.
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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
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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
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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).
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Assessing the Quality and Yield of Nucleic Acids
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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)
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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.
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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)
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Todays Lab Objectives Use the RNeasy Extraction
Kit to isolate total RNA from Arabidopsis
thaliana. Determine RNA yield