DNA ISOLATION

Sunarto Ang

Pusat Kedokteran Tropis
RSUD. A. Wahab Sjahranie
Fakultas Kedokteran - Universitas Mulawarman
Samarinda
DNA Isolation
DNA isolation:
is an extraction process of DNA from various
sources.
The aim:
is to separate DNA present in the nucleus of the
cell from other cellular components.

Basic Protocol:
break the cells open to expose the DNA within.
remove the nuclear membrane lipids by adding a
detergent.
precipitate the DNA with an alcohol. This step will also
remove alcohol-soluble salts.
Application of DNA isolation
1. Scientific:
use DNA in number of Applications, such as introduction
of DNA into cells & animals or plants for diagnostic
purposes (gene cloning)

2. Medicine:
is the most common. To identify point sources for
hospital and community-based outbreaks and to predict
virulence of microorganisms

3. Forensic science:
needs to recover DNA for identification of individuals,
(for example rapists, petty thieves, accident, or war
victims), and paternity determination.

Overview of Procedure
Lyse RBCs & WBCs

Lyse WBCs nuclei & Denature/digest
proteins

Separate contaminants (e.g., proteins, heme)

Precipitate DNA

Resuspend DNA in final buffer


Samples for DNA Isolation
Blood
Semen
Saliva
Urine
Hair (w/Root & Shaft)
Teeth
Bone
Tissue

Cigarette Butts
Envelope & Stamps
Fingernail Clippings
Chewing Gum
Bite Marks
Feces
Blood Collection
1. Blood collected in disodium EDTA tube

2. Samples can be stored at -20
o
C or -70
o
C

3. Fresh samples are kept in freezer for a
few hours to facilitate RBCs hemolysis

4. Allow samples to thaw before starting the
extraction

Methods of DNA Isolation
1. Organic extraction
2. Salting out
3. Selective DNA binding to a solid support

1. Organic extraction
DNA is polar insoluble in organic solvents.
Traditionally, phenol:chloroform is used to extract DNA.

When phenol is mixed with the cell lysate, two
phases form.
DNA partitions to the (upper) aqueous phase
Denatured proteins partition to the (lower) organic
phase.

Genomic DNA isolation: phenol extraction
1:1 phenol : chloroform or
25:24:1 phenol : chloroform : isoamyl
alcohol

Phenol: denatures proteins, precipitates form
at interface between aqueous and organic
layer
Chloroform: increases
density of organic layer

Isoamyl alcohol: prevents
foaming

Genomic DNA is isolated as
pieces up to 1 Mbp!


Salting out
Digested proteins are removed by
salting out with high concentrations of LiCl.

However, if salts are not adequately
removed,
problems could occur with the RFLP
procedure due to alteration of DNA mobility
(band shifting)

Procedure of Salting out
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 65C 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.

Procedure of Salting out
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.


Binding to a support material
Support Materials
Silica
Anion-exchange resin

Advantages
Speed and convenience
No organic solvents

Disadvantage
DNA fragmentation

Plants
Nucleus is protected within a nuclear
membrane which is
surrounded by a cell membrane and a cell wall.

Four steps are used to remove and purify
the DNA
1. Lysis
2. Precipitation
3. Wash
4. Resuspension

LYSIS
In DNA extraction from plants,
this step commonly refers to the breaking of the cell wall
and cellular membranes (most importantly, the plasma and
nuclear membranes)

The cell wall (made of cellulose) is disrupted by
mechanical force (for example, grinding the leaves)

Then the addition of a detergent in the which
breaks down the cell membranes
LYSIS
Detergents are able to disrupt membranes
due to
the amphipathic (having both hydrophilic and
hydrophobic regions) nature of both cellular
membranes and detergent molecules.

The detergent molecules are
able to pull apart the membranes

The end result of LYSIS is
that the contents of the plant cells are distributed
in solution.


PRECIPITATION
A series of steps where DNA is separated
from the rest of the cellular components

The first part of precipitation uses
phenol/chloroform to remove the proteins
from the DNA

Phenol denatures proteins and dissolves
denatured proteins.
Chloroform is also a protein denaturant

PRECIPITATION
The second is the addition of salts
The salts interrupt the hydrogen bonds
between the water and DNA molecules.

The DNA is then precipitated by
isopropanol or ethanol
ethanol induces a structural change in DNA
molecules that causes them to aggregate and
precipitate out of solution.

The DNA is pelleted by spinning with a
centrifuge and the supernatant removed


Washing:
The precipitated DNA is laden with acetate
salts.
It is washed with a 70% ethanol solution to remove
salts and other water soluble impurities but not
resuspend the DNA.

Resuspension:
The clean DNA is now resuspended in a buffer to
ensure stability and long term storage.

The most commonly used buffer for
resuspension is called 1xTE

Break down
the cell wall
and
membranes
Centrifuge to
separate the
solids from
the dissolved
DNA
Precipitate
the DNA
using
isopropanol
Centrifuge to
separate the
DNA from
the dissolved
salts and
sugars
Wash the
DNA pellet
with Ethanol
and dry the
pellet
Dissolve
DNA
Overview of DNA Extraction
Checking the Quality of your DNA
Poor quality DNA will not perform well in
PCR

Assessment of the quality of DNA
extraction:
Mix 10 L of DNA with 10 L of loading buffer
Load this mixture into a 1% agarose gel

Quality from UV Spectrophotometry
DNA absorb maximally at 260 nm.
Proteins absorb at 280 nm.
Background scatter absorbs at 320 nm.
A
260
/A
280
= measure of purity
(A
260
A
320
)/(A
280
A
320
)
1.7 2.0 = good DNA or RNA
<1.7 = too much protein or
other contaminant

Storage Conditions
Store DNA in TE buffer at 4 C for
weeks or at 20 C to 80 C for long
term.

225

C 28

C 20

C 70

C
Recommended
for long-term
storage in ethanol
<4 Months 13 Years <7 Years >7 Years
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