DNA PREPARATION METHODS

Genomic DNA
• Contains a copy of every gene from the
organism
• Can be obtained from any micro-organism,
plant or animal at any time during
development 
• Vector DNA
• Refers to plasmid DNA, lambda DNA etc.
 DNA EXTRACTION METHODS
1) Chemical extraction – also known as the
manual extraction
2) Use of DNA extraction kit
 E.g. Kit for blood samples
 Soil samples
 Bacteria
 Plants and some fungi
 Animal tissues
 DNA Isolation
• Genomic and plasmid DNA isolation require
disruption of the tissue.
• Phenol is used to denature and dissolve proteins,
leaving nucleic acids in aqueous solution.
• Phage particles are isolated from the supernatant
cell cultures
• High molecular wt DNA (> 10 kb) must be treated
carefully to avoid shearing. Therefore phenol
extractions usually achieved by inversions or
gentle rocking of the tube to mix the phases.
 DNA Isolation
• Where amount of DNA is limited, the phenol can be
extracted once with TE buffer (pH 8.0) to enhance
recovery of the DNA.
• Some labs favor use of phenol- chloroform because
DNA less soluble in this medium.
• Samples of DNA which have been treated with
phenol are routinely extracted by chloroform-
isoamyl alcohol to precipitate the remaining protein
and reduce the amount of dissolved phenol which is
in the aqueous phase.
 DNA Isolation
• DNA in aqueous solutions can be precipitated
by mixing with either ethanol or isopropanol
in the presence of sodium or ammonium
acetate and cooling to -200C.
• The DNA is collected by centrifuging except
when it is more than 50 µg when it can be
spooled from the supernatant.
 DNA Isolation
• Regardless of the precipitation method used the DNA
sample is rinsed with 70 %( v/v) ethanol to remove any
traces of salt and dried under vacuum.
• DNA samples large scale plasmid,
• DNA samples from large scale plasmid, genomic or
lambda phage preparations are usually determined
spectroscopically.
• A smooth peak should be observed with maximum
absorption at around 260 nm.
• Small quantities of DNA can be assessed by
electrophoresis of the samples in agarose gel with
known standards.
 GENOMIC DNA ISOLATION
• The technique used depends on the physical
characteristics of the starting material.
• Tissue culture cells and nuclei from blood
samples can be treated directly by incubation
with sodium dodecyl –sulphate (SDS) and
proteinase K.
• Bacteria will require cell wall lysis with lysozyme
and EDTA (Ethylene diamine tetra-acetate)
 GENOMIC DNA ISOLATION
• Some types of animal tissues will require shredding and
grinding at -800 C in liquid nitrogen to form a powder to
enable efficient proteolysis.
• DNA from plants is more difficult to obtain due to the
presence of secondary metabolites and polysaccharides
which interfere with the extraction procedure. This
problem can be overcome by using young leaves.
• Poly-phenols and other secondary plant compounds
damage DNA and/ or inhibit restriction enzymes and
polymerases.
• Also partial or total DNA degradation can occur due to the
presence of endogenous nucleases.
 Genomic DNA isolation
• In a considerable number of plant species, DNA
preparations tend to be brown colored due to
oxidation of polyphenols to quinoic
compounds.
• These are powerful oxidized agents that
damage DNA and proteins.
• As a consequence, yields of high molecular
weight genomic DNA from plants are often
poor.
 Genomic DNA isolation
• Plant tissue also requires grinding in liquid nitrogen
with alumina powder or sand to enhance disruption.
• This is followed by extraction with CTAB
(cetyltrimethyl ammonium bromide) which is a high
salt buffer that dissolves the DNA.
• Disruption of cells must be done in the cold to
minimize degradation of organelles and
macromolecules released from different
compartments
 1. ISOLATION OF DNA BY SDS- PHENOL
EXTRACTION
• This is the simplest and fastest method for
isolation of DNA and is widely used with
filamentous fungi.
• The technique uses SDS with phenol to
denature and dissolve macerated hyphae
leaving the DNA intact which is precipitated
from solution using isopropanol (2-propanol).
 2. ISOLATION OF DNA BY SDS -
PROTEINASE K TREATMENT
• This technique is applicable to most material and
relies on SDS and proteinase K to dissolve the
sample and digest the protein component
respectively without affecting the DNA.
• The sample is then extracted with protein
denaturants ( phenol, phenol- chloroform, or
chloroform) and the aqueous liquid containing
the DNA precipitated with ethanol or isopropanol
in sodium or ammonium acetate solution.
 3. ISOLATION OF DNA BY CTAB
(Cetyltrimethyl ammonium bromide)
• Isolation of DNA from plants is preferably
achieved by treatment with CTAB.
• This is a detergent carries a positive charge which
interacts with the negative charge of DNA in high
salt concentrations and forms a soluble complex.
• In subsequent steps a decrease in the salt
concentration causes precipitation of DNA
leaving other compounds especially
polysaccharides in solution.
 QUALITY OF ISOLATED DNA
• Once isolated, genomic DNA can be digested with restriction
enzymes and be visible as distinct fragments after
electrophoresis.
• The size of the DNA molecules can be estimated by
electrophoresis.
• The samples on the gel should appear as a discrete band
migrating slower than the marker with visible trail in the
direction of migration which would indicate extensive
shearing.
• To minimize on the problem of DNA shearing, the transfer
pipettes should be gentle and Rota-mixers should be avoided.
• When DNA is precipitated with ethanol the solvent must be
at -200 C to avoid shearing.
 COLLECTION AND STORAGE OF PLANT
TISSUE
• The quality and yield of plant DNA preparation are to
a considerable extent influenced by the condition of
the starting material.
• Whenever possible, material should be harvested
immediately before DNA isolation.
• Fresh tissue which has to be stored for a short period
of time should be kept cool but not frozen (e.g
wrapped in paper and kept on ice).
• Storage for longer periods requires freezing directly in
- 800 C (-200 C is not advisable), or freeze drying (i.e
lyophilisation).
 Quantification of nucleic acids
• Quantification of nucleic acids is done to determine
the average concentrations of DNA or RNA present in
a mixture, as well as their purity.
• The accurate measurement is based on sensitivity,
specificity and interference by contaminants.
• Various methods that can be employed to quantify
the nucleic acid concentration are:
• (i) Spectrophotometric analysis
• (ii) Nanodrop method
• (iii) Fluorescence based methods
• (iv) Fluorescence in situ hybridization (FISH)
 Spectrophotometric analysis
• It is a simple and accurate method to assess the concentration
and purity of nucleic acids based on their absorption at
different wavelengths.
• Pure DNA has a peak at OD 260.
• Light absorbed by the nucleic acid in the sample correlates to
the concentration of nucleic acid present. Both DNA and RNA
absorb light at 260 nm, therefore this is a measurement of
total nucleic acid.
• Nucleic acid samples are also typically measured at 280 nm,
which is the absorbance peak for protein.
• The ratio of the 260 nm and 280 nm measurements provides a
determination of the purity of the nucleic acid, with a ratio
near 2.0 (1.8 -2.0) indicating a highly pure nucleic acid sample.
 (ii) Nanodrop method
• Unlike spectrophotometric method requiring 1-2 ml
of the sample, this technique involves micro volume
(1-2 μl) quantification of nucleic acid sample.
• The nucleic acids having the concentration range
from 2-15000 ng/μl can be assessed by this method.
• For this, 1-2μl of sample is loaded between the two
optical surfaces and the software automatically
calculates the concentration and purity of the
nucleic acid and displays sample quality (purity) as a
spectral output.
 (iii) Fluorescence based method
• This method is simple and more sensitive than
spectrophotometric method.
• The method measures the intensity of the dyes that fluoresce
upon interaction with the nucleic acids.
• Various fluorescent dyes such as ethidium bromide (EtBr) and
picogreen can be used for quantification of nucleic acids.
• Quantification of nucleic acids separated by gel electrophoresis
can be done by comparing the stained nucleic acids with stained
standards of known concentration separated on the same gel.
• The dye: DNA complex shows greater fluorescence than the
unbound dye by intercalating between the stacked base pairs.
• The fluorescence intensity of the band estimates the
concentration/amount of DNA.
 DNA ISOLATION PROTOCOLS
• Because the biochemical composition of plant
tissues and species vary considerably, it is virtually
impossible to supply a single protocol which is
optimally suited to for each plant species.
• New plant DNA isolation techniques (most dealing
with problematic species) as well as new
modifications of existing procedures are always
coming up.
• The following is a standard protocol described by
Edward and co-workers, 1991:
 DNA extraction protocol
Procedure
• Collect two leaf discs in a microfuge tube by punching the
discs directly into the tube, using the lid as a punch.
• Add a pinch of carborundum or sand and grind thoroughly
in the microfuge tube using a clean glass grinder.
• Add 400 µl of pre-warmed (600C) *supaquick buffer and
mix gently. Incubate at the 600C water-bath for 10 minutes.
• Add an equal volume of chloroform: isoamylalcohol (24:1)
and mix well (5 min) by inverting the tube several times (do
not vortex). This separates nucleic acids from protein and
cell debris.
• Centrifuge at 10,000g for 10 min.
 DNA extraction protocol
• Carefully transfer 350 µl of the supernatant (aqueous phase)
into a clean microfuge tube.
• Add 0.6 volumes of ice-cold isopropanol. Mix gently by
inversion and leave at -20 0C for 30 min. (alternatively 2
volumes of ice-cold 96 % ethanol can be used).
• Centrifuge at 10,000g for 10 min. and decant to drain the
ethanol.
• Add 500 µl of 70 % ethanol and centrifuge again.
• Invert the tube and drain on tissue paper and allow pellet to
air dry. Take care that pellets do not slip down the tube wall.
• Re-suspend the pellet in 100 µl TE buffer.
 DNA extraction protocol
• Determine DNA concentration.
• A SEQUOIA-TURNER model 450 digital
fluorometer can be used to determine the
concentration fluorometrically.
• Pipette 2 ml fluorometer working solution into
the glass cuvette. Wipe the cuvette and insert
the fluorometer well (always place the cuvette
in the same orientation in the well). Adjust the
reading to 000 with the “ZERO” knob
 DNA quantification
• Remove cuvette and add 2 µl of DNA reference standard. Mix
well and re insert into the fluorometer. Adjust the “SCALE”
knob to set the readout to match the concentration of the
standard (e.g. 100)
• Remove the cuvette and rinse well with water.
• -Add fresh working solution, check zero setting and add 2 µl of
own sample.
• -Mix well and read the concentration.
• A yield of 2-5 µg of DNA is usually obtained.
• Prepare a DNA working solution of 2 ng/ µl by diluting in sterile
water.
• *Supaquick DNA isolation buffer: 200 mM Tris-Cl, pH 7.5; 250
mM NaCl; 25 mM EDTA; 0.5 % SDS.