Purifying Nucleic acids

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Purifying Nucleic acids
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Isolation of Nucleic Acids

• Goals
• removal of proteins
• DNA vs RNA
• isolate specific type of nucleic acid

• Types of Methods
• differential solubility
• adsorption methods
• density gradient centrifugation

• Types
• genomic (chromosomal)
• organellar (satellite)
• plasmid (extra-chromosomal)
• phage/viral (ds or ss)
• complementary (mRNA)

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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
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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Cells
Extract
Bacterial Cells Or tissue culture cells Or
blood Or flies..
HOW?
Organic extraction
Pure DNA
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High MW Genomic DNA Isolation

• Detail of step 3
• Phenol Extraction
• mix sample with equal volume of sat. phenol soln
• retain aqueous phase
• optional chloroform/isoamyl alcohol extraction(s)

• Typical Procedure
• Harvest cells
• 2 Cell Lysis
• 0.5 SDS proteinase K (55o several hours)
• 3 Phenol Extraction
• gentle rocking several hours
• 4 Ethanol Precipitation
• 5 RNAse followed by proteinase K
• 6 Repeat Phenol Extrac-tion and EtOH ppt

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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 debris become
trapped in the interphase between the two layers
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High MW Genomic DNA Isolation

• Detail of step 4
• EtOH Precipitation
• 2-2.5 volumes EtOH, -20o
• high salt, pH 5-5.5
• centrifuge or spool out

• Typical Procedure
• Harvest cells
• 2 Cell Lysis
• 0.5 SDS proteinase K (55o several hours)
• 3 Phenol Extraction
• gentle rocking several hours
• 4 Ethanol Precipitation
• 5 RNAse followed by proteinase K
• 6 Repeat Phenol Extrac-tion and EtOH ppt

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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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Detail of step 5 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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Adsorption Chromatography
Another way to selectively purify DNA Resins
(glass or chemically modified beads) that bind
nucleic acids reversibly are packaged in
columns for easy DNA RNA purification
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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.
Having the ability to destabilize hydrogen
bonding and hydrophobic interactions.
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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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pH SALT
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Purifying one type of DNA away from other DNA
molecules

• Plasmids from bacterial chromosomal DNA

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Isolation of plasmid DNA

• Why plasmid?

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Method

• Boiling lysis or alkaline lysis
• Selectively isolates the plasmid excludes most of
  the chromosomal DNA
• How is it possible to be selective
• Plasmid DNA is almost always extracted as a CCC
• Chromosomal DNA is linear fragments
• Plasmids are much smaller than chromosomal DNA

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Boiling lysis

• Cell lysis with lysozyme
• Chromosomal DNA is separated from the plasmid DNA
  by boiling the lysis mixture followed by
  centrifugation

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Chromosomal DNA
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E. coli
Pellet bacteria
Lyse
Boil and centrifuge
After centrifugation
Soluble plasmid
Glutinous pellet
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Alkaline lysis

• The cells are lysed and their contents denatured
  with alkaline SDS. Proteins and
  high-molecular-weight chromosomal DNA denatured
  under these conditions precipitate leaving the
  supernate containing plasmid DNA and RNA.

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SDS, alkali
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alkali
neutralize
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Separation of Nucleic Acids by CeCl Gradient
Centrifugation
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Plasmid DNA
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Isolation of RNA Special Considerations

• RNAse inhibitors!
• extraction in guanidine salts
• phenol extractions at pH 5-6
• (pH 8 for DNA)
• treatment with RNase-free DNase
• selective precipitation of high MW forms (rRNA,
  mRNA) with LiCl
• oligo-dT column

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Density Gradient Centrifugation

• rate zonal/sucrose (size fractionation)
• electrophoresis more common
• isopycnic/CsCl (density)
• DNA 1.7 g/cm3
• protein 1.3 g/cm3
• RNA gt DNA
• ssDNA gt dsDNA
• GC content

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CsCl Gradients

• Applications
• large scale preparations
• high purity
• RNA cushions
• satellite DNA

Cesium Chloride Gradients
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Assessing the Quality and Yield of Nucleic Acids
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Evaluation of Nucleic Acids

• spectrophotometrically
• quantity
• quality
• fluorescent dyes
• gel electrophoresis

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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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