Practical No : 12

Practical : Isolation of DNA from animal tissues

Objectives : To understand the steps involving DNA extraction from animal materials and

to understand the importance of chemical and conditions used in the solution

methods

Introduction :

DNA extraction involves breaking down cellular membranes, deactivating

enzymes that degrade DNA, and solubilizing the DNA in a suitable buffer. The proteins are hen
digested with a protease and separated from the DNA solution by extraction with organic solvents.
The final and purifying step is to add alcohol and monovalent salt to the extract. The DNA becomes
insoluble, and precipitated from the solution. The mixture is the spun at high speed to pellet
precipitated DNA. The supernatant is removed and the DNA is re-suspended in water or buffer. In
this experiment, you will isolate DNA from human blood. In mammals DNA is present in white
cells only. Therefore the fragile red blood cells are selectively lysed by freezing and thawing the
cell pellets, and the white cells are separated. The DNA is then isolated from white blood cells.

Materials :

Human blood (Collected in a sterile tube containing anticoagulant such as EDTA)

Ice bath

PBS (137 mM NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4.7H2O, 1.4 mM KH2PO4)

Extraction buffer (100 mM NaCl, 10 mM Tris.HCl, pH 8.0, 0.25 mM EDTA, 0.5% SDS)

Proteinase K (20 mg/ml)

Phenol: Chloroform: isoamyl alcohol (25: 24:1 Phenol is equilibrated with 0.1 M Tris.HCl,
pH 8.0 Buffer)

Absolute ethanol

70% ethanol
 Ammonium acetate (7.5 M)

TE buffer (10 mM Tris HCl, 1mM EDTA, pH 8.0)

Polypropylene centrifuge tubes, centrifuge

Method :

Blood sample was collected into a sterile container containing anticoagulant

such as EDTA. Then 5 ml of whole blood was taken into a sterile centrifuge tube and was
centrifuged at 3000 rpm for 5 minutes. The supernatant was discarded ensuring that the buffy coat
remains with the cell pellet. The cell pellet was freezed at -70 ºC for about 20 minutes and then
was thawed on ice for selective lysis of non- nucleated erythrocytes. Then the pallet was re-
suspended in an equal volumes of ice cold solution of PBS and centrifuge at 3000 rpm for 15
minutes. The supernatant was discarded and the above washing step was repeated once more. Next
500 µL of extraction buffer was added and the solution was incubated for half an hour at 37 ºC.
Then 2.5 µL of proteinase K (20 mg/ml) was added and was mixed gently and was incubated at
50 ºC overnight. An equal volume of Phenol- Chloroform -isoamyl alcohol was added and the two
phases were mixed gently. Then was centrifuged at 10,000 rpm for 3 minutes. The viscous aqueous
layer was transferred to a sterile centrifuge tube using a wide bore pipette. The Phenol- Chloroform
-isoamyl alcohol extraction was repeated was centrifuged and was separated the aqueous phase.
0.2 volumes of 7.5 M ammonium acetate were added to the aqueous phase and was mixed gently.
Two volumes of ice cold 100% ethanol was added and was mixed gently. Next DNA was spooled
onto a glass rod (or Pasteur pipette with a heat-sealed end) at -40 ºC for 20 minutes, was centrifuged
at 12,000 rpm for 10 minutes. After that DNA was washed with 70 % ethanol. Then the DNA
pellet was air dried and was re-dissolved in 50- 100 TE buffer. A few microliters of DNA was
diluted with 2 ml of TE buffer and optical absorbance was measured at 260 nm and 280 nm. Next
DNA concentration, the ratio of absorbance at 260 nm and 280 nm were calculated. Finally DNA
was analysed by agarose gel electrophoresis
Observation:

Eppendorf

Extracted DNA from human blood

Discussion:

Deoxyribonucleic acid (DNA) extraction is the process by which DNA is separated from proteins,
membranes, and other cellular material contained in the cell from which it is recovered. This
extraction can be one of the most labor-intensive parts of DNA analysis. Extraction methods may
require an overnight incubation, may be a protocol that can be completed in minutes or a couple
of hours, or may be a recent procedure that employs reagents for which this step can be skipped
completely. DNA extraction methods follow some common procedures aimed to achieve effective
disruption of cells, denaturation of nucleoprotein complexes, inactivation of nucleases and other
enzymes, removal of biological and chemical contaminants, and finally DNA precipitation. Most
of them follow similar basic steps and include the use of organic and nonorganic reagents and
centrifugation methods. Finally, they have developed into a variety of automated procedures and
commercially available kits. Lysis of cells is a common step in most DNA extraction protocols,
and it is commonly achieved through the use of detergents and enzymes. Sodium dodecyl sulfate
(SDS) and Triton™ X-100 (Sigma-Aldrich, St Louis, MO, USA) are examples of popular
detergents used to solubilize cell membranes. Enzymes are also combined with detergents to target
cell surface or cytosolic components. Proteinase K is a commonly used enzyme used in various
protocols to cleave glycoproteins and inactivate RNases and DNases. Other denaturants such as
urea, guanidinium salts, and chemical chaotropes have also been used to disrupt cells and
inactivate cellular enzymes, but these can impact on quality and nucleic acid yield.

DNA precipitation is achieved by adding high concentrations of salt to DNA-containing solutions,

as cations from salts such as ammonium acetate counteract repulsion caused by the negative charge
of the phosphate backbone. A mixture of DNA and salts in the presence of solvents like ethanol
(final concentrations of 70%–80%) or isopropanol (final concentrations of 40%–50%) causes
nucleic acids to precipitate. Some protocols include washing steps with 70% ethanol to remove
excess salt from DNA. Finally, nucleic acids are re-suspended in water or TE buffer (10 mM Tris,
1 mM ethylenediaminetetraacetic acid [EDTA]). TE buffer is commonly used for long-term DNA
storage because it prevents it from being damaged by nucleases, inadequate pH, heavy metals, and
oxidation by free radicals. Tris provides a safe pH of 7–8, and EDTA chelates divalent ions used
in nuclease activity and counteracts oxidative damage from heavy metals.

The DNA extraction process requires careful handling of biological material to prevent sample
contamination and crossover. Tubes should be carefully labeled, especially when transfers are
required. Robots may be employed to extract reference samples and some evidence samples, but
other evidentiary samples may require the direct attention of a DNA analyst.

The simplest cells, such as bacteria cells, are prokaryotes. These prokaryotes comprise a lipid
bilayer outer membrane and a cytoplasm containing a circular chromosome, proteins inorganic
salts and metal ions, sugar molecules, and other elements of cell machinery. Humans, animals, and
plants are composed of eukaryotic cells; these cells also have a lipid bilayer outer membrane and
cytoplasm containing proteins, sugars, lipids, and inorganic ions of various types and function.
However, eukaryotic cells also contain other membrane-enclosed compartments called organelles.
The nucleus of a cell is an organelle that houses 46 chromosomes, and the mitochondria each house
a circular DNA chromosome, all of which direct the production of proteins. The mitochondrial
chromosome is16,569 bp. The mitochondrial proteins are used as some of the metabolic machinery
for digestion of sugars and fats and production of most of the energy for the cell. Other organelles
are involved in the synthesis and modification of proteins, sugars, and fats, and other molecules
used by the cell or in its signaling activities. The genes that code for heritable traits—including
height, blood type, hair color, eye color, skin color, and temperament—are found on chromosomes
in the nucleus of the eukaryotic cell. In plants, the additional chloroplast chromosome contains
genes for photosynthesis. Forensic scientists are largely unconcerned with the genes that code for
proteins or regulatory elements of the DNA; however, forensic scientists are interested in isolating
DNA from the nuclear, mitochondria and chloroplast (if present) chromosomes to evaluate the
sequences, base and repeat polymorphisms, including SNPs and STRs, respectively, that have
proved useful for linking a suspect to a crime scene.

DNA is highly negatively charged because of its phosphate groups. It is stabilized by magnesium
in the cell when unwound. Nuclear DNA consists of 3.2 billion bases in humans. It is organized
into chromosomes in part by coiling around positively charged proteins called histones to form
nucleosomes. Magnesium is also integral to the function of proteases, enzyme proteins that cut up
DNA.

Because of the lipid structure of the cell (and nuclear) membrane(s), presence of proteases and
magnesium, and coiling of DNA around histones, many of the available DNA
extraction procedures have common elements. Indeed, the extraction of DNA generally follows
three basic steps:

1. Lyse (break open) the cells.

2. Separate the DNA from the other cell components.

3. Isolate the DNA.

The cell membrane is disrupted by any of the following methods: using heat to increase fluidity,
dithiothreitol (DTT) to reduce disulfide bonds, or a detergent, such as sodium dodecyl sulfate
(SDS), to disrupt the membrane. Proteins, including nucleases, are inactivated by heat denaturation
or by digestive enzymes, including proteinase K, to cut them up. The temperature must be kept
below 60° C and the period must be kept sufficiently short (15 to 20 minutes) if non-degraded,
high-molecular-weight DNA is required. Either the magnesium needed for nuclease activity or
DNA is immobilized on a solid phase and eluted by buffer/salt. If the DNA remains in the aqueous
phase, it is separated from the other cellular materials including proteins and lipids by centrifuging
the latter to the bottom of the tube or partitioning them in organic solvents.

There are four commonly used extraction procedures for DNA extraction (Hoff-Olsen et al.,
1999):
1. Organic (variations of phenol/chloroform): use of a multistep liquid chemical process that is
labor intensive but produces a high yield and very-clean double-stranded extracted DNA sample

2. Inorganic Chelex or silica methods: simple and cheap one-tube extraction process in which
Mg2+ binds to resin beads and yields a single-stranded DNA product

3. Solid phase extraction methods (e.g., Promega’s DNA IQ (Eminovic et al., 2005, DNA IQ
manual), Applied Biosystems’ PrepFiler (Brevnov et al., 2009, PrepFiler manual), andQiagen’s
QIAamp kits (Castella et al., 2006, Greenspoon et al., 1998)): simple extraction process in which
the DNA binds to paramagnetic or silica beads

4. Differential extraction: a multistep process used to separate sperm from other cells using DTT;
used for analyzing biological evidence from sexual assault cases (Drobnic, 2003)

Figure 01: Different animal DNA extraction methods

Different extraction methods result in different yields and purity of DNA. Some of the extraction
methods have been systematically evaluated for specific applications such as soil and sediment
samples, human microbiome, and fecal samples.

Organic Extraction

In this conventional, widely used method, cells are lysed and cell debris is usually removed by
centrifugation. Then proteins are denatured/digested using a protease and precipitated with organic
solvents such as phenol, or 1:1 mixture of phenol and chloroform, and the protein precipitate is
removed by centrifugation. Purified DNA is usually recovered by precipitation using ethanol or
isopropanol. In the presence of monovalent cations such as Na+, and at a temperature -20°C,
absolute ethanol efficiently precipitates polymeric nucleic acids and leaves behind short-chain and
monomeric nucleic acid components including the ribonucleotides from RNase treatment in
solution. This method uses hazardous organic solvents, is relatively time-consuming, and residual
phenol or chloroform may affect downstream applications such as PCR. Easy-DNA® Kit
(Invitrogen) is an example.

Silica based technology

This is a widely employed method in current kits. DNA adsorbs specifically to silica
membrane/beads/particles in the presence of certain salts and at a particular pH. The cellular
contaminants are removed by wash steps. DNA is eluted in a low salt buffer or elution buffer.
Chaotropic salts are included to aid in protein denaturation and extraction of DNA. This method
can be incorporated in spin columns and microchips, is cost effective, has a simpler and faster
procedure than the organic extraction, and is suitable for automation. Kits based on this method
include Purelink Genomic DNA extraction kit (Invitrogen) and DNeasy Blood and Tissue Kit
(Qiagen).

Magnetic separation
This method is based on reversibly binding DNA to a magnetic solid surface/bead/particles, which
have been coated with a DNA binding antibody or a functional group that interacts specifically
with DNA. After DNA binding, beads are separated from other contaminating cellular
components, washed and finally the purified DNA is eluted using ethanol extraction. This method
is rapid and can be automated. However, it can be more costly than other methodologies. Examples
are Agencourt DNAdvance Kit (Beckman Coulter) and Magnetic Beads Genomic DNA Extraction
Kit (Geneaid).

Anion exchange technology

This method is based on the specific interaction between negatively charged phosphates of the
nucleic acid and positively charged surface molecules on the substrate. DNA binds specifically to
the substrate in presence of low salt, contaminants are removed by wash steps using low or medium
salt buffer, and purified DNA is eluted using a high salt buffer. This technology is more commonly
employed in plasmid isolation kits such as PureLink® HiPure Plasmid DNA Purification Kits
(Invitrogen), Qiagen plasmid mini/midi kits and Genomic-tip, and NucleoBond® PC kits
(Macherey Nagel).

Others

Other methods include salting out, cesium chloride density gradients, and chelex 100 resin. DNA
isolation methods are often modified and optimized for different cell types.
Cetyltrimethylammonium bromide (CTAB) and guanidium thiocynate (GITC) are often included
in protocols for DNA extraction from plant materials, and are discussed more in detail in section
"DNA extraction from plant tissue and cells".

Table 01: Overview of DNA extraction and purification kits for microbes.

Kit Source Yield Usage and advantage

Yield ranges from 15

Provides DNA for restriction
ug - 20 ug DNA from
Bacterial Genomic endonuclease digestions, PCR,
0.8 - 1.5 ml of
DNA Mini-prep Kit Culture and southern blots. Purified
overnight culture,
(BayGene) DNA has an A260/A280 ratio
depending on source
between 1.6 and 1.9. Includes
and OD600 per ml.
 lysozyme diluent & RNase A
solution

Purified DNA is suitable for

sequencing, fingerprinting,
BACMAX DNA Produces 0.6 to 25 ug PCR, and preparation of
purification kit of BAC DNA from 1.5 shotgun libraries. Avoids the
BAC culture
(Epicentre to 100 ml of a single- use of columns or binding
Biotechnologies) copy BAC culture. resins that lower yields and
shear the DNA. No toxic
organic solvents.

Various materials: Fully automated and applicable

Up to 96 samples, in
serum, plasma, for simultaneous purification of
QIAsymphony batches of 24, are
CSF, swabs, viral and bacterial DNA.
Virus/Bacteria kits processed in a single
aspirates, sputum, Provides high quality DNA for
run
BAL, fecal downstream applications.

Isolates DNA free of PCR

Up to 25 μg total DNA inhibitors and suitable for PCR,
ZR Fecal DNA Mammalian feces
is eluted into ≥25 μl arrays, genotyping, methylation
Mini Prep (Zymo (humans, rats,
elution buffer per detection, etc. Omits the use of
Research) mice, cattle)
sample (≤ 150 mg) organic denaturants
(proteinases).

Produces highly purified DNA

Soil or
PowerMax Soil free of PCR inhibitors, which
environmental
DNA Isolation Kit Purifies DNA from 10 can be used for PCR and qPCR.
sample with high
(MO BIO g soil in 30 minutes. High detection sensitivity on
or low microbial
Laboratories) samples with a low microbial
load
load.

Soil,
Provides DNA for PCR, qPCR
PowerSoil DNA environmental
Purifies DNA from 250 and next generation sequencing.
isolation kit (MO samples; fecal,
mg soil in 30 minutes Eliminates PCR inhibitors.
BIO Laboratories) stool and biosolid
Produces high quality DNA
samples.

Yeast, fungi, Yields high quality

UltraClean Provides DNA for PCR,
Gram-negative and DNA up to 50 kb plus.
Microbial DNA restriction digestion. No
Gram-positive Rapidly purifies DNA
Isolation Kit (MO hazardous organic solvents or
bacteria, bacterial from microbial cultures
BIO Laboratories) enzymes are required.
spores and fungi in 20 minutes.

Produces high-quality plasmid

A 1.5 ml overnight
DNA. Preparation of low-copy-
culture can yield from 5
QIAprep Spin number plasmid or cosmids
to 15 ug of plasmid
Miniprep Kit Culture from 1-10 ml overnight E.
DNA. Processes 1-24
(Qiagen) colicultures grown in LB
samples simultaneously
medium. Purification of very
in less than 30 minutes.
large plasmids (>50 kb).
 Provides ultrapure supercoiled
plasmid DNA with high yields
for molecular biology
Yields up to 500 ug
applications. Yields
Plasmid Maxi Kit high-quality plasmids
Culture transfection-grade DNA for
(Qiagen) up to approximately
transfection, in
150 kb.
vitro transcription and
translation, and enzymatic
modifications.

References:
[1] Gross-Bellard M, Oudet P, Chambon P (1973). Isolation of high molecular weight DNA from
mammalian cells. European Journal of Biochemistry 36: 32–38.
[2]Stulnig TM, Amberger A (1994). Exposing contaminating phenol in nucleic acid preparations.
Bio Techniques 16: 402–404.
[3]Glasel JA (1995). Validity of nucleic acid purities monitored by 260nm/280nm absorbance
ratios. Bio Techniques 18: 62–63.
[4]Sambrook J, Fritsch EF, Maniatis T (1989). Molecular Cloning: A laboratory manual. Second
edition. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York.