Unit 6 Nucleic Acid
Extraction Methods
Terry Kotrla, MS, MT(ASCP)BB
Fall 2007
Purpose
► To
release nucleic acid from the cell for use
in other procedures
► Must be free from contamination with
protein, carbohydrate, lipids or other nucleic
acids.
► Used pure nucleic acids for testing.
Isolation
► Routinely
isolated from human, fungal,
bacterial and viral sources.
► Pretreat to make nucleated cells available,
 whole blood
 Tissue samples
 Microorganisms
► Need
sufficient sample for adequate yield.
Organic Isolation
► Must
purify DNA by removing contaminants.
► Accomplished by using combination of high
salt, low pH and an organic mixture of
phenol and chloroform.
► To avoid RNA contamination add RNAse,
enzyme that degrades RNA.
Phenol/Chloroform
► Biphasic
emulsion forms
► Hydrophobic layer on bottom has cell
debris.
► Hydrophilic layer on top has dissolved DNA
► Remove top layer, add cold ethanol, DNA
precipitates out.
Inorganic Isolation Methods
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Also called “salting out”.
Uses low pH and high salt condition to selectively
precipitate proteins.
DNA is left in solution (picture on far left).
Precipitate out DNA with isoproproanol (middle and right
side pictures).
I know this is not scientific but the precipitated out DNA is
usually referred to as “snotty”.
Solid Phase Isolation
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More rapid and effective
Use solid matrix to bind the DNA.
Wash away contaminants.
Elute DNA from column
Textbook page 70, Figure 4.3
Solid Phase Isolation
► The
diagram below explains the attractive
properties of solid phase for DNA and RNA.
Crude Lysis
► Used
for:
 Screening large numbers of samples
 Isolation of DNA in limited amounts
 Isolation from challenging samples, ie, paraffin
embedded tissue
► Usually
not done in clinical laboratory.
► Textbook page 71, Figure 4-4
Isolation of Mitochondrial DNA
► Mitochondrial
DNA is passed from
generation to generation along the maternal
lineage.
► Centrifugation to separate out
► Lyse
► Precipitate with cold ethanol.
Isolation of RNA
► Requires
STRICT precautions to avoid
sample degradation.
► RNA especially labile.
RNAses
► RNases
are naturally occurring enzymes that
degrade RNA
► Common laboratory contaminant (from
bacterial and human sources)
► Also released from cellular compartments
during isolation of RNA from biological
samples
► Can be difficult to inactivate
RNAses
► RNAses
are enzymes which are small
proteins that can renature and become
active.
► MUST be eliminated or inactivated BEFORE
isolation.
► CRITICAL to have a separate RNAse free
area of lab.
Protecting Against RNAse
► Wear
gloves at all times
► Use RNase-free tubes and pipet tips
► Use dedicated, RNase-free, chemicals
► Pre-treat materials with extended heat (180
C for several hours), wash with DEPCtreated water, NaOH or H2O2
► Supplement reactions with RNase inhibitors
Total RNA
► 80-90%
of total RNA is ribosomal RNA.
► 2.5-5% is messenger RNA
Organic RNA Extraction
Lyse/homogenize cells
2. Add phenol:chloroform:isoamyl
alcohol to lysed sample, and
centrifuge
3. Organic phase separates from
aqueous phase
1.
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4.
Organic solvents on bottom
Aqueous phase on top (contains
total RNA)
Cellular debris and genomic DNA
appears as a “film” of debris at the
interface of the two solutions
Remove RNA solution to a clean
tube; precipitate RNA and wash
with ethanol, then resuspend RNA
in water
Affinity Purification of RNA
Lyse cells, and spin to remove
large particulates/cell debris
2. Apply lysate (containing nucleic
acids and cellular contaminants) to
column with glass membrane
3. Wash with alcohol to remove
contaminants; nucleic acids stick
to glass membrane while
contaminants wash through. Treat
with DNase enzyme to remove
contaminating DNA.
4. Apply water to the column;
purified RNA washes off the glass
and is collected
1.
Isolation of PolyA (messenger) RNA
► Only
2.5-5%
► mRNA molecules have a tail of A’s at the 3’
end (polyA tail)
► Oligo(dT) probes can be used to purify
mRNA from other RNAs
► mRNA can be eluted from oligo(dT) matrix
using water or low-salt buffer
► Textbook page 75, Figure 4.7
Electrophoresis
► Analyze
DNA and RNA for quality by
electrophoresis.
► Fluorescent dyes
 Ethidium bromide
 SybreGreen
► Appearance
depends on type of DNA
isolated.
► Will be covered in more detail in unit 7.
Staining with Ethidium Bromide and
Sybr Green
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DNA is electrophoresed and stained.
Left – Ethidium Bromide Right – Sybr Green
The lane in the left side of the picture on the left is a
“ladder” which has fragments of known base pair (bp)
sizes. This allows determination of the size of isolated
fragments.
Spectrophotometry
► Sample
absorbances are determined on the
spectrophotometer at 260nm and 280nm
 nucleic acid (DNA, RNA, nucleotides) absorb light at
260nm
 protein absorbs light at 280 nm 230nm: guanidine
► A260/A280
ratio is a measure of DNA purity
► The absorbance wavelength is directly proportional
to the concentration of nucleic acid.
Determining Concentration
► Formula
Concentration = 260 Reading * Absorbance
unit * Dilution factor
► One optical density or absorbance unit at
260 nm is equal to
 50 ug/mL for DNA
 40 ug/mL for RNA.
► Textbook
page 77 has sample calculations.
Determining Purity
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The OD at 260nm should be 1.6-2.00 times more than the
absorbance at 280nm.
Divide the OD at 260nm by the OD at 280nm to get the
ratio.
If the 260nm/280nm ratio is less than 1.6 for DNA, 2.0-2.3
for RNA this indicates contamination, usually with protein.
DNA -If the OD ratio is higher than 2.0 it may be
contaminated with RNA.
Ratio of the readings : O.D.260/O.D.280 is a measure of
purity.
Pure preparations of DNA and RNA have O.D 260/280 of
1.8 and 2.0 respectively.
Fluorometry
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Fluorometry utilizes fluorescent dyes which specifically bind
DNA or RNA.
It requires a negative control (to set the zero point on the
fluorometer) and a standard of known concentration.
The fluorometer shines light on the sample (excitation) and
then measures level of fluorescent light being emitted to
the side (at a 90º angle) of the excitation light beam.
The fluorescent dyes are relatively specific to nucleic acids
as opposed to protein and other cellular components.
The fluorescence of the dyes increases when they bind
nucleic acids.
Fluorometry
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Fluorometry is about 1,000x more sensitive than
spectrophotometric absorbance (i.e. measurement of
A260) and less susceptible to protein and RNA
contamination.
However it also does not give a crude measurement of
purity (like an A260/A280 ratio) nor does it assure that the
DNA or RNA is not degraded (e.g. like size determination
by gel electrophoesis).
Do not use glass (spectrophotmetry) cuvettes in a
fluorometer because the frosted glass on the side of the
cuvette interferes with detection of fluorescent light.