Genomic DNA:
Purification
Burkhard Tümmler, Medizinische Hochschule Hannover, Hannover, Germany
Frauke Stanke, Medizinische Hochschule Hannover, Hannover, Germany
Genomic deoxyribonucleic acid (DNA) can be prepared
from any source by three steps: cell lysis, deproteinisation,
and recovery of DNA. The basic protocol needs to be
adapted to the demands of the application, the number of
samples to be processed, the requested yield, purity and
molecular weight of the DNA, and the amount and history
of the source. Traditional protocols based on the lysis
with sodium dodecyl sulfate/proteinase K, extraction
with phenol/chloroform/isoamyl alcohol, purifica-
tion with ethanol and storage in Tris/ethylenediamine
tetraacetic acid buffer are recommended for the pur-
ification of long-term stable high-molecular weight
genomic DNA from freshly obtained specimens. Intact
chromosomal DNA is recovered from agarose-embedded
cells. Automated extraction methods are preferred for
forensic applications and high-throughput processing for
biobanking. The technically challenging recovery of
ancient DNA needs to be optimised on a case-to-case basis,
because ancient DNA is damaged and chemically modified
and contains large amounts of polymerase chain reaction
inhibitors.
Basic Protocols
. The analysis of complex genomes requires the prepara-
tion of pure genomic deoxyribonucleic acid (DNA) of
high molecular weight.
The DNA is the substrate for numerous applications in
molecular and cellular biology, as well as in nucleic acid
biochemistry and physical chemistry. Prominent applica-
tions of DNA are:
. analysis by chromatography, electrophoresis, sequen-
cing, blotting or hybridisation;
. amplification of target sequences by the polymerase
chain reaction (PCR);
eLS subject area: Molecular Biology
How to cite:
Tümmler, Burkhard; and Stanke, Frauke (September 2013) Genomic
DNA: Purification. In: eLS. John Wiley & Sons, Ltd: Chichester.
DOI: 10.1002/9780470015902.a0005330.pub2
eLS & 2013, John Wiley & Sons, Ltd. www.els.net
Advanced article
. Basic Protocols
Article Contents
. Preparation of Intact Chromosomal DNA
. DNA Isolation Procedures for Biobanking
. Special Cases: Forensic Medicine and Palaeontology
Online posting date: 20th September 2013
. the construction of recombinant DNA libraries;
. physicochemical characterisation and binding studies;
. in-vitro replication or transcription assays and
. physical, chemical or enzymatic cleavage, modification
or mutagenesis. See also: DNA Structure; Gel Electro-
phoresis; Genome Sequencing; Nucleic Acid Hybridi-
zation; Recombinant DNA
Numerous protocols for the preparation of genomic
DNA have been published.
. Genomic DNA can be prepared from any source by
three steps: cell lysis, deproteinisation and recovery of
DNA.
The main differences among various procedures lie in the
purity, that is, the extent of deproteinisation and the mole-
cular weight of the DNA produced (Ausubel et al., 2012).
Genomic DNA from mammalian sources is most com-
monly prepared using the following protocol: Mammalian
cells are efficiently lysed by 12–18 h of incubation at 50–56
8C with proteinase K in the presence of the ionic detergent
sodium dodecyl sulfate (Gross-Bellard et al., 1973). Pro-
teins are then removed from the sample by extraction with
an equal volume of phenol/chloroform/isoamyl alcohol
(25/24/1v/v/v). Phenol and chloroform efficiently denature
the proteins, which form a layer at the interface between the
aqueous and organic phases. Isoamyl alcohol stabilises the
phase separation. For complete deproteinisation, extrac-
tion may be repeated until no protein precipitate remains at
the aqueous/organic interface. To remove residual phenol
and chloroform, the DNA is precipitated from the aqueous
phase by the addition of 0.5 volume of 7.5 m ammonium
acetate and 2 volume of ice-cold 100% ethanol. The DNA
is recovered by centrifugation, and the pellet is rinsed
with 70% ethanol to desalt the DNA, air-dried and
resuspended in 10 mm 2-amino-2-hydroxymethylpropane-
1,3-diol (Tris) and 10 mm ethylenediamine tetraacetic acid
(EDTA), pH 8 (TE buffer). High molecular weight DNA
will require several days to dissolve. DNA is more stable in
TE buffer than in water and can be stored at 4 8C for at least
20 years without any degradation (own observations).
However, one should note that the EDTA from the TE
buffer chelates bivalent cations. If an Mg2+-dependent
enzymatic reaction, such as the PCR, is performed with a
large volume aliquot of TE buffer containing the DNA
template, a higher than normal MgCl2 concentration may
be needed to ensure full enzymatic activity.
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 Genomic DNA: Purification

The standard procedure will typically yield DNA
molecules that are approximately 50 kb. If DNA of higher
molecular weight (e.g., for the construction of genomic
libraries) is needed, shearing forces during the extraction
step should be minimised using wide bore pipettes and very
gentle mixing, and precipitation of the DNA with ethanol
should be substituted by dialysis (Ausubel et al., 2012).
See also: DNA: Mechanical Breakage; Kilobase Pair (kbp)
Similar protocols consisting of cell lysis, deproteinisa-
tion and alcohol precipitation are also commonly used to
prepare genomic DNA from plants and bacteria (Goldberg
and Ohman, 1984). However, procedures need to be
modified if the organism of interest has a rigid and/or
unusual cell wall and/or produces copious amounts of
exopolysaccharides or phenolic compounds.
For the preparation of bacterial genomic DNA, a rigid
bacterial cell wall may be predigested with lysozyme before
lysis with detergent. Protease incubation is followed by an
extraction with the detergent cetyltrimethylammonium
bromide (CTAB) in the presence of 1 M NaCl whereby
CTAB complexes both with polysaccharide and with
residual protein, while retaining the nucleic acids in solu-
tion. The CTAB–protein/polysaccharide complexes are
then removed by extraction with chloroform/isoamyl
alcohol (Ausubel et al., 2012).
CTAB is also used to isolate DNA from plants (Murray
and Thompson, 1980). First nucleic acids are extracted
from pulverised plant tissue with CTAB in high-salt solu-
tion at 65 8C. After extraction with chloroform/octanol the
nucleic acids are precipitated from the aqueous phase with
CTAB by lowering the NaCl concentration to 0.5 M at
65 8C. CTAB forms an insoluble complex with nucleic
acids at 0.5 M NaCl whereas polysaccharides and other
contaminants remain in the supernatant and are removed.
The CTAB–nucleic acid pellet is solubilised in high-salt TE
buffer, the nucleic acids are precipitated with isopropanol
and residual CTAB is removed by washing the nucleic acid
pellet with 80% ethanol. The pellet is resuspended in TE
buffer. The DNA may then be further purified by treatment
with RNase A and proteinase K.
Table 1 shows commonalities and differences of the
standard procedures for the preparation of genomic DNA
from bacteria, mammals and plants.
The purity of a DNA preparation is commonly determined
by spectrophotometry. A spectrum in the UV range from
220 nm to 330 nm should be recorded to detect contaminants
such as RNA, protein or dust. The commonly applied cri-
terion for DNA purity is the ratio of the absorbance at
260 nm (A260) divided by the absorbance at 280 nm (A280)
after correcting for turbidity (absorbance at 320 nm A320):
DNA purity (A260/A280)=(A2602A320)/(A2802A320)
Good-quality DNA will have an A260/A280 ratio of
1.7–2.0.

Table 1 Flowchart of genomic deoxyribonucleic acid (DNA) preparations

Bacteria Mammals Plants
a b
Cell lysis a. Optional: lysozyme SDS+protease Extraction with high-salt
b. Sodium dodecyl sulfate cetyltrimethylammonium
(SDS)+protease bromide (CTAB)
Deproteinisation and a. Complexation with CTAB Extraction with phenol/ Extraction with phenol/
removal of b. Extraction of CTAB chloroform/isoamyl alcohol chloroform
polysaccharides complexes with
chloroform/isoamyl
alcohol
Recovery of DNA from Precipitation with salt and Precipitation with salt and a. Precipitation with CTAB in
aqueous phase ethanol ethanol low salt
b. Solubilise pellet in high-salt
TE
c. Precipitate with
isopropanol
Desalting Rinse pellet with 70% ethanol Rinse pellet with 70% ethanol Rinse pellet with 80% ethanol
Resuspension Dissolve air-dried DNA in TE Dissolve air-dried DNA in TE Dissolve air-dried DNA in TE
Further purification, if a. Incubation with RNase A
necessary and protease
b. Phenol/chloroform/
isoamyl alcohol extraction
c. Ethanol precipitation and
desalting
d. Dissolve air-dried DNA in
TE
a
Cell lysis of invertebrates works with both CTAB or SDS methods; cell lysis of lower eukaryotes may require predigestion with further enzymes.
b
Predigestion with lysozyme is useful for Gram-positive bacteria.

2 eLS & 2013, John Wiley & Sons, Ltd. www.els.net


Table 2 A selection of commercially available DNA extraction platforms
Platform
AutoMate Express
Biomek 2000 Laboratory Automation
EZ1 Advanced Instruments
Freedom EVO75 (or EVO150) workstation and the Te-MagS magnetic separation module
Maxwell 16 System
MagNA Pure LC 2.0 System
Microlab Nimbus Liquid Handling Workstation
QIAxtractor
Purification of genomic DNA should be executed with
utmost care. Contaminations by PCR products, recombi-
nant phages or plasmids can make the final preparation
worthless for applications such as PCR, cloning or library
construction. All materials used for the analysis of PCR
products or for the preparation of plasmid or phage DNA
should be kept separate from all materials used for the
preparation of genomic DNA. Extreme precautions to
prevent contamination should be taken if the DNA of
interest is degraded and/or present only in trace amounts.
See also: DNA Cloning; Polymerase Chain Reaction (PCR)
Preparation of Intact Chromosomal
DNA
The standard protocols for the purification of DNA by
organic extraction of proteins and alcohol precipitation
outlined above involve shear forces that will break large
DNA fragments to an average length of 50 kb or less.
. Intact chromosomal megabase-sized DNA is purified in
solid agarose to protect the DNA during preparation
(Bautsch et al., 1997).
Intact cells are mixed at 50 8C with a high-quality
low-melting-point agarose, which is then allowed to
solidify in moulds. These solid samples are treated
with detergents and enzymes that lyse the cell wall
and allow proteins and other molecules to diffuse out,
leaving only the nucleic acids. Although the basic method is
always the same, the choice of detergent and enzymes
depends on the constituents of cells and their extracellular
matrix. In particular, the standard protocols need to be
modified on a case-to-case basis for plants and bacteria
with rigid and/or unusual cell walls. The treatment of
mammalian cells only requires proteinase K, the detergent
N-lauroylsarcosine and EDTA. The last is present in all
solutions to inhibit endogeneous nucleases that could
degrade the DNA during incubations. Agarose-embedded
chromosomal DNA stored in TE buffer at 4 8C is stable for
at least 15 years (own observations). See also: Artificial
Chromosomes; Mammalian Artificial Chromosomes
(MACs); Megabase Pair (Mbp); Yeast Artificial Chro-
mosome (YAC) Clones
eLS & 2013, John Wiley & Sons, Ltd. www.els.net
Genomic DNA: Purification
Supplier
Life Technologies
Beckman-Coulter
Qiagen
Tecan
Promega
Roche Applied Science
Hamilton
Qiagen
DNA Isolation Procedures for
Biobanking
Biobanks collect several thousand to hundred thousand
specimens (Hirtzlin et al., 2003). These large sample sets
cannot be processed and stored using laborious manual
procedures.
. DNA extraction for biobanking relies on automated
systems and robotics.
Table 2 lists some commercially available platforms for
automated DNA extraction. Standard protocols handle
the processing of blood, but there are also numerous more
specialised applications such as the DNA extraction from
saliva (Keijzer et al., 2010), human remains (Witt et al.,
2012), plants (Xin and Chen, 2012) or formalin-fixed
paraffin-embedded tissues (Khokhar et al., 2011). See also:
Biobanking: Social, Political and Ethical Aspects
Special Cases: Forensic Medicine and
Palaeontology
The purification of DNA is a straightforward routine
procedure, but it may become a challenging task if the
DNA is ancient, degraded and/or only available in minute
amounts. Research is ongoing in forensic medicine,
anthropology and palaeontology to develop procedures
that maximise the yield of DNA from these special source
materials, and to eliminate inhibitors of PCR such as dyes,
polyphenols, polysaccharides or glove powder.
Amplifiable DNA has been recovered for forensic DNA
analysis with variable success by using the chelating resin
Chelex 100, alkaline lysis, QIAamp spin columns or silica-
gel particles instead of the standard phenol/chloroform
extractions and ethanol precipitation (Cattaneo et al.,
1997; Klintschar and Neuhuber, 2000).
. Automated systems have been increasingly utilised for
DNA extraction by forensic laboratories to handle the
growing numbers of forensic casework samples while
minimising the risk of human errors and assuring high
reproducibility (see Table 2).
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 Genomic DNA: Purification
The automated extraction method has to be very versatile
to reliably prepare high yields of pure genomic DNA from a
broad variety of sample types on different carrier materials
(Witt et al., 2012): To prevent possible cross-contamination
of samples or the loss of DNA, the components of the kit
have to be designed in a way that allows for automated
handling of the samples with no manual intervention being
necessary. DNA extraction using paramagnetic particles
coated with a DNA-binding surface is predestined for an
automated approach. In case of degraded human material,
the yield of PCR-amplified products is higher from younger
than from older specimens, but plateauing occurs after 10
years. See also: Forensic Genetics
. The recovery of ancient DNA from palaeontological
specimens is a demanding task, because ancient DNA is
damaged and chemically modified and contains large
amounts of PCR inhibitors that interfere with DNA
amplification that are copurified with the DNA.
Several methods for ancient DNA extraction exist,
including ethanol or isopropanol precipitation (Kalmar
et al., 2000), concentration of DNA using membranes
(Leonard et al., 2000) and binding DNA to silica (Rohland
and Hofreiter, 2007). Depending on the nature of the
sample, the DNA extraction method should represent the
optimal compromise between DNA release, DNA degra-
dation during extraction and separation of DNA and
inhibitors. Meanwhile, protocols are available for the
extraction of DNA from soils, sediments, ancient plants,
paleofeces and animal remains such as hairs, bones and
teeth (see the monograph by Shapiro and Hofreiter, 2012).
Even the nondestructive extraction of DNA from museum
specimens of extinct species has become feasible. Speci-
mens are soaked in extraction buffer, and DNA is then
purified from the soaking solution using adsorption to
silica (Shapiro and Hofreiter, 2012).
Successfully extracted ancient DNA is a valuable source
to generate primary data on phylogeny and evolution. For
example, the rates of sequence evolution could be eval-
uated from DNA that had been isolated from subfossil
bones of penguins, dating back to more than 7000 years bp
(Lambert et al., 2002). This spectacular case demonstrates
that under favourable conditions, DNA can be purified
from samples that are several thousand years old. See also:
Ancient DNA: Phylogenetic Applications; Ancient DNA:
Recovery and Analysis; Humans: Archaic and Modern
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DNA laboratory. Annals of Anatomy 194: 3–6.
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the past and the present. Journal of Biomedicine and Bio-
technology 2009: 574398.