NUCLEIC ACID EXTRACTION

SAHIL KULKARNI
SENIOR RESEARCH FELLOW
HAFFKINE INSTITUTE

Where is DNA present in the cell????

Purpose
To release nucleic acid from the cell for

use in other procedures

Must be free from contamination with
protein, carbohydrate, lipids or other
nucleic acids.
Used pure nucleic acids for testing.

Isolation
Routinely isolated from plant ,human, fungal, bacterial
and viral sources.
Pre treat to make nucleated cells available,
whole blood
Tissue samples
Microorganisms
Need sufficient sample for adequate yield.

There are three basic and one optional steps in a

DNA/RNA extraction:
Breaking the cells open, commonly referred to as cell
disruption or cell lysis, to expose the DNA/RNA within. This is
commonly achieved by grinding or sonicating the sample.
Removing membrane lipids by adding a detergent.
Removing proteins by adding a protease (optional but almost
always done).
Precipitating the DNA/RNA with an alcohol usually icecold ethanol or isopropanol. Since DNA/RNA is insoluble in these
alcohols, it will aggregate together, giving a pellet upon
centrifugation. This step also removes alcohol-soluble salt.

Two major types of methods :

Liquid liquid extraction

1.Phenol-chloroform extraction
2.Trizol
3. ((C16H33)N(CH3)3Br, cetyltrimethylammonium bromide,
hexadecyltrimethylammonium bromide, CTAB)
Adsorbtion based

1. silica coloumn
2. Diatomaceous Earth

Phenol-chloroform extraction
It is a liquid-liquid extraction technique
in biochemistry.
Liquid-liquid extraction, also known
as solvent extraction and partitioning,
is a method to separate compounds
based on their relative solubilities in
two different immiscible liquids. It is
an extraction of a substance from one
liquid phase into another liquid phase.
It is widely used in molecular biology for
isolating DNA, RNA and protein.
Equal volumes of a phenol : chloroform
mixture and an aqueous sample are mixed,
forming a biphasic mixture.

How it works
This method relies on phase separation by centrifugation of a mix of the
aqueous sample and a solution containing water-saturated phenol,
chloroform and a chaotropic denaturing solution (guanidinium
thiocyanate) resulting in an upper aqueous phase and a lower organic
phase (mainly chloroform).

Nearly all of the RNA is present in the aqueous phase, while DNA and
protein partition in the interphase and organic phase, respectively.
In a last step, RNA is recovered from the aqueous phase by precipitation
with 2-propanol or ethanol.

DNA will be located in the interphase thus the technique can be used for
DNA purification alone.

Guanidinium thiocyanate denatures proteins, including RNases, and

separates rRNA from ribosome and dna from histones.

In the presence of chloroform or BCP (bromochloropropane), these

solvents separate entirely into two phases that are recognized by their
color: a clear, upper aqueous phase (containing the nucleic acids) and
a bright pink lower phase (containing the proteins dissolved in
phenol and the lipids dissolved in chloroform).
The major downside is that Phenol and chloroform are both
hazardous and inconvenient materials, and the extraction is often
more laborious, so in recent years many companies now offer
alternative ways to isolate DNA.

Column-based nucleic acid purification

It is a solid phase extraction method to quickly purify nucleic
acids.
This method relies on the fact that the nucleic acid may bind
(adsorption) to the solid phase (silica or other) depending on the
pH and the salt content of the buffer, which may be a Tris-EDTA
(TE) buffer or Phosphate buffer (used in DNA microarray
experiments due to the reactive amines).

Three stages are:

The sample is added to the column and the nucleic acid binds

thanks to the high pH and salt concentration of the

binding solution, which may contain buffer, a denaturing
agent (such as guanidine hydrochloride)
The column is then washed (5 mM KPO4 pH 8.0 or similar,

80% EtOH)
The column can be eluted with buffer or simply water

How does this method of purification work?

A sample (this may be anything from purified cells to a tissue
specimen collected in the field only moments earlier) is lysed.
The resultant mix of proteins, DNA, phospholipids, etc., is then run
through the channel where the DNA is adsorbed by silica surface in
the presence of solutions with high ionic strength.
The highest DNA adsorption efficiencies are shown to occur in the
presence of buffer solution with pH at or below pKa of the surface
silanol groups. Although the exact method for this interaction is not
well known, one possible explanation involves reduction of the
silicas surfaces negative charge due to the high ionic strength of the
buffer.

This decrease in surface charge leads to a decrease in the electrostatic

repulsion between the negatively charged DNA and the negatively charged
silica. Meanwhile, the buffer also reduces the activity of water by formatting
hydrated ions.
This leads to the silica surface and DNA becoming dehydrated. These
conditions lead to an energetically favorable situation for DNA to adsorb to
the silica surface.

A better explanation of how DNA binds to silica is based on the action of

Guanidium HCl (GuHCl), which acts is a chaotrope. A chaotrope denatures
biomolecules by disrupting the shell of hydration around them.
This allow a positively charged ion to form a salt bridge between the
negatively charged silica and the negatively charged DNA backbone in high
salt concentration.
The DNA can then be washed with high salt and EtOH, and ultimately
eluted with low salt.

QIAGEN QIAamp VIRAL RNA

Kit contains :

Mini spin column

Collection tube

Buffers AVL

AW1

AW2

AVE

Carrier RNA

Procedure
Pipette 560 l of prepared Buffer AVL containing
carrier RNA into a 1.5 ml micro centrifuge tube.
Add 140 l body fluid to the Buffer AVL carrier RNA
Mix by pulse vortexing for 15 s.
Incubate at room temperature (15-25C) for 10 min.
Briefly centrifuge the tube to remove drops from the
inside of the lid.

Role of AVL
The sample is first lysed under the highly denaturing
conditions provided by buffer AVL to inactivate RNases
and to ensure isolation of intact viral RNA

Why we add carrier RNA?

Firstly , it enhances binding of viral nucleic acids to the
QIAamp mini membrane, especially if there are very few
target molecules in the sample.
Secondly the addition of large amounts of carrier RNA
reduces the chance of viral RNA degradation in the rare
event that Rnase molecules escape denaturation by
chaotropic salts and detergents in buffer AVL.

If carrier RNA is not added to buffer AVL this may lead to

reduced viral RNA recovery.

Add 560 l of ethanol (96-100%) to the sample, and

mix by pulse-vortexing for 15 s.
only ethanol should be used since other alcohols may result in
reduced RNA yield and purity.

After mixing, briefly centrifuge the tube to remove

drops from the lid.
Carefully apply 630 l of the solution to the Mini Spin
Column-in a 2ml Collection tube-without wetting the
rim.
Close the cap, and centrifuge at 8000 rpm for 1 min.
Place the spin column into a clean 2ml collection tube
and discard the tube containing the filtrate.
Carefully open the Mini Spin column and repeat the
step .

Carefully open the Mini Spin Column and add 500 l of

Buffer AW1.
WASH BUFFER- PROTEIN DENATURING CHEMICALS

Close the cap and centrifuge at 8000rpm for 1 min.

Place the Mini Spin Column in a clean 2 ml collection
tube and discard the tube containing the filtrate.
Carefully open the Mini Spin Column and add 500 l of
Buffer AW2.Close the cap and centrifuge at 13,000 rpm
for 4 min.
INCREASES THE AFFINITY BINDING OF RNA, AND CREATES
AN ENVIRONMENT FOR RNA BINDING TO SILICA SURFACE

Place the Mini Spin Column into a clean, labeled 1.5ml

micro centrifuge tube.

 Discard the old collection tube containing the filtrate.

Carefully open the Spin Column & add 60 l of Buffer AVE
equilibrated to room temperature.
It is elution buffer nothing but Rnase, Dnase free water.

Close the cap and incubate at room temperature for 1 min.

Centrifuge at 8000rpm for 1 min.
Stored the viral RNA at -20 to -70 degree.
RNA loss is a major cause of extraction failure and commonly
occurs during extraction of RNA from samples using column.
More than 30-40% of the RNA is lost due to insufficient binding
of the RNA, or incomplete elution of the RNA from the column.
Hence, a non-binding, non-elution extraction method, which
eliminates the RNA loss and maximizes the yield of RNA should
be developed.

Quantification Of extracted nucleic

acid
The concentration of an RNA or DNA sample can be
checked by the use of UV spectrophotometry
The nitrogenous bases in nucleotides have an
absorption maximum at about 260 nm
Proteins have a UV absorption maximum of 280 nm,
due mostly to the tryptophan residues
Phenol has an absorbance maximum of 270 but the
absorbance spectrum overlaps considerably with that
of nucleic acids

Quantification contd
The ratio of the absorbance at 260 nm/ absorbance at 280

nm is a measure of the purity of a DNA sample; it should

be between 1.65 and 1.85.
The ratio of the absorbance at 260 nm/ absorbance at 280
nm is a measure of the purity of a RNA sample; it should be
between 1.90 and 2
Yield estimation
DNA concentration (g/ml) = (OD 260) x (dilution factor) x

(50 g DNA/ml)/(1 OD260 unit)

RNA concentration (g/ml) = (OD 260) x (dilution factor) x
(40 g RNA/ml)/(1 OD260 unit)

Applications:
De novo: Oligonucleotide synthesis
Amplification: PCR
Nucleic acid simulations
Cloning
DNA sequencing
Expression cloning
Southern blot
northern blot
Fluorescent in situ hybridization
several Bioinformatics methods, such as RNA structure prediction

THANK YOU

sahilkulkarni@gmail.com