Transcript Nucleic Acid Isolation

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
Complementary
Base pairs
Purines
Pyrimidines
DNA
DNA template
is used to build
complementary
RNA strand
RNA polymerase transcribes gene into mRNA
RNA
Protein
mRNA
rRNA
tRNA
translation
Amino acids specified by code
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
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 (EDTA)
Salts
Reducing agents
CTAB (cetyltrimethylammonium bromide)
PVP (polyvinylpyrollidine)
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.)
Adsorption Chromatography Method
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 Chaotropic salts disrupt
molecular structure based
their negative charge
on hydrogen bonds;
hydrophilic interactions
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
Degraded DNA may be
unsuitable for most uses
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.
How can we use isolated DNA?
• Restriction Digest
• Hybridization with specific probes using
Southern Blot
• PCR amplification of particular genes
• Recombination, cloning
• Sequencing