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valentina.iadevaia1 [email protected]

DNA
EXTRACTION
METHODS
DR VALENTINA IADEVAIA
 Definition

DNA Extraction is the isolation and

purification of DNA (deoxyribonucleic
acid)
Examples
DNA extraction is used to
isolate…

◦ Mitochondrial DNA
◦ Genomic DNA
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DNA can be extracted from…

◦ Cells or tissues
◦ Environmental samples
◦ ...
◦ any nucleated cell

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 Purpose of DNA Extraction
To obtain DNA in a relatively purified form which can be used
for further investigations such as:
◦ PCR (polymerase chain reaction)
◦ RFLP (restriction fragment length polymorphism)
◦ Southern Blotting
◦ DNA seq
◦ Cloning
◦ Transfection
◦ Transformation
◦ Biochemistry assay
DNA Analysis
Downstream techniques can:

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◦ Reveal how organisms are

related

◦ Identify cryptic species

◦ Locate mutations in DNA

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◦ Transgenic organisms
James Watson and Francis Crick (1953)
5' 3'

3' 5'
 What are the essential
components of a DNA
extraction Procedure?
•Maximize DNA recovery
•Remove inhibitors
•Remove or inhibit nucleases
•Maximize the quality of DNA
How Much DNA Can We Recover?
•A Diploid Cell contains approximately 6 pg of DNA
•Sperm contains approximately 3 pg of DNA
•The average WBC of an adult is 5 - 10 X 106 cells
per ml of blood. Therefore, the theoretical
recovery of DNA per ul of blood is 30 - 60 ng.
 How Much DNA Do We Need?
•The PCR procedure on requires a minimum of
50 ng of high molecular weight double stranded
DNA. This is the equivalent of approximately 2
ul of blood. The number of intact sperm ( 3
pg/sperm) is approximately 20,000.
 A technique to lyse the
cells gently and solubilize
the DNA
Most DNA extraction
protocols consist of two
parts Enzymatic or chemical
methods to remove
contaminating proteins,
RNA, or macromolecules

Basic Protocol
 Isolation of Nucleic Acids
Goals: Types of Methods:
• removal of proteins • differential solubility
• DNA vs RNA • ‘adsorption’ methods
• isolation of a specific • density gradient
type of DNA (or RNA) centrifugation

Types of DNA: General Features:

• genomic (chromosomal) • denaturing cell lysis
• organellar (satellite) (SDS, alkali, boiling,
• plasmid (extra- chaotropic)
chromosomal) •  enzyme treatments
• phage/viral (ds or ss) - protease
• complementary (mRNA) - RNase (DNase-free)
- DNase (RNase-free)
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
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.

What are the Most Commonly used DNA
Extraction Procedures?

1. Organic (Phenol-Chloroform) Extraction

2. Non-Organic (Proteinase K and Salting out)
3. Adsorption method (silica-gel membrane)

The method utilized may be sample dependant,

technique dependant, or analyst preference
What are the Most Commonly used DNA
Extraction Procedures?
 High MW Genomic DNA Isolation
Typical Procedure
Phenol Extraction
1 Cell Lysis
• mix sample with equal
– 0.5% SDS + volume of sat. phenol soln
proteinase K (37o C 2
• retain aqueous phase
hours)
• optional chloroform/isoamyl
2 Phenol Extraction alcohol extraction(s)

3 Ethanol Precipitation
4 RNAse followed by  aqueous phase
proteinase K (nucleic acids)
5 Repeat phenol extrac-  phenol phase
tion and EtOH ppt (proteins)
 ORGANIC EXTRACTION
Perhaps the most basic of all procedures in molecular biology
is the purification of DNA by organic extraction.
 High MW Genomic DNA Isolation
Typical Procedure EtOH Precipitation
1 Cell Lysis • 2-2.5 volumes EtOH, -20o
– 0.5% SDS + • high salt, pH 5-5.5
proteinase K (55o • centrifuge or ‘spool’ out
several hours)
2 Phenol Extraction
3 Ethanol Precipitation
4 RNAse followed by
proteinase K
5 Repeat Phenol Extrac-
tion and EtOH ppt Phenol
Extraction
 ORGANIC EXTRACTION
REAGENTS
•Cell Lysis Buffer - Non-ionic detergent (SDS), Tris-Cl, EDTA
- designed to lyse outer cell membrane and nuclear
membrane.
•EDTA (Ethylenediaminetetraacetic disodium salt) is a
chelating agent of divalent cations such as Mg2+. Mg2+is a
cofactor for Dnase nucleases. If the Mg2+is bound up by
EDTA, nucleases are inactivated.
DNA Isolation
What does the detergent do?

Each cell is surrounded by a cell

membrane
DNA is in the nucleus of a cell, which is
also surrounded by a membrane
 The membranes must be broken
open in order to get the DNA out
of the cell
Detergent:
Breaks apart membranes by attaching to the lipids (fats) & proteins in
the membranes
The cell and nuclear membranes have been broken apart,
as well as all of the organelle membranes,
such as those around the mitochondria and chloroplasts.
So what is left?

Proteins
Carbohydrates (sugars)
DNA
 ORGANIC EXTRACTION
REAGENTS
•Proteinase K - it is usual to remove most of the protein by
digesting with proteolytic enzymes such as Pronase or
proteinase K, which are active against a broad spectrum of
native proteins, before extracting with organic solvents.
•Protienase K is approximately 10 fold more active on denatured
protein.
•Proteins can be denatured by SDS or by heat.
 ORGANIC EXTRACTION
REAGENTS
•Phenol/Chlorform - The standard way to remove proteins from
nucleic acids solutions is to extract once with phenol, once with a 1:1
mixture of phenol and chloroform, and once with chloroform. This
procedure takes advantage of the fact that deproteinization is
more efficient when two different organic solvents are used
instead of one.
•The final extraction with chloroform removes any lingering traces
of phenol from the nucleic acid preparation.
•Phenol is highly corrosive and can cause severe burns.
 ORGANIC EXTRACTION REAGENTS
•Phenol - often means phenol equilibrated with buffer (such as TE) and
containing 0.1% hydroxyquinoline and 0.2% b-mercaptoethanol (added as
antioxidants). The hydroxyquinoline also gives the phenol a yellow
color,making it easier to identify the phases (layers).
•Chloroform - often means a 24:1 (v/v) mixture of chloroform and isoamyl
alcohol. The isoamyl alcohol is added to help prevent foaming.
•The Phenol/Chloroform/Isoamyl Alcohol ratio is 25:24:1
 Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly Aqueous

with an equal
volume of organic Centrifuge Collect aqueous phase
solvent
Interphase
e.g. phenol, chloroform,
or phenol:chloroform Organic

Perform additional extractions for increased

purity

Crude lysate The aqueous phase contains water-

containing nucleic soluble molecules, including nucleic
acids and other cell acids. Proteins and lipids become
constituents trapped in the organic phase, and
are thus separated away. Insoluble
debris become trapped in the
interphase between the two layers
◦Phenol denatures proteins and
dissolves denatured proteins.
◦Chloroform is also a protein
denaturant
 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).
Concentrating DNA Alcohol Precipitation

•The most widely used method for concentrating DNA is

precipitation with ethanol. The precipitate of nucleic acid,
forms in the presence of moderate concentrations of
monovalent cations (Salt, such as Na+), is recovered by
centrifugation and redissolved in an appropriate buffer such
as TE.
•The technique is rapid and is quantitative even with nanogram
amounts of DNA.
 Concentrating DNA Alcohol Precipitation
•The salts interrupt the hydrogen bonds between the
water and DNA molecules
•The DNA is then precipitated from the protein in a
subsequent step with isopropanol or ethanol
•In the presence of cations, ethanol induces a
structural change in DNA molecules that causes
them to aggregate and precipitate out of solution.
 Concentrating DNA Alcohol
Precipitation
•The four critical variables are the purity of the DNA, its
molecular weight, its concentration, and the speed at which
it is pelleted.
•DNA a concentrations as low as 20 ng/ml will form a
precipitate that can be quantitatively recovered.
•Typically 2 volumes of ice cold ethanol are added to
precipitate the DNA.
 Concentrating DNA Alcohol
Precipitation
•Very short DNA molecules (<200 bp) are
precipitated inefficiently by ethanol.
•The optimum pelleting conditions depend on the
DNA concentration. Relatively vigorous
microcentrifuge steps such as 15 minutes at or
below room temperature at 12,000 rpm are
designed to minimized the loss of DNA from
samples with yields in the range of a few
micrograms or less.
 Concentrating DNA Alcohol
Precipitation
•Solutes that may be trapped in the precipitate may
be removed by washing the DNA pellet with a
solution of 70% ethanol.
•To make certain that no DNA is lost during
washing, add 70% ethanol until the tube is 2/3 full.
Recentrifuge.
•After the 70% ethanol wash, the pellet does not
adhere tightly to the wall of the tube, so great
care must be taken when removing the supernatant.
 Concentrating DNA Alcohol
Precipitation
•Isopropanol (1 volume) may be used in place of
ethanol (2 volumes) to precipitate DNA.
Precipitation with isopropanol has the advantage
that the volume of liquid to be centrifuged is
smaller.
•Isopropanol is less volatile than ethanol and it is
more difficult to remove the last traces; moreover,
solutes such sodium chloride are more easily
coprecipitated with DNA when isopropanol is used.
 Extraction/Precipitation Method
Step 4: Nucleic Acid Precipitation

Before After

Supernatant 70% EtOH

Centrifuge Wash Centrifuge

Pellet
Dissolve
• Pellet down nucleic acids. pellet (H2O,
Add alcohol and salt TE, etc.)
to precipitate nucleic • Wash pellet with 70% ethanol to
acids from the remove
aqueous fraction residual salts and other
contaminants.
• Discard ethanol and allow pellet
to dry.
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 Non-Organic DNA Extraction

However, if salts
are not
Digested
adequately
proteins are
Does not use removed,
removed by
organic reagents problems could
salting out with
such as phenol occur with the
high
or chloroform. RFLP procedure
concentrations
due to alteration
of LiCl (NaCl).
of DNA mobility
(band shifting)
Non-Organic DNA Extraction Procedure
1. Cell Lysis Buffer - lyse cell membrane, nuclei are intact,
pellet nuclei.
2. Resuspend nuclei in Protein Lysis Buffer containing a
high concentration of Proteinase K. Lyse nuclear
membrane and digest protein at 65oC for 2 hours.
Temperature helps denature proteins, and Proteinase K
auto digests itself
3. To remove proteinaceous material, LiCl is added to a
final concentration of 2.5 M, and incubated on ice.
Proteins precipitate out and are pelleted by
centrifugation.
Non-Organic DNA Extraction Procedure

4. DNA remains in solution. Transfer

supernatant to a new tube, care must be
taken not to take any of protein pellet.
5. DNA is precipitated by the addition of
room temperature isopropanol. LiCl will not
precipitate with DNA.
6. Precipitated DNA is washed with 70%
ethanol, dried under vacuum and
resuspended in TE buffer.
 Adsorption 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 is recovered.
 Adsorption method
• Nucleic acids selectively absorb to silica or resins
in the presence of certain chaotropic agents or
salts Plasmid Miniprep Protocol
• applications: 1. Solubilize bacteria in
• plasmid preps alkali solution
• fragments after 2. Neutralize with Na-
electrophoresis acetate
• PCR templates 3. Centrifuge, discard pellet
4. Mix supernatant with
resin + chaotropic agent
5. Wash resin
6. Elute DNA with low salt
buffer
 Adsorption Method
Step 1: Prepare crude lysate Step 2: Adsorb to silica surface

Apply to Centrifuge
column
Nucleic acids
Silica-gel membrane

Extraction Buffer composition Flow through

favors DNA adsorption to silica: (discard)
• Low pH
• High ionic strength
Nucleic acids bind to the
membrane, while contaminants
pass through the column.

Having the ability to

destabilize hydrogen
bonding and
hydrophobic
interactions.
 Adsorption Method
Step 3: Wash away residual contaminants

Centrifuge
Wash buffer
Nucleic acids Nucleic acids

Flow through

Step 4: Elute nucleic acids (discard)

Centrifuge
Elution buffer

Nucleic acids

Elution Buffer composition is

unfavorable to surface binding:
High pH
Low ionic strength Nucleic acids
Evaluation of Nucleic
Acids
• spectrophotometrically
• quantity
• quality
• fluorescent dyes
• gel electrophoresis
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)
conc. DNA (mg/ml) = (A260 x R x 50)/1000
 Using Spectroscopy to analyze DNA
DNA absorbs UV light with a major peak at 260 nm
Optical Density

This absorption is useful because

it varies with the structure of
DNA (&RNA)
i.e. extinction coefficient
Wave Length depends on the structure

dsDNA ssDNA
Low Higher
extinction extinction
coefficient coefficient
How pure is your sample?
• The A260/A280 ratio is ~1.8 for dsDNA. 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.
How pure is your sample?
• spectrophotometrically
• quantity
• quality
• fluorescent dyes
• gel electrophoresis

A260 1.0  50 g/ml

DNA
A260/A280 1.6 - 1.8
A260 1.0  40 g/ml
RNA
A260/A280 ~2.0
Nanodrop 2000c
 What is it a NanoDrop?
•Manufacture and Cell analytical laboratory
equipment worldwide
•Used in biotechnology, pharmaceutical, and life
science fields
•NanoDrop ND2000cSpectrophotometer is best
known small liquid sample instrument
 NanoDrop ND2000c
•The NanoDrop 2000c is the only
spectrophotometer that combines microvolume
pedestal technology and cuvette capability in a
single instrument.
• The pre-configured methods for all common
nucleic acid and protein concentration and
purity measurements, in both microvolume and
cuvette modes.
•Dual-mode UV-Vis spectrophotometer with
microvolume and cuvette capability:
 NanoDrop ND2000c
•Features the same patented sample retention
system* as the NanoDrop 2000 spectrophotometer
for measurement of 0.5 – 2 µL DNA, RNA, and protein
samples
•Measure very low and high concentration samples
(0.4 – 15,000 ng/µL dsDNA)
•Cuvette capability allows for kinetics (time or time /
temperature studies) and cell culture (OD 600)
measurements
•Cuvette position includes temperature control (37° C)
and stirring
•Custom methods capability in both modes
Classic NanoDrop

Applications
•Nucleic Acid
•Protein A280
•Proteins & Labels
•UV-Vis
•Cell Cultures
•plus Diagnostics
 Instrument Specifications
•NanoDrop 2000/2000c – pedestal mode
•Instrument Type: Spectrophotometer •Detection limit: 2 ng/μL dsDNA
•Minimum Sample Size: 0.5 μL •Maximum Concentration:
•Pathlength: 1 mm (auto-ranging to 0.05 mm) 15,000 ng/μL (dsDNA)

•Light Source: Xenon flash lamp •Measurement Time: < 5 seconds

•Detector Type: 2048-element linear silicon CCD •Footprint: 14 cm x 20 cm
array •Weight: 2.0 kg
•Wavelength Range: 190-840 nm •Sample pedestal Material
•Wavelength Accuracy: +1 nm •of Construction: 303 stainless
•Spectral Resolution: <1.8 nm (FWHM @Hg steel and quartz fiber
253.7 nm) •Operating Voltage: 12 VDC
•Absorbance Precision: 0.002 absorbance (1 mm •Operating Power Consumption:
path) 12-18 W, (max 30 W)
•Absorbance Accuracy: ± 2% (at 0.76 •Software Compatibility:
absorbance at 257 nm) Windows® XP and Vista (32
•Absorbance Range: 0.02 -300 (10 mm bit)
equivalent)
NanoDrop 2000c – Beam height: 8.5
Heating: 37 ± 0.5°C
cuvette mode mm

Stirrer: 150-850 Pathlength: 10, 5, Detection Limit: 0.4

RPM 2, 1 mm ng/μL dsDNA

Maximum
Measurement
Concentration: 750 Weight: 2.1 kg
Time: < 3 seconds
ng/μL (dsDNA)
Using the ND2000c
1. Turn on PC

2. Open NanoDrop program

3. Select “DNA” from menu

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Pedestal Basic Use
1. Raise the sampling arm and pipette the sample onto the lower measurement pedestal.

2. Lower the sampling arm and initiate a spectral measurement using the software on the
PC. The sample
column is automatically drawn between the upper and lower pedestals and the measurement
is made.
3. When the measurement is complete, raise the sampling arm and wipe the sample from
both the upper and
lower pedestals using a dry, lint-free laboratory wipe. Simple wiping prevents sample
carryover in
subsequent measurements for samples varying by more than 1000 fold in concentration.
1-2
Cuvette Measurements
The NanoDrop 2000c will accept 10 mm cuvettes up to 48 mm tall. When measuring
samples using micro, semimicro,
or ultra-micro cuvettes, we recommend using masked cuvettes. Masked cuvettes ensure
that all light hitting
the detector has passed through the sample. Unmasked cuvettes can allow light to hit the
detector that has not
passed through the sample. Expected differences between unmasked plastic cuvettes can
lead to significant
measurement error, especially at low concentrations.
Un-masked Eppendorf Uvette® Starna masked micro cuvette
When measuring samples with analytical wavelengths in the UV (<340 nm), use cuvettes
made of quartz, as these
pass UV wavelengths. Although some manufacturers offer "UV-transparent" plastic
disposable cuvettes, even the
best of these are not transparent at wavelengths below 220 nm; most plastic and glass
cuvettes block UV
wavelengths entirely.

Un-masked Eppendorf Uvette® Starna masked micro cuvette

 Using the ND2000c
Lift arm

Place 1 μL water on pedestal to initialize

instrument

BLANK instrument using water or elution

buffer

Place 1-2 μL of DNA sample on pedestal

Click “MEASURE” on instrument screen

Recover sample if precious, or clean

pedestals using a lab wipe

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NanoDrop Data Screen
How does it work?
Fiber optic cable in
measurement arm

Sample (1-2 μL)

Receiving fiber (fiber optic

cable)

Xenon
Spectrophotometer lamp
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