DNA Extraction

Dilshan Dissanayaka
Lecturer
Department of Biosystem Technology
Faculty of Technology
University of Ruhuna
 Introduction
• DNA extraction
• Applications

Steps of DNA extraction process

Contents •
•
Disruption
Separation
• Purification
• Concentration
• Elusion

Types of DNA extraction

What is the DNA Extraction…?

DNA extraction is a routine

procedure used to isolate
DNA from the nucleus of
cells
What can this DNA be used for?

DNA can be used for molecular analyses

including,
▪ PCR
▪ Electrophoresis
▪ Sequencing
▪ Fingerprinting
▪ Cloning
 Basic steps of DNA extraction
There are four basic steps of DNA extraction,

1. Disruption of the cellular structure to create a lysate

2. Separation of the soluble DNA from cell debris and
other insoluble material
3. Purification of DNA from a cell extract
4. Concentration of DNA samples
5. Elusion
 Basic steps of DNA extraction
1. Disruption of the cellular structure to create a lysate

▪ The first step in any nucleic acid purification reaction is

releasing the DNA into solution

▪ The goal of lysis is to rapidly and completely disrupt cells in a

sample to release nucleic acid into the lysate

▪ There are four general techniques for lysing materials,

I. Physical methods
II. Enzymatic methods
III. Chemical methods
IV. Combinations of the three
1. Disruption of the cellular structure to
create a lysate
I. Physical methods

A common method of physical disruption is freezing &

grinding samples with a mortar and pestle under liquid
nitrogen to provide a powdered material that is then
exposed to chemical or enzymatic lysis conditions
1. Disruption of the cellular structure to crea
te a lysate
I. Physical methods

Other devices use bead beating or shaking in the presence of

metallic or ceramic beads to disrupt cells or tissues, or
sonication to disrupt tissues and lyse cells

beads
1. Disruption of the cellular structure to crea
te a lysate
II. Chemical Methods

Chemical methods can be used alone with easy-to-lyse

materials, such as tissue culture cells or in combination with
other methods
1. Disruption of the cellular structure to crea
te a lysate
II. Chemical Methods

Chemicals commonly used include detergents [e.g., sodium

dodecyl sulphate (SDS)] and chaotropes (e.g., guanidine salts and

alkaline solutions)

SDS aid the process of

lysis by removing lipid
molecules and thereby
cause disruption of the
cell membranes
1. Disruption of the cellular structure to crea
te a lysate
II. Chemical Methods
Chaotropes

Destabilize hydrogen bonds, van der Waals

forces and hydrophobic interactions,
leading to destabilization of proteins,
including nucleases
1. Disruption of the cellular structure to crea
te a lysate
III. Enzymatic Methods

❖ Enzymatic methods are often used with more structured

starting materials in combination with other methods with
tissues, plant materials, bacteria and yeast
❖ The enzymes utilized help to disrupt tissues and tough cell
walls
❖ Depending on the starting material, typical enzymatic
treatments can include:
lysozyme, zymolase and liticase, proteinase K, collagenase and
lipase
1. Disruption of the cellular structure to crea
te a lysate
III. Enzymatic Methods
Lysozyme

❖ An enzyme that is present in egg white and in

secretions such as tears and saliva
❖ Digests the polymeric compounds that give the cell
wall its rigidity
 Basic steps of DNA extraction
2. Separation of the soluble DNA from cell debris and
other insoluble material

❖ Final step in the preparation of a cell extract is the removal

of insoluble cell debris
❖ Clearing is accomplished by centrifugation, filtration
2. Separation of the soluble DNA from cell
debris and other insoluble material
❖ Components such as partially digested cell wall fractions
can be pelleted by centrifugation leaving the cell extract as
a reasonably clear supernatant

❖ Filtering can be a rapid method, but samples with a large

amount of debris can clog the filter
 Basic steps of DNA extraction
3. Purification of DNA from a cell extract

❖ A variety of methods can be used to purify the DNA from

this mixture,
a) Removing contaminants by organic extraction and enzyme
digestion
b) Using ion-exchange chromatography
c) Using silica

Degradation of contaminants Seperation of DNA from contaminants

 3.
3. Purification of DNA from a cell extract
a) Removing contaminants by organic extraction and
enzyme digestion
❖ The standard way to deproteinize a cell extract is to add
phenol or a 1 : 1 mixture of phenol and chloroform
❖ These organic solvents precipitate proteins but leave the
nucleic acids (DNA and RNA) in aqueous solution
 3.
3. Purification of DNA from a cell extract

a) Removing contaminants by organic extraction and

enzyme digestion
01 Problem

With some cell extracts the protein content is so great that a

single phenol extraction is not sufficient to completely purify
the nucleic acids. How to solve this problem?
 3.
3. Purification of DNA from a cell extract

a) Removing contaminants by organic extraction and

enzyme digestion
Solutions

❑ Carrying out several phenol extractions one after the other

This is undesirable as each mixing and centrifugation step

results in a certain amount of breakage of the DNA molecules
 3.
3. Purification of DNA from a cell extract

a) Removing contaminants by organic extraction and

enzyme digestion
Solutions

✓ The answer is to treat the cell extract with a protease such

as pronase or proteinase K before phenol extraction. These
enzymes break polypeptides down into smaller units,
which are more easily removed using phenol
 3.
3. Purification of DNA from a cell extract

a) Removing contaminants by organic extraction and

enzyme digestion
02 Problem

Some RNA molecules, especially messenger RNA (mRNA), are

removed by phenol treatment, but most remain with the DNA
in the aqueous layer. How to remove this?
 3.
3. Purification of DNA from a cell extract

a) Removing contaminants by organic extraction and

enzyme digestion

Solutions

✓ The only effective way to remove the RNA is with the

enzyme ribonuclease, which rapidly degrades these
molecules into ribonucleotide subunits.
 3.
3. Purification of DNA from a cell extract
b) Using ion-exchange chromatography to purify DNA
from a cell extract

❖ Separates molecules according to how tightly they bind to

electrically charged particles present in a chromatographic
matrix or resin
❖ DNA and RNA are both negatively charged, as are some
proteins, and so bind to a positively charged resin
 3.
3. Purification of DNA from a cell extract
b) Using ion-exchange chromatography to purify DNA
from a cell extract

❖ The electrostatic attachment is disrupted by salt

❖ To remove more tightly bound molecules requires higher
concentrations of salt
 3.
3. Purification of DNA from a cell extract
b) Using ion-exchange chromatography to purify DNA
from a cell extract

By gradually increasing
the salt concentration,
different types of
molecule can be
detached from the
resin, one after
another
 3.
3. Purification of DNA from a cell extract
c) Using silica to purify DNA from a cell extract

In the presence of
guanidinium
thiocyanate, DNA
binds tightly to
silica particles,
providing an easy
way of recovering
the DNA from a cell
extract
 3.
3. Purification of DNA from a cell extract

c) Using silica to purify DNA from a cell extract

Different types of silica based purification of DNA,

i. Silica particles added directly to the cell extract
ii. Silica-coated magnetic beads
 3.
3. Purification of DNA from a cell extract

c) Using silica to purify DNA from a cell extract

i. Silica particles added directly to the cell extract

 3.
3. Purification of DNA from a cell extract

c) Using silica to purify DNA from a cell extract

ii. Silica-coated magnetic beads

 3.
4. Concentration of DNA samples

❖ The most frequently used method of concentration is

ethanol precipitation
❖ In the presence of a salt (monovalent cations such as
sodium ions, Na+) and at a temperature of −20 ◦C or less,
absolute ethanol efficiently precipitates polymeric nucleic
acids
❖ With a thick solution of DNA the ethanol can be layered
on top of the sample, causing molecules to precipitate at
the interface
 3.
4. Concentration of DNA samples

Fibres of DNA can Precipitated

be withdrawn with a DNA collected by
glass rod centrifugation
 5. Elusion

✓ DNA is soluble in low-ionic-strength solution such as TE buffer or nuclease-free

water.

✓ When such an aqueous buffer is applied to a silica membrane, the DNA is released
from the silica, and the eluate is collected.

✓ The purified, high-quality DNA is then ready to use in a wide variety of demanding
downstream applications (PCR, coupled in vitro transcription/translation systems,
transfection and sequencing reactions).
 Storage
✓ Among the most often used preservation method of samples collected
for DNA analyses is freezing.

✓ Freezing at −80 °C or in liquid nitrogen (−196 °C) is most often used for
long term storage.

✓ For short term storage −20 to −28 °C is preferred

 Types of DNA extraction

Mainly 3 different types of DNA Extractions

1. Total cell DNA

2. Plasmid DNA
3. Phage DNA
 Total cell DNA

❖ Total cell DNA will often be required as a source of

material from which to obtain genes to be cloned
❖ It consists of the genomic DNA of the organism along with
any additional DNA molecules, such as plasmids, that are
present

❖ Total cell DNA may be DNA from,

1. a culture of bacteria
2. a plant
3. animal cells
4. any other type of organism that is being studied
Difference of total cell DNA extraction
between organisms

A. Cell growth medium

B. Cell breakage stage/Cell lysis stage
C. Biochemical content of the cells
 Difference of total cell DNA extration
between organisms
A. Cell growth medium

❖ Most common growth media for microorganisms

(bacteria) are nutrient broths (liquid nutrient
medium) or lysogeny broth medium

❖ Cell culture is the general term used for the removal

of cells, tissues or organs from an animal or plant and
their subsequent placement into an artificial
environment favorable to their survival and/or
proliferation
 Difference of total cell DNA extration
between organisms
B. Cell breakage stage/Cell lysis stage
❖ The chemicals used for disrupting bacterial cells do
not usually work with other organisms
➢ Example,
▪ Lysozyme,
✓ has no effect on plant cells
❖ Specific degradative enzymes are available for most
cell wall types, but often physical techniques (e.g.,
grinding frozen material with a mortar and pestle) are
more efficient
❖ Most animal cells have no cell wall at all and can be
disrupted simply by treating with detergent
Difference of total cell DNA extration
between organisms
C. Biochemical content of the cells

Most bacteria, the main

biochemicals present in a cell extract
are protein, DNA and RNA, and
consequently a phenol extraction
and/or protease treatment, followed
by the removal of RNA with
ribonuclease, leaves a pure DNA
sample
Difference of total cell DNA extration
between organisms
C. Biochemical content of the cells

❖ Plant tissues are particularly difficult in this respect as

they often contain large amounts of carbohydrates
that are not removed by phenol extraction

➢ Example

✓ The detergent cetyltrimethylammonium bromide

(CTAB) - which forms an insoluble complex with
nucleic acids
 1. Total DNA preparation from a
culture of bacterial cells

❖ The procedure for total DNA preparation from a culture of

bacterial cells can be divided into four stages,
A. A culture of bacteria is grown and then harvested
B. The cells are broken open to release their contents
C. This cell extract is treated to remove all components
except the DNA
D. The resulting DNA solution is concentrated
1. Total DNA preparation from a
culture of bacterial cells
 1. Total DNA preparation from a
culture of bacterial cells
A. Growing and harvesting a bacterial culture

❖ Most bacteria can be grown without too much difficulty in a

liquid medium (broth culture)
❖ The culture medium must provide a balanced mixture of
the essential nutrients at concentrations that will allow the
bacteria to grow and divide efficiently
 1. Total DNA preparation from a
culture of bacterial cells
A. Growing and harvesting a bacterial culture

The composition of two typical media for the growth of

bacterial cultures
Thank you