Chapter-3

Principles and Methods of DNA Extraction

 DNA extraction has two main aims:

maximizing the yield

extracting DNA that is pure enough

 Then quantifying the DNA

 Basic principles

 Finding a suitable DNA isolation system to satisfy forensic

DNA testing needs is important for the successful
completion of testing.
 Extraction procedures may vary according to the type of
biological evidence such as cell types, and the quantity of
biological sample collected.

 The method of choice is often the one that yields sufficient

quantity, good quality, and high purity of DNA.

 An insufficient quantity of DNA may result in a partial

2 DNA profile or the failure to obtain a profile.
 Additional considerations for selecting proper DNA
extraction methods include adaptability to automation,
throughput, simplicity, risk of contamination, and cost
effectiveness.
 DNA isolation methods can differ in many respects
because of the variation of the biochemical compositions
of tissues.
 However, certain basic principles, reagents, and isolation
procedures apply to various types of samples are similar.
 Thus, the most common DNA extraction protocols
include the following components.
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1. Cell and Tissue Disruption
 In most DNA isolation protocols, enzymatic digestions, such as
with proteinase K, have been used for cell and tissue disruption.
 Tissues can also be disrupted by boiling and alkali treatment.

 Materials such as bone and teeth can be frozen in liquid nitrogen

and then ground to a fine powder with a mortar.

2. Lysis of Membranes and Organelles

 The lysis of membranes and release of DNA from nuclei or
mitochondria are performed during or immediately after tissue
disruption.

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The lysis buffer usually consists :
1) detergents such as anionic compounds, and sodium dodecyl
sulfate (SDS) to destroy membranes, denature proteins, and
dissociate proteins from the DNA;
2) a buffer system, such as Tris-HCl to maintain the pH in a range
that avoids the activities of degrading enzymes;
3) high salt concentrations to dissociate nuclear proteins such as
histones from the DNA;
4) reducing agents such as mercaptoethanol or dithiothreitol (DTT)
to inhibit oxidization processes that can damage DNA; and
5) chelating agents such as ethylene diamine tetra acetic acid
(EDTA) or Chelex® to capture divalent metal ions that are
cofactors of endogenous DNases that catalyze the hydrolysis of
DNA.
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3. Removal of Proteins and Cytoplasmic Contaminants
 Most cell constituents interfering with DNA isolation reside in the
cytoplasm.
 Proteins are dissociated from the DNA by the action of detergents
and high salt concentrations in the lysis buffer.
 Dissolved proteins are then usually removed by one or more rounds
of extraction with phenol, phenol–chloroform mixtures, or
chloroform–iso amyl alcohol mixtures, followed by centrifugation
to separate the phases.
 Another strategy to remove cytoplasmic contaminants is to employ
the reversible binding of DNA to a solid matrix such as silica that
selectively binds DNA.
 The proteins and cytoplasmic contaminants can be removed by
washing steps.
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4. Contamination
 Studying organisms is largely depend on studying DNA variation within
and among populations.
 However, the practicability of such studies is often limited by one’s
ability to isolate good DNA.
 The problems include DNA degradation due to the presence of native
DNases
 This can occur between a person and a sample, between samples,
between samples with amplified DNA, or through contamination from
other organisms.
 To prevent the occurrence of contamination, certain procedures should
be followed.
7 The evidence samples should be processed and extracted in separate
 Evidence and reference samples should be processed separately in
different rooms to avoid between-sample contamination.

 In situations where space is limited, the evidence sample should

be processed before the reference sample.

 Solutions and test tubes used for extraction should be DNA-free

and aerosol-resistant pipette tips should be used during the
extraction process.

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5. Storage of DNA Solutions
 Highly purified and high molecular weight DNA is usually stored in
TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) to chelate
magnesium ions and thereby inhibit magnesium dependent DNases.
 Such a DNA solution may be stored at 4 oC or –20oC but for long-
term storage, –80oC is recommended.
 Frequent freezing or thawing cycles should be avoided because the
fluctuations may cause breaks of single- and double-stranded DNA.
 Certain substances such as heavy metals and free radicals should also
be avoided because they can cause breakage of phosphodiester bonds
in the molecules.
 Furthermore, contamination of nucleases may lead to subsequent
9 degradation of DNA.
 Methods of DNA Extraction
1. Extraction with Phenol-Chloroform
 This method is also called organic extraction.

 Major steps include the following:

 Cell Lysis and Protein Digestion- These steps can be achieved by

digestion with proteolytic enzymes such as proteinase K before extraction
with organic solvents.
 Extraction with Organic Solvents- The removal of proteins is carried
out by extracting aqueous solutions containing DNA with a mixture of
phenol: chloroform : iso amyl alcohol (25:24:1).
 Phenol is used to extract the proteins from the aqueous solution.

 However, although phenol has a slightly higher density than water, it may
be difficult to separate from the aqueous phase so chloroform is employed
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 The phenol–chloroform mixture forms the lower organic phase and
is easily separated from the aqueous phase.

 Iso amyl alcohol is often added to the phenol–chloroform mixture

to reduce foaming.

 During partition, DNA is solubilized in the aqueous phase while

lipids are solubilized in the organic phase.

 Proteins are located at the interface between the two phases

(Figure.1).

 Concentrating DNA — Two common methods for concentrating

DNA are ethanol precipitation and ultrafiltration.

 In the first method, the DNA is precipitated from the aqueous

solution with ethanol and salts.
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or
ga
ni
ce
xt
ra
c t io
n
 Figure.1. DNA extraction using organic solvent.
 The DNA is contained in the aqueous phase while cellular materials
such as lipids are contained in the organic solvent phase.
13 Proteins remain in the barrier between the two phases.
 The Microcon® /Centricon® are centrifugal ultrafiltration devices
that can concentrate DNA samples (Figure .2).
 Phenol–chloroform extraction yields large sized, double stranded
DNA, and can be used for either restriction fragment length
polymorphism (RFLP)- or polymerase chain reaction (PCR)-based
analysis.
 However, the organic extraction method is time-consuming, involves
the use of hazardous reagents, and requires transferring samples
among tubes.

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 Figure 2 Concentration of DNA solutions using filtration devices.
 DNA samples are loaded into the reservoir, the liquid is filtered by
centrifugation and the DNA becomes trapped in the membrane, The
cartridge is then inverted to recover the trapped DNA by centrifugation.
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 2. Extraction by Boiling Lysis and Chelation
 This technique, also called the Chelex® extraction method was
introduced in the early 1990s and usually includes the following steps:
 Washing — This step removes contaminants and inhibitors that may
interfere with DNA amplification.
 Boiling — Cells are suspended in solution and then lysed by heating
to boiling temperature to break open the membranes and release the
DNA.
 A chelating resin (Chelex® 100) is employed during the extraction
process.
 The Chelex® 100 act as chelators by binding to polyvalent metal ions
such as magnesium.
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  Magnesium is a cofactor of endogenous DNase.
 Thus, sequestering magnesium in the solution using Chelex®
100 protects DNA from degradation by DNase (Figure.3).
 Centrifugation - Brief centrifugation is performed to pull the
Chelex® 100 resin and cellular debris to the bottom of the tube.
 The supernatant is used for the DNA analysis.
 This method is simple, rapid, and uses only a single tube for
extraction, thus reducing the risks of contamination and sample
mix-ups.

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 However, the heating step of this method disrupts and denatures
proteins and also affects the chromosomal DNA.
 The resulting DNA extracted from the solution becomes single
stranded.
 Thus, the DNA extracted is not suitable for RFLP analysis because
RFLP requires double stranded DNA samples.
 The DNA obtained by lysis and chelation can only be used for PCR-
based DNA analysis.

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 3. Silica-Based Extraction
 The method of adsorbing DNA molecules to solid silica surfaces has
been used for extraction.
 The method is based on the phenomenon that DNA is reversibly
adsorbed to silica in the presence of high concentrations of salts
(Figure .4).
 Silica (silicon dioxide, SiO2) is the oxide of silicon.

 Salts were originally used to disrupt the three-dimensional structures

of proteins.

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 Common salts employed for DNA extraction include guanidinium
salts such as guanidinium thiocyanate (GuSCN) and guanidinium
hydrochloride (GuHCl).
 GuSCN is a more potent and also facilitates cell lysis and DNA
adsorption.
 This technique usually includes the following steps:
 Cell Lysis and Protein Digestion — This is carried out by proteinase
K digestion. The cell membranes are broken open and DNA is
released.
 DNA Adsorption onto Silica — This step employs silica as the
stationary phase in a column configuration to bind the DNA contained
in the cell lysate.

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 Adsorption of the DNA to the silica occurs in the presence of high
concentrations of chaotropic agents (some protocols adjust pH
conditions to enhance adsorption).

 Under these conditions, cellular materials and other contaminants that

can inhibit DNA amplification reactions are not retained on the
column.

 The adsorbed DNA is double stranded but partial denaturation may

still occur.

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Figure 4: Silica-based DNA extraction. Cells are lysed in the presence
of proteinase. The DNA then binds to the silica matrix.
A washing step removes unbound cellular materials and salts from the
matrix.
The purified DNA is then eluted for use in downstream applications.
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 Washing - This step removes chaotropic agents and other
contaminants.
 The adsorbed DNA cannot be eluted from the silica matrix by certain
solvents, for example, an ethanol-based wash solution but the
chaotropic agents and contaminants can be removed via an ethanol-
based wash from the column.
 Elution of DNA- The adsorbed DNA can be eluted by rehydration
with aqueous low-salt solutions
 The eluted DNA is double stranded and can be used for a wide
variety of applications.

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4. Differential Extraction
 This method is very useful for the extraction of DNA from
biological evidence derived from sexual assault cases, for
example, vaginal swabs and body fluid stains.
 These types of evidence often contain mixtures of sperm cells
from a male contributor and epithelial cells from a female victim.
 Mixtures of individual DNA profiles can complicate data
interpretation.
 This method selectively lyses the nonsperm and sperm cells in
separate steps based on the differences in cell membrane properties
of spermatozoa and other types of cells.
 Thus, the DNA from sperm and nonsperm cell fractions can be
isolated.
 First, the differential extraction procedure involves preferentially
lysing the nonsperm cells with proteolytic degradation using
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proteinase.
 Sperm plasma membrane contains proteins cross-linked by disulfide
bonds.
 The membrane exhibits a much higher mechanical stability than
nonsperm cells and is thus resistant to proteolytic degradation.
 The nonsperm DNA is released into the supernatant and the liquid
containing it (the nonsperm fraction) is extracted, yielding a fraction
that predominantly contains DNA from nonsperm cells.
 To lyse the sperm cells, it is necessary to cleave the disulfide bonds in
addition to proteolytic digestion.
 The application of DTT, a reducing agent, is an approach that can be
used for cleavage.
 In the presence of DTT and proteinase K, the sperm plasma
membrane is then lysed. Subsequently, DNA from the sperm cells can
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be extracted.
 However, the nonsperm cell DNA and sperm cell DNA may not be
completely separated, for example, if the sperm cells have already lysed
due to poor sample conditions.
 Some sperm DNA may be present in the nonsperm cell fraction.

 Additionally, if a mixture has an abundance of non sperm cells and fewer

sperm cells, non sperm cell DNA may be detected in the sperm fraction.

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 DNA quantification and purity determination
 Reliable measurement of DNA concentration is important for
many applications
 DNA quantity and quality can be assessed using several
different methods include:
 Absorbance by spectrophotometer or Nanophotometer.
 Agarose gel electrophoresis.
 Absorbance: is the most common easies to determine DNA
yield and purity.

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 Quality of DNA using spectrophotometer
•An instrument employed to measure the amount of light
that a sample absorbs.

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 The rings of the bases
(A, C, G, T, U) are made
up of alternating single
and double bonds.
Such ring structures
absorb in the U.V.
 Each of the four
nucleotide bases has a
slightly different
absorption spectrum, and
The spectrum of DNA is
the average of them.

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 DNA UV absorbance at 260nm.
 protein >> at 280nm.
 Carbohydrate >> at 230nm.
 Any insoluble light-scattering components……. absorbance at
320 nm.
Note:
 Nucleic acids absorb light at 260nm, the A260 reading should
be between 0.1–1.0.
 The spectrophotometer is most accurate when measurements
are in the range of 0.1–1.0.
 However, DNA is not the only molecule that can absorb UV-
light at 260nm.
 Since RNA also has a great absorbance at 260nm will
contribute to the total measurement at 260nm

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 The ratio of the absorbance at 260nm/280nm is a measure of the
purity of a DNA; it should be between1.7and 2.0.
 If < 1.7, the nucleic acid preparation may be contaminated with
protein. Use protinaseKto remove protein.
 If > 2.0 indicates RNA contamination. RNase should be used to
remove the contaminating RNA.
 DNA Purity (A260/A280)= (A260 reading - A320
reading)/(A280 reading–A320 reading)

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 The ratio of the absorbance at 260nm/320 nm is ameasure of
the purity of a DNA sample from organics and/or salts; it
should be about 2.0.
Low A260/A320 ratio indicates contamination by organics
and/or salts.
The absorbance reading indicates how much the sample is
pure.

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Quantification of DNA by spectrophotometry.
 Using TE buffer as the diluent,
 Make an appropriate dilution of your DNA depending on the
size of the cuvettes available (e.g. for1ml cuvettes, dilute10
micro liter DNA solution in 990 microliters of TE).
 Determine the absorbance of DNA at 260nm using TE as the
reference solution (i.e.as ablank).

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 Using a conversion factor:
 One OD at260 nm is equivalent to

 Multiply the absorbance reading by

 The conversion factor and
 The dilution factor to find the concentration of nucleic acid.

 Pure DNA Concentration (microg/ml)= (A260

reading–A320 reading)x dilution factor x50
microg/ml
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  Total yield is obtained by multiplying the DNA
concentration by the final total purified sample
volume.

 DNA Yield (microgram/ml)= DNA Concentration

x Total Sample Volume

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 DNA concentration by
Nanophotometer
 Don’t need dilution
 The volume required for measurement 3-
5microliters
 The concentration given in nano gram\
microliters.

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 Quality from agarose gel electrophoresis
 Quality of DNA extracted is assessed using the following
simple protocol:
Mix 3μL of DNA with 12μL of loading Dye
Load this mixture into a 1% agarose gel
Stain with ethidium bromide
Electrophorese at 70–80 volts, 45–90 minutes.

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 Checking for Degradation DNA
Running your sample through an
agarose gel is a common method
for examining the extent of DNA
degradation.
Smearing indicates
DNA degradation or
Too much DNA loaded.

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 Good quality of genomic
DNA

Good quality DNA should

migrate as a high molecular
weight band, with little or no
evidence of smearing.

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