The Analysis of Food Samples for the Presence of

Genetically Modified Organisms

Session 4

Extraction and Purification of DNA

M. Somma

WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE

REGIONAL OFFICE FOR EUROPE BUREAU REGIONAL DE L'EUROPE

WELTGESUNDHEITSORGANISATION ВСЕМИРНАЯ ОРГАНИЗАЦИЯ ЗДРАВООХРАНЕНИЯ

REGIONALBÜRO FÜR EUROPA ЕВРОПЕЙСКОЕ РЕГИОНАЛЬНОЕ БЮРО
Extraction and Purification of DNA 2

Table of Contents

Session 4

Extraction and Purification of DNA

Introduction 3

Extraction methods 4
Purification methods 4
CTAB extraction and purification method 6
Quantification of DNA by spectrophotometry 9
Principles of spectrophotometric determination of DNA 9
Determination of the concentration of nucleic acids 11

Experimental 13

References 17

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 3

Introduction
Extraction and purification of nucleic acids is the first step in most molecular biology
studies and in all recombinant DNA techniques. Here the objective of nucleic acid
extraction methods is to obtain purified nucleic acids from various sources with the
aim of conducting a GM specific analysis using the Polymerase Chain Reaction
(PCR). Quality and purity of nucleic acids are some of the most critical factors for
PCR analysis. In order to obtain highly purified nucleic acids free from inhibiting
contaminants, suitable extraction methods should be applied. The possible
contaminants that could inhibit the performance of the PCR analysis are listed in
Table 1. In order to avoid the arising of a false negative result due to the presence of
PCR inhibitors in the sample, it is highly recommended to perform a control
experiment to test PCR inhibition. For this purpose, a plant-specific (eukaryote or
chloroplast) or species-specific PCR analysis is commonly used.

Table 1. Some inhibitors of the PCR process

Inhibitor Inhibiting concentration
SDS > 0.005%
Phenol > 0.2%
Ethanol > 1%
Isopropanol > 1%
Sodium acetate > 5 mM
Sodium chloride > 25 mM
EDTA > 0.5 mM
Hemoglobin > 1 mg/ml
Heparin > 0.15 i.m/ml
Urea > 20 mM
Reaction mixture > 15%

As a wide variety of methods exist for extraction and purification of nucleic acids, the
choice of the most suitable technique is generally based on the following criteria:
• Target nucleic acid
• Source organism
• Starting material (tissue, leaf, seed, processed material, etc.)
• Desired results (yield, purity, purification time required, etc.)
• Downstream application (PCR, cloning, labelling, blotting, RT-PCR, cDNA
synthesis, etc.)
The principles of some of the most common methodologies used today for the
extraction and purification of nucleic acids are described in the following sections.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 4

Extraction methods
The extraction of nucleic acids from biological material requires cell lysis, inactivation
of cellular nucleases and separation of the desired nucleic acid from cellular debris.
Often, the ideal lysis procedure is a compromise of techniques and must be rigorous
enough to disrupt the complex starting material (e.g. tissue), yet gentle enough to
preserve the target nucleic acid. Common lysis procedures include:
• Mechanical disruption (e.g. grinding, hypotonic lysis)
• Chemical treatment (e.g. detergent lysis, chaotropic agents, thiol reduction)
• Enzymatic digestion (e.g. proteinase K)
Cell membrane disruption and inactivation of intracellular nucleases may be
combined. For instance, a single solution may contain detergents to solubilise cell
membranes and strong chaotropic salts to inactivate intracellular enzymes. After cell
lysis and nuclease inactivation, cellular debris may easily be removed by filtration or
precipitation.

Purification methods
Methods for purifying nucleic acids from cell extracts are usually combinations of two
or more of the following techniques:
• Extraction/precipitation
• Chromatography
• Centrifugation
• Affinity separation
A brief description of these techniques will be given in the following paragraphs
(Zimmermann et al., 1998).

Extraction/Precipitation

Solvent extraction is often used to eliminate contaminants from nucleic acids. For
example, a combination of phenol and chloroform is frequently used to remove
proteins. Precipitation with isopropanol or ethanol is generally used to concentrate
nucleic acids. If the amount of target nucleic acid is low, an inert carrier (such as
glycogen) can be added to the mixture to increase precipitation efficiency. Other
precipitation methods of nucleic acids include selective precipitation using high
concentrations of salt (“salting out”) or precipitation of proteins using changes in pH.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 5

Chromatography

Chromatography methods may utilise different separation techniques such as gel

filtration, ion exchange, selective adsorption, or affinity binding. Gel filtration exploits
the molecular sieving properties of porous gel particles. A matrix with defined pore
size allows smaller molecules to enter the pores by diffusion, whereas bigger
molecules are excluded from the pores and eluted at the void volume. Thus,
molecules are eluted in order of decreasing molecular size. Ion exchange
chromatography is another technique that utilises an electrostatic interaction between
a target molecule and a functional group on the column matrix. Nucleic acids (highly
negatively charged, linear polyanions) can be eluted from ion exchange columns with
simple salt buffers. In adsorption chromatography, nucleic acids adsorb selectively
onto silica or glass in the presence of certain salts (e. g. chaotropic salts), while other
biological molecules do not. A low salt buffer or water can then elute the nucleic
acids, producing a sample that may be used directly in downstream applications.

Centrifugation

Selective centrifugation is a powerful purification method. For example

ultracentrifugation in self-forming CsCl gradients at high g-forces has long been used
for plasmid purification. Frequently, centrifugation is combined with another method.
An example of this is spin column chromatography that combines gel filtration and
centrifugation to purify DNA or RNA from smaller contaminants (salts, nucleotides,
etc.), for buffer exchange, or for size selection. Some procedures combine selective
adsorption on a chromatographic matrix (see above paragraph “Chromatography”)
with centrifugal elution to selectively purify one type of nucleic acid.

Affinity separation

In recent years, more and more purification methods have combined affinity
immobilisation of nucleic acids with magnetic separation. For instance, poly(A) +
mRNA may be bound to streptavidin-coated magnetic particles by biotin-labelled
oligo(dT) and the particle complex removed from the solution (and unbound
contaminants) with a magnet. This solid phase technique simplifies nucleic acid
purification since it can replace several steps of centrifugation, organic extraction and
phase separation with a single, rapid magnetic separation step.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 6

CTAB extraction and purification method

The cetyltrimethylammonium bromide (CTAB) protocol, which was first developed by
Murray and Thompson in 1980 (Murray and Thompson, 1980), was successively
published by Wagner and co-workers in 1987 (Wagner et al., 1987). The method is
appropriate for the extraction and purification of DNA from plants and plant derived
foodstuff and is particularly suitable for the elimination of polysaccharides and
polyphenolic compounds otherwise affecting the DNA purity and therefore quality.
This procedure has been widely applied in molecular genetics of plants and already
been tested in validation trials in order to detect GMOs (Lipp et al., 1999; 2001).
Several additional variants have been developed to adapt the method to a wide
range of raw and processed food matrices (Hupfer et al., 1998; Hotzel et al., 1999;
Meyer et al., 1997; Poms et al., 2001).

Principles of CTAB method: lysis, extraction and precipitation

Plant cells can be lysed with the ionic detergent cetyltrimethylammonium bromide
(CTAB), which forms an insoluble complex with nucleic acids in a low-salt
environment. Under these conditions, polysaccharides, phenolic compounds and
other contaminants remain in the supernatant and can be washed away. The DNA
complex is solubilised by raising the salt concentration and precipitated with ethanol
or isopropanol. In this section, the principles of these three main steps, lysis of the
cell membrane, extraction of the genomic DNA and its precipitation will be described.

Lysis of the cell membrane. As previously mentioned, the first step of the DNA
extraction is the rupture of the cell and nucleus wall. For this purpose, the
homogenised sample is first treated with the extraction buffer containing EDTA
Tris/HCl and CTAB. All biological membranes have a common overall structure
comprising lipid and protein molecules held together by non-covalent interactions.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 7

Figure 1. Simplified representation of the cell membranes1

As shown in Figure 1, the lipid molecules are arranged as a continuous double layer
in which the protein molecules are “dissolved”. The lipid molecules are constituted
by hydrophilic ends called “heads” and hydrophobic ends called “tails”. In the CTAB
method the lysis of the membrane is accomplished by the detergent (CTAB)
contained in the extraction buffer. Because of the similar composition of both the
lipids and the detergent, the CTAB component of the extraction buffer has the
function of capturing the lipids constituting the cell and nucleus membrane. The
mechanism of solubilisation of the lipids using a detergent is shown in Figure 2.

Figure 2. Lipid solubilisation

Figure 3 illustrates how, when the cell membrane is exposed to the CTAB extraction
buffer, the detergent captures the lipids and the proteins allowing the release of the
genomic DNA. In a specific salt (NaCl) concentration, the detergent forms an
insoluble complex with the nucleic acids. EDTA is a chelating component that among
other metals binds magnesium. Magnesium is a cofactor for DNase. By binding Mg
with EDTA, the activity of present DNase is decreased. Tris/HCl gives the solution a
pH buffering capacity (a low or high pH damages DNA). It is important to notice that,

1
Pictures in current and following page: "Genetic Science Learning Center, University of Utah,
http://gslc.genetics.utah.edu."

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 8

since nucleic acids can easily degrade at this stage of the purification, the time
between the homogenisation of the sample and the addition of the CTAB buffer
solution should be minimised. After the cell and the organelle membranes (such as
those around the mitochondria and chloroplasts) have been broken apart, the
purification of DNA is performed.

Figure 3: Disruption of the cellular membrane and extraction of genomic DNA

Extraction. In this step, polysaccharides, phenolic compounds, proteins and other

cell lysates dissolved in the aqueous solution are separated from the CTAB nucleic
acid complex. The elimination of the polysaccharides as well as phenolic
compounds is particularly important because of their capability to inhibit a great
number of enzymatic reactions. Under low salt concentration (< 0.5 M NaCl), the
contaminants of the nucleic acid complex do not precipitate and can be removed by
extraction of the aqueous solution with chloroform. The chloroform denatures the
proteins and facilitates the separation of the aqueous and organic phases. Normally,
the aqueous phase forms the upper phase. However, if the aqueous phase is dense
because of salt concentration (> 0.5 M), it will form the lower phase. In addition, the
nucleic acid will tend to partition into the organic phase if the pH of the aqueous
solution has not been adequately equilibrated to a value of pH 7.8 - 8.0. If needed,
the extraction with chloroform is performed two or three times in order to completely
remove the impurities from the aqueous layer. To achieve the best recovery of
nucleic acid, the organic phase may be back-extracted with an aqueous solution that
is then added to the prior extract. Once the nucleic acid complex has been purified,
the last step of the procedure, precipitation, can be accomplished.

Precipitation. In this final stage, the nucleic acid is liberated from the detergent. For
this purpose, the aqueous solution is first treated with a precipitation solution
comprising a mixture of CTAB and NaCl at elevated concentration (> 0.8 M NaCl).
The salt is needed for the formation of a nucleic acid precipitate. Sodium acetate may

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 9

be preferred over NaCl for its buffering capacity. Under these conditions, the
detergent, which is more soluble in alcohol than in water, can be washed out, while
the nucleic acid precipitates. The successive treatment with 70% ethanol allows an
additional purification, or wash, of the nucleic acid from the remaining salt.

Quantification of DNA by spectrophotometry

DNA, RNA, oligonucleotides and even mononucleotides can be measured directly in
aqueous solutions in a diluted or undiluted form measuring the absorption A (also
defined as optical density, OD) in ultraviolet light (but also in the visible range). If the
sample is pure (i.e. without significant amounts of contaminants such as proteins,
phenol or agarose), the spectrophotometric measurement of the amount of ultraviolet
irradiation absorbed by the bases is simple and accurate. For this method, aqueous
buffers with low ion concentrations (e.g. TE buffer) are ideal. The concentration of
nucleic acids is usually determined by measuring at 260 nm against a blank.
Interference by contaminants can be recognised by the calculation of a “ratio”. Since
proteins absorb at 280 nm, the ratio A260/A280 is used to estimate the purity of nucleic
acid. Pure DNA should have a ratio of approximately 1.8, whereas pure RNA should
give a value of approximately 2.0. Absorption at 230 nm reflects contamination of the
sample by substances such as carbohydrates, peptides, phenols or aromatic
compounds. In the case of pure samples, the ratio A260/A230 should be approximately
2.2.
An alternative method, the ethidium bromide agarose plate method, is useful when
only small quantities of nucleic acid are available; the amount of nucleic acid can be
estimated, when compared to a range of concentration standards, from the intensity
of fluorescence emitted by the ethidium bromide when irradiated with UV light.

Principles of spectrophotometric determination of DNA

A spectrophotometer makes use of the transmission of light through a solution to
determine the concentration of a solute within the solution. The apparatus operates
on the basis of a simple principle in which light of a known wavelength passes
through a sample and the amount of light energy transmitted is measured with a
photocell on the other side of the sample.
As shown in Figure 4, the design of the single beam spectrophotometer involves a
light source, a prism, a sample holder and a photocell. Connected to each are the
appropriate electrical or mechanical systems to control the illumination intensity, the
wavelength and for the conversion of energy received at the photocell into a voltage

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 10

fluctuation. The voltage fluctuation is then displayed on a meter scale, or is recorded

via connection to a computer for later investigation.

Figure 4. Schematic light transmission

All molecules absorb radiant energy at a specific wavelength, from which it is

possible to extrapolate the concentration of a solute within a solution. According to
the Beer-Lambert law there is a linear relationship between the absorbance A (also
called optical density, OD) and the concentration of the macromolecule given by the
following equation:

A = OD = εlc (1)

Where ε is the molar extinction coefficient, c is the concentration; and l is the

pathlength of the cuvette. Proteins and nucleic acids absorb light in the ultraviolet
range within wavelengths of between 210 and 300 nm. As previously explained, the
maximum absorbance of DNA and RNA solutions is at 260 nm whereas the
maximum absorbance of protein solutions is at 280 nm. Since, both DNA and RNA
solutions do partially absorb light at 280 nm, and protein solutions partially absorb
light at 260 nm, the ratio between the readings at 260 nm and 280 nm (A260/A280)
provides an estimate of the purity of the nucleic acids. Pure preparations of DNA and
RNA have A260/A280 values of 1.8 and 2.0 respectively. For a 10 mm pathway and a
260 nm wavelength, an absorption A = 1 corresponds to approximately 50 µg/ml of
dsDNA, approximately 37 µg/ml of ssDNA, 40 µg/ml of RNA or approximately 30
µg/ml of oligonucleotides. If there is contamination with protein, the A260/A280 will be
significantly less than the values given above and accurate quantification of the
amount of nucleic acid will not be possible. It is important to mention the fact that
impurities in DNA solutions caused by RNA cannot be confidently identified by

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 11

spectrophotometry. An absorbance of 325 nm can be used to indicate the presence

of debris in the solution or that the cuvette itself is dirty.

Determination of the concentration of nucleic acids

Choice of the cuvette. The amount of nucleic acid solution used for the
measurement of the absorbance A, depends on the capacity of the cuvette. A
suitable cuvette should be chosen depending on sample concentration range, dilution
factor and available sample volume. In most of the procedures used for the detection
of GMOs the volume of genomic DNA collected is between 50 and 100 µl. Several
types of microvolume cuvettes with a capacity of 5 to 70 µl are utilised for the
spectroscopic quantification of small volumes of nucleic acids.

Set up. In order to calibrate the spectrophotometer, it is important:

• to set the correct cell pathlength
• to set the correct factor (select between dsDNA, ssDNA, RNA)
• to measure a blank solution (set reference) constituted by either water or a buffer
solution (A260 = 0)
• to ensure that the set reference is renewed periodically
• to measure a known amount of pure nucleic acid in order to check the reliability of
set reference

Measurement of an unknown sample. Depending on the capacity of the cuvette

used, specific amounts of DNA solution are used for the concentration evaluation
(e.g. for cuvette of capacity lower than 0.2 ml, 5 µl of DNA is diluted in 195 µl of
water). After calibrating the spectrophotometer and the addition of the nucleic acid
solution, the cuvette is capped, the solution mixed, and the absorbance measured. In
order to reduce pipetting errors, the measurement should be repeated at least twice
and at least 5 µl of the DNA solution should always be used. A260 readings lower
than 0.02 or between 1 and 1.5 (depending on the instrument used) are not
recommended because of the possibility of a high margin of error.
The concentration c of a specific nucleic acid present in a solution is calculated using
the following equations:
• Single-stranded DNA: c(pmol/µl) = A260/0.027
• Double-stranded DNA: c(pmol/µl) = A260/0.020
• Single-stranded RNA: c(pmol/µl) = A260/0.025
• Oligonucleotide: c(pmol/µl) = A260100/1.5NA+0.71NC+1.20NG + 0.84NT

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 12

where A260 is the absorbance measured at 260 nm.

An example of absorbance readings of highly purified calf thymus DNA suspended in

1x TNE buffer assuming that the reference DNA is dsDNA with A260 = 1 for 50 µg/ml
in a 10 mm pathlength cuvette is shown in Table 2. The concentration of DNA was
nominally 25 µg/ml.

Table 2. Absorbance reading of highly purified calf thymus DNA in 1x TNE buffer

Wavelength Absorbance A260/A280 Conc. (µg/ml)

325 0.01 - -
280 0.28 - -
260 0.56 2.0 28
230 0.30 - -

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 13

Experimental
Equipment

REMARK
All of the equipment used must be sterilised prior to use and any residue of DNA
must be removed. In order to avoid contamination, barrier pipette tips that are
protected against aerosol should be used.

• Instruments for size reduction like a sterile surgical blade or a mortar

• Water bath or heating block
• Microcentrifuge
• Micropipettes
• Vortex mixer
• 1.5 ml microcentrifuge tubes
• Weigh boats or equivalents
• Spatulas
• Balance capable of 0.01 g measurement
• Loops
• Rack for microcentrifuge tubes
• Optional: vacuum desiccator to dry DNA pellets

Reagents

REMARK
All chemicals should be of molecular biology grade. Deionised water and buffers
should be autoclaved prior to use. In addition all chemicals should be DNA and
DNase free.

• Cetyltrimethylammonium bromide (CTAB) CAS 124-03-8

• Chloroform
• Isopropanol
• Na2EDTA CAS 6381-92-6
• Ethanol
• NaCl

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 14

• Proteinase K
• RNase A
• Tris[hydroxymethyl] aminomethane hydrochloride (Tris-HCl)
• Sterile deionised water

CTAB-buffer

20 g/l CTAB 4g
1.4 M NaCl 16.4 g
0.1 M Tris-HCl 3.15 g
20 mM Na2EDTA 1.5 g
• add 100 ml of deionised water
• adjust pH to a value of 8.0 with 1M NaOH
• fill up to 200 ml and autoclave
• store buffer at 4°C for max. 6 months

CTAB-precipitation solution

5 g/l CTAB 1g
0.04 M NaCl 0.5 g
• add 100 ml of deionised water
• adjust pH to a value of 8.0 with 1 M NaOH
• fill up to 200 ml and autoclave
• store solution at 4°C for max. 6 months

NaCl 1.2 M

• dissolve 7.0 g of NaCl in 100 ml deionised water

• autoclave and store at room temperature

Ethanol-solution 70 % (v/v)

70 ml of pure ethanol are mixed with 30 ml of sterile deionised water.

RNase A 10 mg/ml store at –20°C

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 15

Proteinase K 20 mg/ml store at –20°C

Procedure

The procedure requires sterile conditions. Contamination may be avoided during

sample preparation by using single-use equipment, decontamination solutions and by
avoiding the formation of dust.

• transfer 100 mg of a homogeneous sample into a sterile 1.5 ml microcentrifuge

tube
• add 300 µl of sterile deionised water, mix with a loop
• add 500 µl of CTAB-buffer, mix with a loop
*
• Add 20 µl Proteinase K (20 mg/ml), shake and incubate at 65°C for 30-90 min
*
• Add 20 µl RNase A (10 mg/ml), shake and incubate at 65°C for 5-10 min

• centrifuge for 10 min at about 16,000 xg

• transfer supernatant to a microcentrifuge tube containing 500 µl chloroform,
shake for 30 sec
• centrifuge for 10 min at 16,000 xg until phase separation occurs
• transfer 500 µl of upper layer into a new microcentrifuge tube containing 500 µl
chloroform, shake
• centrifuge for 5 min at 16,000 xg
• transfer upper layer to a new microcentrifuge tube
• add 2 volumes of CTAB precipitation solution, mix by pipetting
• incubate for 60 min at room temperature
• centrifuge for 5 min at 16,000 xg
• discard supernatant
• dissolve precipitate in 350 µl NaCl (1.2 M)
• add 350 µl chloroform and shake for 30 sec
• centrifuge for 10 min at 16,000 xg until phase separation occurs
• transfer upper layer to a new microcentrifuge tube
• add 0.6 volumes of isopropanol, shake

*
These additional optional steps are now commonly introduced to the CTAB extraction
method to enhance the yield of genomic DNA from highly complex matrices.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 16

• centrifuge for 10 min at 16,000 xg

• discard the supernatant
• add 500 µl of 70% ethanol solution and shake carefully
• centrifuge for 10 min at 16,000 xg
• discard supernatant
• dry pellets and re-dissolve DNA in 100 µl sterile deionised water

The DNA solution may be stored in a refrigerator for a maximum of two weeks, or in
the freezer at - 20°C for longer periods.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 17

References
Hotzel, H., Müller, W. and Sachse, K. (1999). Recovery and characterization of
residual DNA from beer as a prerequisite for the detection of genetically modified
ingredients. European Food Research Technology 209, 192-196.

Hupfer, C., Hotzel, H., Sachse, K. and Engel, K.H. (1998). Detection of the genetic
modification in heat-treated products of Bt maize by polymerase chain reaction.
Zeitschrift für Lebensmittel-Untersuchung und -Forschung A 206, 203-207.

Lipp, M., Bluth, A., Eyquem, F., Kruse, L., Schimmel, H., Van den Eede, G. and
Anklam, E. (2001). Validation of a method based on polymerase chain reaction
for the detection of genetically modified organisms in various processed
foodstuffs. European Food Research Technology 212, 497-504.

Lipp, M., Brodmann, P., Pietsch, K., Pauwels, J. and Anklam, E. (1999). IUPAC
collaborative trial study of a method to detect genetically modified soy beans and
maize in dried powder. Journal of AOAC International 82, 923–928.

Meyer, R. and Jaccaud, E. (1997). Detection of genetically modified soya in

processed food products: development and validation of PCR assay for the
specific detection of glyphosate-tolerant soybeans. In Amadò, R. Battaglia
(Eds.). Proceedings of the ninth European conference on food chemistry (Vol. 1).
Authenticity and adulteration of food-the analytical approach. 24-26 September
1997. Interlaken 1, 23-28. ISBN : 3-9521414-0-2.

Murray, M.G. and Thompson, W.F. (1980). Rapid isolation of high molecular weight
plant DNA. Nucleic Acids Research 8, 4321–4325.

Poms, R.E., Glössl, J. and Foissy, H. (2001). Increased sensitivity for detection of
specific target DNA in milk by concentration in milk fat. European Food Research
Technology 213, 361-365.

Wagner, D.B., Furnier, G.R., Saghay-Maroof, M.A., Williams, S.M., Dancik, B.P. and
Allard, R.W. (1987). Chloroplast DNA polymorphisms in lodgepole and jack pines

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4
Extraction and Purification of DNA 18

and their hybrids. Proceedings of the National Academy of Science USA 84,
2097–2100.

Zimmermann, A., Lüthy, J. and Pauli, U. (1998). Quantitative and qualitative

evaluation of nine different extraction methods for nucleic acids on soya bean
food samples. Zeitschrift für Lebensmittel-Untersuchung und -Forschung A 207,
81–90.

The Analysis of Food Samples for the Presence of Genetically Modified Organisms Session 4