DNA

EXTRACTION
What is DNA?
Importance of DNA extraction
The steps in DNA Extraction
The different methods of DNA
extraction
Chemicals used in DNA
extraction

Note:try to look for common

example/s of the importance….
What is DNA?

 Deoxyribonucleic acid (DNA) is a molecule

composed of two chains that coil around
each other to form a double helix carrying
the genetic instructions used in the growth,
development, functioning, and reproduction
of all known organisms.
 DNA Extraction
DNA isolation is a process of purification
of DNA from sample using a
combination of physical and chemical
methods.
Isolation of DNA by breaking the cell
membrane and nuclear membrane with
the help of chemicals, enzymes or
physical disruptions.
 Three Basic Steps of DNA
extraction
 Step 1: Lysis
In this step, the
cell and the
nucleus are
broken open to
release the DNA
inside.
Can be done in two
ways:
• First, mechanical disruption breaks open the cells.
This can be done with a tissue homogenizer (like a
small blender), with a mortar and pestle, or by
cutting the tissue into small pieces and particularly
important when using plant cells because they
have a tough cell wall.
• Second, lysis uses detergents and enzymes such
as Proteinase K to free the DNA and dissolve
Step 2: Precipitation

When you complete the lysis step, the

DNA has been freed from the nucleus,
but it is now mixed with mashed up cell
parts. Precipitation separates DNA from
this cellular debris. First, Na+ ions
(sodium) neutralize the negative
charges on the DNA molecules which
makes them more stable and less water
soluble. Next, alcohol (such as ethanol
or isopropanol) is added and causes
DNA to precipitate out of the aqueous
solution because it is not soluble in
Step 3: Purification

Now that DNA has been separated from

the aqueous phase, it can be rinsed with
alcohol to remove any remaining
unwanted material and cellular debris. At
this point the purified DNA is usually re-
dissolved in water for easy handling and
storage.
Different types of DNA extraction
methods
1.Chemical based DNA extraction method
A.Organic solvent-based DNA extraction
-Phenol-chloroform Method
B. Inorganic solvent-based DNA extraction
-Proteinase K Method
-Salting out Method
-Enzymatic Method
2.Solid phase DNA extraction method
-Silica column based DNA extraction method
3. Other DNA extraction Method
-DNA extraction using the anionic resins
-DNA extraction by magnetic beads
-CsCl density gradient method
Phenol-chloroform method
This method is one of the best methods of
DNA extraction. The yield and quality of
DNA obtained by the PCl method is very
good if it’s perform well.
The major chemicals of PCl method are
lysis buffer, Phenol and Chloroform.
The PCl method is widely accepted. Even
the forensic department trust PCl method
rather than a Kit method. The quantity of
DNA obtain is very high. It can obtain 800-
900 mg of DNA with great quality.
Proteinase K Method
The proteinase K DNA extraction
method facilitates high DNA yield but
it is time-consuming. Also if not
maintained well in a cold chain, the
proteinase K cannot be utilized for a
longer period of time.
The lower stability of the enzyme is
another major issue in this method.
Salting out DNA extraction
method
This method is safer than the PCl
method. The use of salts such as
sodium chloride, potassium acetate
and ammonium acetate helps in the
DNA extraction. However, the method
is more aggressive in combination
with proteinase K.
Use of the different salt in DNA
extraction can increase the yield but
the purity is not good enough.
Enzymatic Method
 This method is a combination of a salt method as
well as enzymatic method. Here the extraction
buffer is used before going further on enzymatic
digestion.
 The extraction buffer composition may vary from
lab to lab, however, the major components are Tris,
EDTA, NaCl, sodium lauryl and SDS. Here phenol,
chloroform or isoamyl alcohol is not used. Instead,
the enzyme proteinase K is utilized for digesting
the sample.
 This method of DNA extraction is rapid and easy.
We can use ready-to-use DNA extraction buffer.
Even the yield is very high, the quality of DNA is a
major concern in this method.
Silica column based DNA
extraction method
This method is very unique and different
from other DNA extraction methods.
The silica-based method works on the
unique chemistry of interaction between
silica and DNA. A positively charged silica
particles bind with the negatively charged
DNA and hold it during centrifugation.
It is widely accepted because of its good
quality DNA yield and minimal simple
operating system. The method is fast
reliable, accurate and consumes less time.
DNA extraction using anionic
resins
The Chelex the product of Bio-Rad
laboratories is the best example of the
anionic resin used in the DNA
extraction. It is made up of the styrene-
divinylbenzene copolymers.
The positively charged chelex binds to
the negatively charged phosphate of
DNA and helps in the extraction of DNA.
Another resin used is the
Diethylaminoethyl.
DNA extraction by magnetic
beads
Positively charged magnetic beads
attract the negatively charged DNA.
The DNA is separated under the
magnetic field.
DNA extraction buffer is needed in this
technique.
CsCl density gradient method
of DNA extraction
 In this method, the DNA is separated based
on the density of it with the centrifugation.
 In the high-speed centrifugation, at the
isopycnic point where the density of the DNA
and the gradient (CsCl) become same, the
DNA band will appear.
 However, the method is tedious and hard to
perform as it required high-speed
centrifugation for more than 10 hours.
 Another major limitation of density gradient
centrifugation is the use of the carcinogenic
EtBe.
 Chemical used in DNA Extraction

Chloroform – chloroform prevents cutting of DNA

during isolation. It solubilizes lipids and a lot of
protein to remove them from the DNA.
Isopropanol – its main role is to precipitate the DNA,
by engaging with the H2O molecules not giving
chance for the DNA to get dissolve in the H2O.
Ethanol- is a dehydrating agent. When a molecule is
to be precipitated it should be dehydrated as the H2O
molecules forming a film around it prevent their
interaction.
CTAB (Cetyl Trimethyl Ammonium Bromide)- CTAB
is a best detergent to use during extraction of highly
polymerized DNA from plant material. This detergent
simultaneously solubilizes the plant cell wall.
Tris- DNA is pH sensitive, Tris buffer maintains the pH
of the solution. Also, it interacts with
lipopolysaccharides of the cell membrane and
makes them permeable.
EDTA- is a chelating agent and can be used to block
DNAse activity.
SDS (Sodium Dodecyl Sulphate)- is an anionic
detergent which helps cell membrane and nuclear
envelope to break open.
NaCl- the Na+ ion of NaCl creates the ionic bond with
the negative charge of DNA and neutralize it. It will
help DNA comes together and protect from
denaturation.
MgCl2- it protects the DNA. MgCl2 block the negative
charge of the lipoproteins of the cell membrane.
Phenol- it precipitates the protein impurities.
THANK YOU.