Nucleic Acid Isolation &

Purification

Naimesh N Patel
M.Pharm Sem-ΙΙ
Pharmaceutics
KBIPER
Content : -
Define Biotechnology.
Major Types of Nucleic Acid in Cell.
Why do we need Isolated Pure DNA & RNA?
Methods for Isolation & Purification of DNA.
Method for Isolation & Purification of RNA.
Method for Isolation of Specifically mRNA.
BIOTECHNOLOGY
Fusion of two words : -
Biological
Technology
It refers to as a link between the biological science,
physical science, chemical science and technological
achievement, commonly reffered as the clever science
of biology.
Various basic biological disciplines involved in the
origin of biotechnology are : -
 Genetics

Biochemistry Engineering technology

Cell biology BIOTECHNOLOGY Biophysics

Microbiology Molecular biology

Major Types of Nucleic Acid

Deoxyribonucleic acid(DNA)
Ribonucleic acid(RNA)
 The Central Dogma of Genetics

non-coding RNA (rRNA, tRNA, siRNA, etc.)

Transcription
Replication

mRNA

Translation
Why do we need to isolated pure DNA?
 Detect, enumerate species
 Detect, enumerate, clone genes
 Detect/sequence specific DNA regions
 Create new DNA “constructs” (recombinant DNA)
The purification of nucleic acids broadly
involves following stages : -
 Breaking or opening of the cells to expose nucleic acid.
 Inactivation of DNA- and RNA-degrading enzymes
(DNases, RNases)
 Separation of nucleic acids from cellular components.
 Extraction/Precipitation Methods
 Adsorption Chromatography Method
 Recovery of nucleic acids in a pure form.
1. Breaking or Lysis of cells
a) Bacterial cells
b) Animal cells
c) Plant cells
1. Breaking or Lysis of cells(cont…)
a) Bacterial cells : -

• The bacterial cells (e.g. E.coli) can be lysed by

combination of enzymatic and chemical treatments.
• The enzyme lysozyme and the chemical ethylenediamine
tetraacetate(EDTA) are used for this purpose.
• This is followed by the addition of detergent such as
sodium dodecyl sulfate(SDS).
1. Breaking or Lysis of cells(cont…)
b) Animal cells : -
Animal cells, paticularly cultured animal cells, can be
easily opened by direct treatment of cells with
detergent(SDS).

c) Plant cells : -
Plant cells with strong cell walls require harsh
treatment to break open. The cells are frozen and then
ground in a mortar and pestle. This is an effective way of
breaking the cellulose walls.
2.Separation of DNA from cellular compartment

A) Extraction/Precipitation Method
I. Purification of DNA by removing cellular compartment
II. Direct purification of DNA
B) Adsorption Chromatography Method
A) PRECIPITATION METHOD
I. Purification of DNA by Removing Cellular Components:-

 The cellular extract is centrifused at a low speed to remove the

debris(e.g. pieces of cell wall) that forms a pellet at the bottom of
the tube.
 The supernatant is collected and treated with phenol to precipitate
protien at the interface between the organic and aqueous layers.
 The aqueous layer, containing the dissolved nucleic acid, is collected
and treated with the enzyme ribonuclease(RNase).
 The RNA is degraded while the DNA remain intact.
 This DNA can be precipitated by adding ethanol and isolated after
centrifugation, and suspended in an appropiate buffer.
A) PRECIPITATION METHOD(Cont…)
II. Direct Purification of DNA
 In this approach ,the DNA itself is selectively removed from the
cellular extract and isolated.
 In this method, the addition of detergent cetyltrimethyl ammonium
bromide(CTAB) results in the formation of an insoluble complex
with nucleic acid.
 This complex, in the form of a precipitate is collected after
centrifugation and suspended in a high-salt solution to release
nucleic acids.
 By treatment with RNase, RNA is degraded.
 Pure DNA can be isolated by ethanol precipitation.
Overview of the Extraction/Precipitation Method
 Extraction/Precipitation Method
Step 1: Disruption of cell walls by grinding

Step 1+2: mechanical disruption and

homogenization in extraction buffer

Grind sample into a fine powder to

shear cell walls and membranes

Step 2: Lysis of cells in extraction buffer

A homogenizer allows cells to be

mechanically disrupted within the
extraction buffer
Mix thoroughly with extraction
buffer to dissolve cell membranes
and inhibit nuclease activity Crude lysate
 Extraction/Precipitation Method
Detergents
Purposes of the Extraction Buffer Chaotropic salts
1. Dissolve cellular membranes Metal chelators
2. Inactivation of DNase and RNase Salts
3. Assist in the removal of contaminants Reducing agents
CTAB
PVP

Use of Detergents to Lyse Cells: Like Dissolves Like

Mixed micelle
Plasma membrane
(phospholipid bilayer) Detergent molecules

SDS
 Extraction/Precipitation Method
Step 3: Organic extraction
Mix thoroughly with Aqueous

an equal volume of
organic solvent Centrifuge Collect aqueous phase
e.g. phenol, chloroform,
or phenol:chloroform Interphase

Organic

Perform additional extractions for increased purity

Crude lysate containing The aqueous phase contains water-

nucleic acids and other soluble molecules, including nucleic
cell constituents acids. Proteins and lipids become
trapped in the organic phase, and are
thus separated away. Insoluble plant
debris become trapped in the
interphase between the two layers
 Extraction/Precipitation Method

Step 4: Nucleic Acid Precipitation

Before After

Supernatant 70% EtOH

Centrifuge Wash Centrifuge

Pellet

Add alcohol and salt to
precipitate nucleic acids
from the aqueous fraction
Dissolve pellet
(H2O, TE, etc.)
• Pellet down nucleic acids.
• Wash pellet with 70% ethanol to remove
residual salts and other contaminants.
• Discard ethanol and allow pellet to dry.
B) ADSORPTION CHROMATOGRAPHY METHOD

Basic Principle
Nucleic acids within a crude lysate
are bound to a silica surface

The silica surface is washed with a

solution that keeps nucleic acids bound,
but removes all other substances

The silica surface is washed with a solution

unfavorable to nucleic acid binding. The solution,
containing purified DNA and/or RNA, is recovered.
 Adsorption Chromatography Method
Step 1: Prepare crude lysate Step 2: Adsorb to silica surface

Apply to column Centrifuge

Nucleic acids
Silica-gel membrane

Extraction Buffer composition favors Flow through

DNA and RNA adsorption to silica: (discard)
• Low pH
• High ionic strength
• Chaotropic salt Nucleic acids bind to the membrane,
while contaminants pass through the
column.

Surface silanol groups are weakly

acidic, and will repel nucleic acids
at near neutral or high pH due to
their negative charge
 Adsorption Chromatography Method
Step 3: Wash away residual contaminants

Centrifuge
Wash buffer
Nucleic acids Nucleic acids

Flow through
(discard)
Step 4: Elute nucleic acids

Centrifuge
Elution buffer
Nucleic acids

Elution Buffer composition is

unfavorable to surface binding:
High pH
Low ionic strength Nucleic acids
Using Nucleases to Remove Unwanted DNA or RNA

Add DNase

+ DNase (protein)

Add RNase

+ RNase (protein)

Depending on when nuclease treatment is performed, it may be necessary to

repeat purification steps for protein removal (e.g. phenol/chloroform extraction).
Applications of Isolated DNA
After DNA is extracted, it is used as a template in
further molecular techniques such as…

 PCR (polymerase chain reaction) :- Technique for generating

large quantities of a specified DNA.

 RFLP (restriction fragment length polymorphism)

 Southern Blotting:-Identification of desired DNA from

thousands of molecules.
3. Separation of RNA from cellular compartment
The problem(s) with RNA:

RNA is chemically unstable -- spontaneous cleavage of phosphodiester

backbone via intramolecular transesterification

RNA is susceptible to nearly ubiquitous RNA-degrading enzymes (RNases)

 RNases are released upon cell lysis

 RNases are present on the skin
 RNases are very difficult to inactivate
 -- disulfide bridges conferring stability
 -- no requirement for divalent cations for activity
3. Separation of RNA from cellular compartment(Cont…)

Common sources of RNase and how to avoid them

Contaminated solutions/buffers

USE GOOD STERILE TECHNIQUE

TREAT SOLUTIONS WITH DEPC (when possible)
MAKE SMALL BATCHES OF SOLUTIONS

Contaminated equipment

USE “RNA-ONLY” PIPETS, GLASSWARE, GEL RIGS

BAKE GLASSWARE, 300°C, 4 hours
USE “RNase-free” PIPET TIPS
TREAT EQUIPMENT WITH DEPC
3. Separation of RNA from cellular compartment(Cont…)

Top 10 sources of RNAse contamination

(Ambion Scientific website)

1) Ungloved hands
2) Tips and tubes
3) Water and buffers
4) Lab surfaces
5) Endogenous cellular RNAses
6) RNA samples
7) Plasmid preps
8) RNA storage (slow action of small amounts of RNAse
9) Chemical nucleases (Mg++, Ca++ at 80°C for 5’ +)
10) Enzyme preparations
 3. Separation of RNA from cellular compartment(Cont…)

Inhibitors of Rnase

DEPC: diethylpyrocarbonate

alkylating agent, modifying proteins and nucleic acids

fill glassware with 0.1% DEPC, let stand overnight at room temp

solutions may be treated with DEPC -- add DEPC to 0.1%, then autoclave (DEPC
breaks down to CO2 and ethanol)
 3. Separation of RNA from cellular compartment(Cont…)

Inhibitors of Rnase

Vanadyl ribonucleoside complexes

competitive inhibitors of RNAses,
but need to be removed from the final preparation of RNA

Protein inhibitors of RNAse:-

horseshoe-shaped, leucine rich protein,
found in cytoplasm of most mammalian tissues
must be replenished following phenol extraction steps
3. Separation of RNA from cellular compartment(Cont…)

Break the cells/solubilize components/inactivate RNAses by

the addition of guanidinium thiocyanate (very powerful
denaturant)

Extract RNA using phenol/chloroform (at low pH)

Precipitate the RNA using ethanol/LiCl

Store RNA:
1. in DEPC-treated H20 (-80°C)
2. in formamide (deionized) at -20°C
4. Selective separation of mRNA from cellular
compartment

The purification of mRNA can be achived by affinity

chromatography using oligo(dT)-cellulose.
This is based on the principle that oligo(dT)-cellulose can
specifically bind to the poly(A) tails of eukaryotic mRNA.
Thus , by this approach, it is possible to isolate mRNA from
DNA, rRNA and tRNA.
4. Selective separation of mRNA from cellular
compartment(Cont….)

 As nucleic acid solution is passed through an affinity

chromatography column, the oligo(dT) binds to poly(A) tails of
mRNA.
 By washing the column with high-salt buffer , DNA, rRNA and
tRNA can be eluted, While the mRNA is tightly bound .
 This mRNA can be eluted by washing with low salt buffer.
 The mRNA is precipitated with ethanol and collected by
centrifugation.
References :-
1. Dr. Satyanaran U. and Dr. Chakrapani U. “Biochemistry
“ , 2nd edition , Chapter 24 to 27, Page no.523 to 578.

2. Dr. Vyas S. P. and Dr. Dixit V. K. “Pharmaceutical

Biotechnology” CBS Publisher & Distributors, New
Delhi, 1st edition, Chapter 10, Page no. 341 to 400.

3. Ambion Scientific Website.

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