DNA ISOLATION

Kamalraj Subban
Research Associate
Prof. N. Ravi Sundaresan Lab
MCB, IISc, Bangalore
INTRODUCTION
• DNA as a genetic materials
• All living organisms possess genomic DNA (gDNA)
• gDNA contains genes necessary for survival and normal function of each
organism
• These genes encode proteins such as enzymes, receptors, and structural
proteins
• It is a single piece of coiled DNA containing many genes regulatory
elements and other nucleotide sequences.
 •DNA (deoxyribonucleic acid) and RNA
(ribonucleic acid) store and transfer
genetic information in living organisms.
• DNA:
– major constituent of the nucleus
– stable representation of an organism’s complete
genetic makeup
How Can We Recover DNA From a Variety of
Why do we isolate genomic DNA? • Many Sources of Biological Evidence?
Forensic science

applications require isolate Blood

and purified DNA. ⮚Detection of pathogens Semen Saliva Urine Hair Teeth Bone
(bacteria, viruses and fungi) Tissue
• Example applications:
Cigarette Butts Envelope & Stamps
⮚Human identity testing
⮚Tissue typing for organ Fingernail ClippingsChewing Gum
transplant ⮚Genetic research Bite Marks
Feces
⮚Development of diagnostics
What is the Goal? • The RFLP procedure on requires a minimumof
50 ng of high molecular weight
1. Maximize DNA recovery 2. Remove doublestranded DNA.
inhibitors • This is the equivalent of approximately 2ulof
blood. The number of intact sperm (
3. Remove or inhibit nucleases 4. Maximize 3pg/sperm) is approximately 20,000
the quality of DNA • The PCR reactions call for on average 1 ng of
How Much DNA Can We Recover?
DNA (single or double stranded).
• This is the equivalent of 1/20 of 1 ul of
1. A Diploid Cell (6 pg of DNA) blood, or 350 sperm. 2. Sperm contains (3 pg of
DNA)
3. 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.
History of DNA extraction
• The first DNA extraction by Friedrich Miescher in 1869. He had isolated the cell material
and named it as the “nuclei” later on his student named it as a “nucleic acid”. Although he
accidentally developed a method for isolation of nucleic acid, he was notsure that what he
isolated was DNA or not.

• 1958: Meselson and Stahl developed a full-function protocol for DNA extraction.
Thedensity gradient centrifugation protocol was the first protocol described by
isolatingDNA from E.coli bacteria.
•
• The protocol of the proteinase K enzyme method of DNA extraction was developedby
Lahiri and Nurenberger in 1991. They also modified the protocol by using the Nonidet
P40 and SDS.

• The phenol-chloroform isoamyl alcohol method was developed by Joseph

Sambrookand David W. Russell.
DNA purification: overview Lysis of cell wall/ cell
membrane:
 •Chemical disruption
•enzymatic disruption
•Mechanical disruption
Lysis of nuclear
membrane:
•Chemical lysis
•Enzymatic lysis
Removing cell debris
•Centrifugation
IMPORTANCE OF LYSIS BUFFER FOR DNA EXTRACTION
•It lyses the nuclear membrane as well as a cell membrane.
•It maintains the pH during the DNA extraction.
•Lysis buffer maintains the integrity of the DNA (protect DNA
from lysis)
•It separates DNA from other cell debris.
 •It protects DNA from acidic degradation.
LYSIS OR EXTRACTION BUFFER

Blood dna extraction: Plant DNA extraction: Bacterial DNA extraction:

10mM Tris (0.061gm) 10mM KCl 2% CTAB (4.0 g) 10% SDS (10 ml)
(0.037gm) 10mM MgCl2 100 mM Tris (pH 8.0) (20 ml) 90 ml TE buffer
(0.048gm) 0.5M NaCl(1.461gm) 20 mM EDTA (2 ml)
1.4 M NaCl (16.4 g) Plasmid DNA extraction:
2mM EDTA(0.037gm) 4% polyvinylpyrrolidone (PVP) (8.0 g)
Mix all components in sterile D/W and set pH 7.6 Autoclave it and wait to come at room temperature.
0.1% ascorbic acid (0.2 g) 100mM Tris HCl 10mM EDTA
0.5% SDS (0.250gm). 10 mM β-mercaptoethanol (140 µL)
DNA isolation from blood samples
Isolation of plasmid DNA
 Samples
Mechanical disruption
Proteinase K
Hydrophobic Hydrophilic
AFTER ADDING THE DETERGENT, WHAT DOYOU HAVE IN
YOUR SOUP
 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!

Phenol-chloroform method of DNA extraction:
 DNA extraction step Chemical Lysis of cell wall/ cell
membrane and
Tris, MgCl2, EDTA, NaCl, SDS,
Lysis of nuclear membrane CTAB, Triton X100
Digestion of protein CTAB, SDS, phenol, chloroform, Nonidet P40,
different chaotropic,
urea, guanidium isothiocyanate,
guanidium thiocyanate, N-Lauroyl
sarcosine
Precipitation of DNA Isopropanol, ethanol, methanol, NaCl, sodium
acetate
Washing of DNA Any alcohol

Dissolving DNA TE buffer, distil water

ROLE OF CHEMICALS
Tris: DNA is pH sensitive, Tris buffer maintains the pH of the solution. Also, it interacts withthe
lipopolysaccharides of the cell membrane and makes them permeable, this will helpinlysis of
the cell membrane.
EDTA: EDTA is a chelating agent and can be used to block DNase activity. DNase is an
enzymewhich lyses the DNA. However, every enzyme required cofactor to work properly.
The chelator EDTA blocks the activity of DNase by blocking the cofactor binding site. It will work
best in combination with Tris.
SDS: Sodium dodecyl sulphate is an anionic detergent which helps cell membrane andnuclear
envelope to break open.
NaCl: the Na+ ion of NaCl creates the ionic bond with the negative charge of
DNAandneutralize it. It will help DNA comes together and protect from denaturation.
MgCl2: overall, it protects the DNA. MgCl2 block the negative charge of the lipoproteins ofthe
cell membrane. After the lysis of cell, there is no compartment in the cell henceitprotects DNA
by mixing with other cell organelles.
Non-Phenol chloroform based extraction of DNA
Binding to a support material
 Most modern DNA purification methods are based on purification of DNA from
crudecell lysates by selective binding to a support material.
Support Materials
• Silica
• Anion-exchange resin
Advantages
• Speed and convenience
• No organic solvents
• Amenable to automation/miniaturization
Disadvantage
• DNA fragmentation
Silica column-based DNA extraction method

• The method was first described by McCormick in 1989. However, the idea
wasdeveloped in 1979, when silica was used in DNA purification by Vogelstein. • The
silica-based DNA extraction method works on the unique chemistry of interactionbetween
silica and DNA. A positively charged silica particles bind with the negatively chargedDNA and
hold it during centrifugation.

Genomic DNA analysis

https://www.addgene.org/protocols/gel-electrophoresis/
DNA QUANTIFICATION
NanoDrop 2000™
Nucleic acid digestion and
ligation
 Restriction digestion
• Restriction digestion is a methods used in molecular biology to prepare DNA for analysis or other processing •

Restriction enzymes are enzymes isolated from bacteria that recognise specific sequence in DNA • RE cut the DNA to

produce fragments called restriction fragments.

• RE play a very important role in the construction of recombinant DNA technology such as gene cloning and map
location of restriction site in DNA
 Nomenclature

More than 800 REs are Known and more then 400 of them are available commercially
 Subunit composition, cleavage position, sequence
specificity and cofactor requirements
• Type II restriction enzymes are the most widely used in molecular biology applications •

Restriction endonucleases bind and cleave DNA at specific target sequences

• The first mechanism is simple random walk diffusion, in which the protein moves on and off the DNA molecule,
randomly searching for its target site
 • The protein then tests this site and, if it is not the target site, completely dissociates from the DNA and
continues searching.

• This is trial and error searching, and is also known as a macroscopic random diffusional search. • They

require only Mg2+ as a cofactor and ATP is not needed for their activity.

• Type II endonucleases are widely used for mapping and reconstructing DNA in vitro because they recognize
specific sites and cleave just at these sites.

Recognize specific sites and cleave Eco RI

Sliding:
Hopping Jumping
One dimensional sliding. The enzyme (red)
locates DNA
(blue) and binds non specifically before sliding
in
either a 5’-3’ or 3’-5 direction, testing DNA
sequences
before locating a specific site (red bar) and
subsequently
dissociating from DNA.

Subsequently dissociating from DNA

 a threshold distance away from the DNA
(dotted arrow).

enzyme

Three dimensional hopping. The enzyme (red) locatesDNA

(blue) and binds non-specifically, before then
dissociating and reassociating with the same stretch of DNA.
The enzyme remains within the DNA domain. This is
DNA

repeated until a target is located or the enzyme diffuses

 Testing DNA sequences before locating a specific site
The steps involved in DNA binding and cleavage by a type II
restriction endonuclease:

These enzymes have nonspecific contact with DNA and initially

bind to DNA as dimmers.•
The target site is then located by a combination of linear
diffusion or “sliding” of the enzyme along the DNA over short
distances, and hopping/jumping over longer distances.
Once the target restriction site is located, the recognition
process (coupling) triggers large conformational changes of
the enzyme and the DNA, which leads to activation of the
catalytic center.

Catalysis results in hydrolysis of phosphodiester bond and

product release

Structures of free, nonspecific and specific DNA bond form of BamHI

Positions of the two metal ions at the active site. The
arrowsindicate nucleophilic attack and leaving group
protonation.
 Restriction Enzymes Sites
Specific restriction enzymes cut
at specific
DNA sequences.
For example:
EcoRI is an enzyme that cuts at
the following sequence:
GAATTC

EcoRI was discovered in E. coli

bacteria.

The resulting pieces of DNA are

called “restriction fragments.”
Different enzymes…different sites…different cuts
❑ HindIII was discovered inH.
influenza

❑ PstI was discovered in P.

stuartii

❑EcoRV was discovered inE.

coli
Protruding ends are also called
“sticky” ends. Why might these
be useful?
• Restriction fragment length polymorphism (RFLP)
Recombinant DNA Technology
 Recombinant insulin
Recombinant hepatitis B vaccine

Restriction fragments: sticky ends

Purpose: making recombinant DNA restriction enzyme, the fragments will have matching sticky
When you cut two separate molecules of DNA withthe same ends.

Digestion Procedure
• Digestion: the act of breaking down into pieces
Prepare Master Mix
Add buffer and enzyme in correct proportions.

Add restriction digest master mix to DNA

Mix thoroughly by flicking tube.
 Incubate
Temperature and time depend on enzyme to be used.

View result by gel electrophoresis

Restriction Digest Analysis • Aim: the

digested fragments must be separated and identified.

• Fragments are separated by agarose gel electrophoresis.

[Agarose is a large polysaccharide].

• Gel electrophoresis: your DNA will move through spaces in agarose

against a current

• DNA has a negative charge and will migrate towards the cathode

Restriction Digest Analysis

• Digestion of a gene and expected fragment sizes

XhoI

Restriction Digest Analysis

 How to join

DNA
DNA Ligation
 DNA Ligation
• Joining linear DNA fragments together with covalent bonds is called ligation •

Recombining fragments of DNA from different sources into a new DNA • Enzyme used to

ligate DNA fragments is DNA ligase

• DNA ligases include the sealing of nicks

• The role of DNA ligase is to seal nicks in the backbone of double-stranded DNA.

• This joining process is essential for the normal synthesis of DNA and for repairing
damaged DNA.

• It has been exploited by genetic engineers to join DNA chains to form recombinant DNA
molecules.
DNA ligase
• DNA ligase catalyses the formation of phosphodiester bond between two deoxynucleotide residues of two DNA
strands.

• it require free hydroxyl group at the 3 -end of one DNA chain and a phosphate group at the 5 -end of the other and
 requires energy in the process.
• Two types of DNA ligase

• E.coli and other bacterial DNA ligase utilizes NAD+ as energy donor,

• T4 bacteriophage, T4 DNA ligase uses ATP as cofactor.

• The most widely used DNA ligase is isolated from T4 bacteriophage.

• T4 DNA ligase needs ATP as a cofactor. The enzyme

from E. coli uses cofactor NAD. Except this, the
catalysis mechanism is somewhat similar for both the
ligases.

• The role of cofactor is splitting and forming an

enzyme-AMP complex which further aids in
formation of phosphodiester bonds between hydroxyl
and phosphate groups by exposing them.
Ligase
Function

Ligase
Ligase-adenylate intermediateCofactor (ATP or NAD+)
Ligase(red) reacts with cofactor (ATP or AMP is transferred from Ligase-adenylate to the 5’ Pat
NAD+)transferring AMP to form Ligase-adenylate the nick site.
intermediate and inducing a closed conformationof the
enzyme. R represents the second productof Ligase
reaction: PPi (ATP Ligase) orr NMN(NAD+ Ligase).
The 3’ OH attacks the 5’ P adenylated DNA, creating
aphosphodiester bond, releasing enzyme.
Ligation reaction

Ligation 12hr, 22˚C

Application:
• DNA ligase enzyme is used by cells to join the “okazaki fragments” during DNA replication process. • In

molecular cloning, ligase enzyme has been routinely used to construct a recombinant DNA. • Followings are

some of the examples of application of ligase enzyme in molecular cloning. • Joining of adapters and linkers

to blunt end DNA molecule.

• •Cloning of restricted DNA to vector to construct recombinant vector

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