Molecular Diagnostics

Nucleic Acid Extraction Methods

Dr. Rezwana Ahmed
Assistant Professor
Department of Pharmaceutical Sciences
North South
University
Nucleic Acids Extraction
• The purpose of extraction of nucleic
acids is to release the nucleic acid from
the cells for use in subsequent
procedures such as PCR, cloning,
checking for gene expression, etc.

• The target nucleic acid should be free

of contamination from proteins,
carbohydrates, lipids, or other nucleic
acid, i.e., DNA free of RNA or RNA
free of DNA.
Preparing the Sample
• The initial steps in nucleic acid extraction depends on the
nature of the starting material.

• As the DNA is present inside the nucleated cells, the first

objective is to obtain the nucleated cells from the clinical
sample, for example from blood.
Preparing the Sample
Nucleated Cells in Suspension (e.g. blood)

• Nucleated cells in suspension, such as white blood cells

(WBCs) must be first isolated from blood or bone marrow
specimens.

• This is done by differential density gradient

centrifugation.
Differential density gradient centrifugation:
• Differential density gradient centrifugation is a method used
to separate cell populations based on differential cell
densities.
• A medium of a specified density is layered with a sample,
such as whole blood, and then centrifuged.
• Based on the density of the medium relative to the density of
the cells, certain populations will settle above, below, or in
between the medium.
Differential density gradient centrifugation:
Steps:
1. Whole blood is mixed with isotonic saline and placed on
top of Ficoll. Ficoll is a highly branched sucrose polymer
that does not penetrate biological membranes.
2. The mixture is centrifuged and the nucleated WBCs settle
into a layer in the Ficoll gradient that is below the less
dense plasma components and above the red blood cells
(RBCs).
Differential density gradient centrifugation:

3. The layer containing the WBCs is removed from the

tube and washed carefully.

4. Then the nucleic acid extraction procedure

is followed.
Extraction of DNA
Principle:
First, cell lysis is done to release the cellular materials by
breaking the cell and nuclear membranes.

Next, the target material (DNA or RNA) is separated and purified

from the rest of the cellular components.

Finally, the concentration and purity of the target material (DNA or

RNA) is determined.
DNA Extraction Procedures
Organic Extraction Method
• This method is one of the best methods of DNA
extraction.
• The yield and quality of DNA obtained by
this method is very good.
• It is also cheaper.
Organic Extraction Method

• The major chemicals of this extraction method are

lysis buffer, Phenol and chloroform, and ethanol
(or isopropanol)
Organic Extraction Method

1. Cells in suspension are lysed using NaOH and SDS.

2. Acetic acid and salt are added to the lysed cells
to create a hydrophilic clear cell lysate.
3. Phenol and chloroform mixture are added to the cleared
lysate and mixed gently. Centrifuge this mixture.
4. A biphasic emulsion forms and 2 distinct layers are visible.
The hydrophilic layer is on the top and the hydrophobic
layer is at the bottom. Lipids and other hydrophobic
components will dissolve in the lower hydrophobic phase.
DNA will dissolve in the upper aqueous phase. Amphiphilic
components, such as some proteins as well as some cell
debris, will collect as a white precipitate at the interface
between the two layers.
5. The is then precipitated using ethanol or
DNAisopropanol in a high concentration of salt. The DNA
precipitate is collected by centrifugation. Remove the
ethanol or isopropanol.
6. Wash the DNA pellet to remove excess salt by rinsing the
pellet in 70% ethanol, centrifuging and discarding the
ethanol supernatant.
7. The DNA pellet is dissolved in buffer, usually 10 mM Tris, 1
mM EDTA (TE), or nuclease-free water.
Different chemicals used:
• NaOH and SDS (a detergent) : The alkaline mixtures
ruptures the cells, and the SDS detergent breaks apart the lipid
membrane and solubilizes cellular proteins. NaOH also
denatures the DNA into single strands.

• Acetic Acid: The acetic acid neutralizes the pH, allowing

the DNA strands to renature.

• Salts – NaCl / KCl: The cations Na+ or K+ binds to negative

phosphate groups of DNA and makes it more stable in
aqueous solution. It will help the DNA to come together. In
the absence of Na+ or K+, DNA molecules repel each other and
do not allow grouping of DNA molecules.
Different chemicals used:

• Phenol: In the cell, proteins normally folds in such a way

that the polar part of the proteins remain on peripheral areas
and non-polar part moves to the core of the proteins. As
phenol is less polar than water, proteins gets denatured
slightly and dissolve in phenol. DNA being more polar
dissolves in upper aqueous phase.

• Chloroform: It increases the viscosity of the organic phase

and avoids phase shifting leading to more accurate phase
separation.
Different chemicals used:

• Isoamyl alcohol: Sometimes isoamyl alcohol is also

added with the phenol and chloroform, it acts as an anti-
foaming agent.

• Ethanol precipitation: Ice cold ethanol is added to

the solution containing DNA and cell debris. Proteins gets
dissolved in ethanol. Upon centrifugation, DNA is obtained
in the form of pellet on the bottom of tube. Isopropanol can
also be used instead of ethanol. Isopropanol is more
effective in precipitation but it is less volatile than ethanol
so more time is required for air drying.
Different chemicals used:

• Tris: It is a buffering agent used to maintain

a constant pH ( = 8.0).

• EDTA: DNase present inthecellscan degrade

the DNA. EDTA acts as a chelating agent and
binds
Mg2+
ionsto which acts as co-factor for DNase. The
unavailability of Mg2+ ions leads to the deactivation of
DNase activity and hence saves the DNA from degradation.
Inorganic Extraction Method
• Phenol is a caustic reagent so its use in lab is undesirable.
Hence inorganic extraction methods were developed.
• Inorganic DNA extraction is sometimes called “salting out”.
It makes use of high salt conditions to selectively precipitate
proteins, leaving the DNA in solution.
Inorganic Extraction Method
• The DNA can then be precipitated using isopropanol,
pelleted, washed and resuspended in TE buffer or nuclease-
free water.
• One of the major limitations of the salting-out method is the
purity, though enough yield can be obtained, the quality
obtained might not be good.
Solid-Phase Extraction
• Nowadays all the DNA extraction kits available are based on
the unique chemistry of the solid-phase
DNA
extraction.

• Silica-based products can effectively bind DNA. The principle

of silica matrices purification is based on the binding of the
negatively charged DNA backbone towards the silica particles
via chaotropic agents.

• The main advantage is that it is rapid and gives “PCR ready

DNA” for downstream applications. No precipitation steps are
required therefore this method is superior among all, however,
it can be expensive.
Solid-Phase Extraction
The stages of the method are lyse, bind, wash, and
elute.

Sample
Solid-Phase Extraction

1. For lysis, the cells in the sample is incubated with a cell lysis buffer.
A small amount of proteinase k is also added to the sample.
Solid-Phase Extraction

2. For binding, a buffer solution is added to the lysed sample.

The sample in binding solution is then transferred to a spin column, and
the column is put either in a centrifuge.
The centrifuge forces the solution through a silica membrane that is
inside the spin column and nucleic acids will bind to the silica
membrane, as the rest of the solution passes through.
Solid-Phase Extraction
2. The silica surface has negative charges and remains hydrated. DNA has a
phosphate backbone which has negative charge and feels the repulsion.
The chaotropic buffer disrupts the hydration shell of the silica surface.
It also reduces the negative charges on the silica surface.
The positive ions in the buffer aid in the formation of the salt bridge between
the DNA and silica surface. Guanidium HCl and Guanidinium thiocyanate
are commonly used as chaotropic salt in this type of DNA extraction
method.
Solid-Phase Extraction

3. To wash, a new buffer is added onto the column, then centrifuged

through the membrane. This buffer is intended to maintain binding
conditions, while removing the binding salts and other remaining
contaminants.
Solid-Phase Extraction

4. Finally, elution is the process of adding an aqueous solution,

allowing the DNA to leave the column and return to solution. The
elution is carried out using TE buffer or water at pH 8.4. It removes
the positively charged ions and disrupts the salt bridge between the
DNA and the silica surface.
Extraction of RNA
Special Requirements for RNA extraction

• Working with RNA in the laboratory requires strict

precautions to avoid RNA degradation.
• RNA is especially labile due to the ubiquitous presence of
RNAses.
• These enzymes are small proteins that can renature, even
after autoclaving, and become active. So RNAses must be
eliminated or inactivated before isolation of RNA.
Special Requirements for RNA extraction

Special needs for RNA extraction:

• It is useful to allocate a separate RNAse-free (RNF) area

of the laboratory for storage of materials and specimen
handling.
• Gloves must always be worn.
Types of RNA

There are several types of naturally occurring RNA in

cells e.g.

• ribosomal RNA (rRNA)

• messenger RNA (mRNA)
• transfer RNA and small nuclear RNAs
Organic Extraction of Total RNA
Organic Extraction of Total RNA

• The cell lysis step for RNA extraction done in

is detergent with RNAse inhibitors.

• After cell lysis, a mixture of

acidic phenol:chloroform:isoamyl alcohol solution is added.
Organic Extraction of Total RNA
• After phase separation, the upper aqueous phase containing
the RNA is removed to a clean tube.

• The RNA is precipitated by addition of ethanol or

isopropanol. The RNA precipitate is then washed in 70%
ethanol and resuspended in RNF buffer or water.

***Optimum pH plays a critical role in the separation

process as DNA partitions to the organic phase under
acidic condition (pH 4–6) or to the aqueous phase at
neutral pH(pH7-8).
Extraction of polyA (messenger) RNA

• Most of eukaryotic mRNA molecules possess a

polyadenylated (polyA) tail of about 250 nucleotides at 3’
end.
• Hence Oligomers of polyT or polyU sequence (10-20
nucelotides) will bind to the polyA tail of mRNA.
Extraction of polyA (messenger) RNA
• These oligomers are immobilized on a matrix resin column.
The cell lysate is passed through the column and the mRNA
binds to the oligomers.
Binding Wash Collected mRNA
Elute
Extraction of polyA (messenger) RNA

• The column is washed to remove other

unbound materials.

• Finally, warmed buffer is added that destabilizes

the bond of the mRNA with resin to collect the mRNA.
Binding Wash Collected mRNA
Elute
Detection of Nucleic
Acids
Electrophoresis
• DNA and RNA can be analyzed for quality by resolving an
aliquot of the isolated sample on an agarose gel.
• Fluorescent dyes such as ethidium bromide or SybrGreen I bind
specifically to the nucleic acids and are used to visualize the
sample preparation.
Concentration of Nucleic
Acids
• Spectrophotometric analysis can be performed for
checking the concentration of nucleic acids.
Concentration of Nucleic
Acids
Nucleic acids absorb light at 260 nm. Using the Beer-
Lambert Law, concentration can be determined from the
absorptivity constants (50 for DNA, 40 for RNA). The
relationship of concentration to absorbance is expressed as:

A absorbance, ε = molar absorptivity constant (L/molcm),

b=path length (cm), and c=concentration (mg/L).
The absorbance at this wavelength is thus directly proportional
to the concentration of the nucleic acid in the sample.
Thus concentration can be easily found using the above
equation.
Concentration of Nucleic
Acidsbenefit of using spectrophotometric analysis
• The secondary
for nucleic acid quantitation is the ability to determine sample
purity using the 260 nm:280 nm calculation. Contaminants
such as proteins will absorb at 280nm.
• The ratio of the absorbance at 260 and 280 nm (A260/280) is
used to assess the purity of nucleic acids.

 For pure DNA, A260/280 is widely considered ~1.8

 For pure RNA, A260/280 is ~2.0

• If the ratio is appreciably lower in either case, it may indicate

the presence of protein, phenol or other contaminants that
absorb strongly at or near 280 nm.