THE COMPARISON OF FUNCTIONALITY
OF THE METHODS USED FOR THE ASSESSMENT
OF DNA SAMPLES QUALITY
Joanna Dudczak*1, Marta Ligaj2
Poznan University of Economics and Business, Faculty of Commodity Science,
1
Department of Commodity Science and Ecology of Industrial Products,
2
Department of Natural Science and Quality Assurance,
* Corresponding author, Al. Niepodległości 10, 61-875 Poznań, Poland,
[email protected]
Abstract: Genetic material is the basis for the functioning and development
of all life forms. Despite the general stability of its structure, DNA is sensitive
to external factors exposure which can cause dangerous for the organism irreversible
changes. The monitoring of those changes is difficult, but is essential, because
it allows the study of the influence of given factors on DNA damages and assess
the quality of nucleic acids samples before their further analysis. The latter question
is the subject of the presented research. Although there have been many methods
of DNA examination and isolation developed so far, there are no evidence that they
are appropriate. The reliable results of the assessment of the quality of DNA samples
can be gathered only when using techniques which won’t cause DNA damage.
Therefore, it is significant to use methods which can make it possible to control DNA
samples condition in an easy and quick way.
In the research there have been applied simultaneously spectrophotometric,
electrophoretic and electrochemical methods to compare their effectiveness
in the assessment of the DNA samples quality. On the basis of the measurements
it has been observed that the most effective and most sensitive technique used
to examine DNA structure damage is SWV (Square Wave Voltammetry). All samples
which have been improperly stored or exposed to induced damage resulted
from too high temperature, revealed visible irregularities. Electrophoretic methods
allowed only to assess the degree of DNA fragmentation thread and may come
as complement to SWV. Spectrophotometric methods might be used to rate
232
of the purity and concentration of nucleic acids samples, but they are no useful
in the assessing of its damage: the samples characterized by the right values
of absorbance showed irregularities visible on the voltammograms obtained thanks
to SWV.
Keywords: DNA sample quality, DNA damage detection strategies, SWV.
1. INTRODUCTION
The stability of DNA structure is not durable enough to make
it immutable. There are many factors, both in the surrounding and inside
of the organism, which may lead to changes in DNA. Those changes are called
damages and may lead to amendments in the functioning of genetic material.
Living organisms have a repairing system and changes mentioned before
are removed [Hoeijmakers 2001, Liu 2006]. There are not many unrepaired
places left and generally they do not have a big influence on the functioning
of the organism.
If many errors occur during the transcription, an incorrectly code RNA
thread can be synthesized, and if such a situation takes place during
the replication process, it can lead to apoptosis or to the development of cancer
or genetic diseases [Wang and Huang 2005, Allison 2007, Jun 2010].
Not all from these changes induce negative effects, some of them can
be preserved and may lead to biological evolution.
The genetic material may also be subject to damage during isolation
and storage, so that nucleic acids samples need a protection and control before
further research [Słomski 2008]. DNA damages can be caused by physical
and chemical factors. When it comes to the former ones you can list for
example high temperature, ionizing and UV radiation. Higher temperature may
lead to cleavage of N-glycosidic bonds between base and carbohydrate.
As a result, there is a place in the structure deprived of the base named abasic
233
sites. The height of temperature is essential, literature data show that DNA
denaturation temperature is 85°C, but first abnormalities in DNA occur already
in 55°C [Yan, Iwasaki 2003].
Another essential factor which has an influence on the genetic material
structure is ionizing radiation which affects a molecule in a direct way by
triggering base ionization or radiolysis of water molecules in a cell. What is
the most frequent, nevertheless, is that the ionizing radiation stimulates the
creation of free radicals and this is, so called, the indirect effect [Bartosz 2004,
Czajka 2006].
UV radiation is another factor that affects DNA structure. Under its
influence, pyrimidine dimers – thymidine or thymine with cytosine are created.
The photodimers go into the replication cycle and they become so called
footprints which indicate the direct cell exposure to the UV radiation
[Wolnicka-Głubisz and Płonka 2007]. The proper spatial and chemical genetic
material structure is essential to create complementary pairs of bases.
Any modification of chemical groups which are part of nitrogen bases may
lead to damages. Each base has nucleophile centers exposed to endogenous
factors. Those centers are rich in electrons and that’s why they react
with electrophiles which revealed an electron shortage. The most frequently
observed reactions are: alkylation, hydrolysis and oxidation [Wyatt and Pitman
2006, Zhang and Yang 2007]. Alkylation is a process of moving the alkyl
group. Its effect depends on the kind of the group which was added
and on the place where it was built in. When it comes to nitric bases,
the most preferred is the N7 position of the guanine ring because it shows
the biggest nucleophilic potential. The N7 alkylation may lead to the formation
of cross-links, brake of DNA strand, removal/insertion of nucleotides
and the block of replication process [Debont and Laerbeke 2004, Drablos et al.
2004, Yang and Garcia 2009]. Hydrolytic deamination is related
234
to the separation of amino group with the liberation of ammonia in the presence
of water. This reaction may go along with the change of amino group into
ketone group in cyclical compounds. All the changes resulted from hydrolytic
deamination cause spot mutations [Sun, Li and Li 2005, Vongchampa et al.
2005]. Oxidation takes place when oxygen joins other compounds.
These reactions are triggered by reactive oxygen forms which join mostly
carbon. They may cause the detachment of hydrogen from methyl group
in C5 carbon of nitric bases rings. Oxidation causes modifications mostly
of bases but also pentose’s what results in DNA brake [Tuteja et al. 2009].
The discovery of the damage of DNA structure is an essential
part of many research. In this work, in order to measure of DNA damages were
used three analytical methods. Spectrophotometry is a simple instrumental
technique that utilizes electronic transient phenomena in the compounds
resulted from the absorption of electromagnetic radiation. The measurements
of light absorption, reflection and emission are taken directly. Thanks to this
method it is possible to find and mark numerous substances of organic
and inorganic origin. The spectrophotometric methods are characterized
by high sensibility, precision and selectivity [Szczepaniak 2002, Keer
and Brich 2008]. Another method which allows controlling the quality
of samples of isolated genetic material is electrophoresis. It’s one of the most
popular techniques of compounds separation and it lets us reach a high level
of resolution. The term electrophoresis is used to describe the movements
of ions and charged particles in the electric field. It’s an electrokinetic
phenomenon which causes the movement of particles under the influence
of electromagnetic field [Jun 2010]. The velocity of its movement depends
on the charge, shape, size and environmental resistance. Using the above
mentioned relations it is possible to quickly separate different macromolecules.
The application of electric field in case of aqueous solution provokes
235
the movement of both ions in the electrolyte and macroions – directed
to electrode with the sign opposite to particle charge. The assessment
of the nucleic acid samples quality was made also with the usage
of electrochemical methods. Electrochemistry is a branch of instrumental
analysis which makes observation and interpretation of chemical and physical
phenomena taking place on the electrode – electrolyte interface. Changes
occurring there can be related to electrical layer and the con-version
of electrical energy into chemical energy and vice versa [Rogowska 2007].
Several groups can be distinguished among those methods. This paper focuses
on voltammetry techniques which were used to carry out the presented
research. Voltammetry is based on the measurement of amperage which
is dependent on the application of the potential controlled by the potentiostat.
In the squarewave voltammetric technique, the current at a working electrode
is measured while the potential between the reference electrode and a working
one is swept linearly in time. The potential waveform can be viewed
as a superposition of regular squarewave onto an underlying staircase.
The current is sampled at two times, once at the end of the forward potential
pulse and again at the end of backward potential pulse. As a result of having
current sampling at two different instances per squarewave cycle, both have
diagnostic value, and therefore are preserved. The measurement is registered
and creates the curve called voltammogram [Qiu, Qu 2011]. The voltammetry
techniques can differ in terms of the program of polarizing voltage gradation
and the methods of signal measurement. It is common to use sensitive pulsating
techniques among which the SWV (Square Wave Voltammetry) is the most
frequently applied. The wave of the applied potential is stepwise and is
composed of pulses generated in short intervals. The effect of the measurement
is showed by the resultant of positive and negative current which
is the consequence of oxidation and reduction of the analyzed compounds.
236
The SWV measurement is very sensitive, quick and inexpensive [Diculescu et
al. 2005, Kumari et al. 2008].
2. MATERIALS AND METHODS
The genomic DNA was isolated from liver tissue and Escherichia coli
cells. DNA from a liver was isolated by salting out method [Miller, Dykes
and Polesky 1988]. Bacterial DNA was extracted by boiling: 200 μl
of overnight culture. It was centrifuged at 12000 rpm for 5 min, the pellet
was resuspended in 500 μl of sterile water and centrifuged as previously.
The pellet was resuspended again in 50 μl of sterile water, boiled at 100°C
for 5 min, cooled in ice and centrifuged as previously. The supernatant
was treated as the source of DNA. During the research also were used dsDNA
from
salmon
sperm
(Fluka),
dsDNA
from
calf
thymus
(Sigma)
and oligonucleotide at the sequence: 5’GTCAACTTCCGTACCGAGC
(Tib MolBiol, Poznań). The nucleic acids samples were improperly stored
for given different time and then examined with the use of spectrophotometric,
electrophoretic and electrochemical methods.
The analytical background used during electrochemical measurements
was 0.05 M phosphate buffer with 0.01 M KCl (pH 7.0). The voltammetry
measurements were taken with the potentiostat AUTOLAB PGSTAT 12
provided with the software GPES 4.9. The cell contains 3 electrodes:
the working electrode made of carbon paste (CPE), the reference electrode
(Ag/AgCl, 3 M KCl) and the auxiliary electrode (a platinum wire).
SWV measurements were carried out after the adsorption of DNA samples
on the working electrode supported by the potential. The following procedure
was applied: conditioning of the CPE (1.7 V, 60 s), immobilization of nucleic
acid (0.5 V, 120 s), rinsing (30 s) and SWV measurement. The experiment
was performed in a volume of 1 ml with continuous stirring (200 rpm).
237
The SWV parameters were: amplitude of 40 mV, step potential of 15 mV
and frequency of 50 Hz. The rest of the research was done with horizontal
electrophoresis apparatus (Biometra) and the spectrophotometer Heλios.
3. RESULTS AND DISCUSSION
One of the most frequently employed methods for the assessment
of nucleic acids purity and concentration is the spectrophotometric analysis.
It is used because nucleic acids reveal the maximum light absorption
at a wavelength of 260 nm. DNA samples may be polluted by the proteins
with the maximum light absorption at a wavelength of 280 nm or by small
compounds (such as salts, SDS or phenol) which absorb light with shorter
wavelengths the most intensively. To assess the purity of nucleic acids samples
there was a light absorption measurement taken at a wavelength of 230, 260
and 280 nm. On the basis of the measurements are calculated the absorbance
relations of A230/A260 and A260/A280. The first one should not be higher
than 0.5 whereas the second one cannot be lower than 1.7 for the samples
with appropriate purity [Farkas 1993].
On the basis of the data included in the Table 1 can clearly see that
dsDNA from the calf thymus (inappropriately stored) and dsDNA isolated
from the liver are characterized by high purity level. The parameters which are
definitely worse are shown for the samples of dsDNA from the salmon sperm
and the inappropriately stored oligonucleotide.
The next applied method used to assess the DNA samples quality
was agarose gel electrophoresis. This technique allows checking of the nucleic
acids fragmentation. On the basis of the results presented in the figure 1 it can
be observed that oligonucleotide on the first path is shown as a diffuse band
which means that separated fragments have different length. It may mean
the insufficient purification after the synthesis of the oligonucleotide
238
or is a result from an inappropriate storage. The DNA isolated from E. coli
is invisible on the gel (second path), which may result from the significant
degradation of the examined material due to improper storage (1 month
at room temperature). The third path presents the salmon sperm DNA stored
for 11 years in the powdered form under refrigeration. Despite the prolonged
time of storage the sample did not undergo the total degradation, although
fragmentation of DNA strands is visible as blurring path.
Table 1. The spectrophotometric analysis of the examined nucleic acid samples
Sample
λ230
λ260
λ280
λ230/λ260
λ260/λ280
1.913
1.745
1.085
1.09
1.60
2.447
1.816
1.158
1.35
1.57
0.100
0.242
0.142
0.41
1.70
calf thymus dsDNA
0.119
0.337
0.178
0.35
1.90
dsDNA isolated from the liver
0.065
0.171
0.080
0.38
2.14
0.154
0.317
0.157
0.48
2.02
oligonucleotide improperly
stored
salmon sperm dsDNA
improperly stored
calf thymus dsDNA
improperly stored
dsDNA isolated from the liver
after purification
Source: Authors’ own study
The calf thymus DNA was dissolved in water and stored for 7 days
in the room temperature, there is a visible significant fragmentation
(Fig. 1, path 4), and we cannot see the band with homogenous length.
239
The DNA sample isolated from calf thymus is shown on the 6th path and it is
characterized by a visible band which means the lack of fragmentation
and the accurate length. The last examined sample was DNA isolated
with salting out method from the liver and it was characterized by not clearly
visible contrail and after being purified three times with the ethanol
precipitation there was the lack of contrail. The analyzed DNA was
of an appropriate length and it did not show the fragmentation, on the gel the
band with high molecular weight is visible.
Figure 1. Separation of analyzed samples of nucleic acids by electrophoresis.
From the left: path 1 - oligonucleotide (stored), path 2 – DNA isolated from E. coli,
path 3 – salmon sperm DNA (stored), path 4 – calf thymus DNA (stored),
path 5 – 100bp DNA leader (Fermentas), path 6 – calf thymus DNA (stored),
path 7 and 8 – DNA isolated from the liver. Source: Authors’ own work
The complement of the research was the analysis of samples with the use
of electrochemical method. The results of the measurements are presented
on the figures 2, 3 and 4. They clearly indicate that time and temperature
has a distinct influence on the quality of nucleic acids sample.
All the voltammograms show an additional peak in the lower potential
(+0.75 V) and some changes in signals with the maximum current
240
at the potential from +1.0 to +1.1 and +1.3 V, typical for guanine and adenine
respectively.
Figure 2. SW voltammogram of oligonucleotide stored 4 weeks at room temperature
(on the left) and dsDNA isolated from Escherichia coli stored for 1 month at room
temperature (on the right). Source: Authors’ own work
Figure. 3. SW voltammogram of salmon sperm dsDNA salmon sperm stored in an
irregular manner (11 years at +4°C) (on the left) and calf thymus dsDNA stored
correctly – solid line, and incorrectly – dashed line (on the right). Source: Authors’
own work
241
The irregularities are, nevertheless, related to the conditions of sample
storage. If it comes in the case of sample isolated from E.coli and stored
for 1 month in the room temperature, the adenine signal was invisible (Fig. 2).
Similar disorder on a square wave voltammogram revealed a calf thymus
dsDNA stored one week in the room temperature (Fig. 3) and salmon sperm
dsDNA stored for 11 years in the powdered form under refrigeration (Fig. 4).
Figure 4. SW voltammogram of dsDNA isolated from liver tissue before (solid line)
and after purification (dashed line). Source: Authors’ own work
The research revealed that the guanine characterized most stable
oxidation signal on the voltammograms. The differences in the placement
and height of this signal were dependent on the type of DNA
which was examined. Adverse changes observed on the voltammograms made
for the analyzed DNA samples stored in inappropriate conditions could have
been caused by the factors damaging the nucleic acids structure. As indicated
by the results particularly susceptible to damage during improper storage
of DNA is adenine. This resulting in loss of its signal on the voltammograms.
After analysis of damaged samples an additional peak in the lower potential
(+0.75 V) was frequently observed. The occurrence of this signal means
the damage of nucleobeses in the DNA structure [Ligaj et al. 2014].
242
4. CONCLUSIONS
The performed comparative comparison of three methods used to quality
control of the DNA samples leads to conclusions that the most precise
and sensitive one is the voltammetric analysis. In one case there was not any
DNA presence detected when the spectrophotometric and electrophoresis
techniques were used, whereas its presence was detected on the basis of SWV
signals analysis. The voltammetry measurements indicated that the usage
of SWV in nucleic acids analysis allows observing even the minor changes
in its structure which was impossible with the use of two other methods.
It was observed that both time and conditions of storage influence
on the electrochemical response of the DNA samples. The longer storage
of the samples in inappropriate temperature conditions leads to observation
of greater irregularities on the voltammograms. All of the samples improperly
stored showed, apart from the signals typical for adenine and guanine,
the presence of signal in the potential of +0.75 V, which mean the damage
of nucleobases. The electrophoretic methods let us assess only the length
of nucleic acids fragments, they can act as a good complement
of electrochemical techniques, because using them both enables to assess
the degree of bases damage and strands fragmentation. The spectrophotometric
methods, which are confirmed by the obtained results, can be used to detect
the pollution, but they are not useful for DNA damage assessment because
for the samples which showed the right absorbance parameters were visible
irregularities on the voltammograms obtained thanks to SWV.
243
REFERENCES
Allison A. L. 2007, Fundamentals of Molecular Biology (in Polish: Podstawy biologii
molekularnej), Wydawnictwo Uniwersytetu Warszawskiego.
Bartosz G. 2004, The Second Face of Oxygen. Free Radicals in Nature
(in Polish: Druga twarz tlenu. Wolne rodniki w przyrodzie), Wydawnictwo
naukowe PWN, Warszawa.
Czajka A. 2006, Oxygen Free Radicals and Body Defence Mechnisms (in Polish:
Wolne rodniki tlenowe a mechanizmy obronne organizmu), Nowiny Lekarskie,
vol. 75, issue 6, pp. 582-586.
Debont R., Laerbeke N. 2004, Endogenous DNA Damage in Humans: a Review
of Quantitative Data, Mutagenesis, vol. 19, pp. 169-185.
Drablos F., Feyzi E., Aas P., Vaagbo C., Kavli B., Bratlie M., Pena-Diaz J., Otterlei
F., Slupphaug G., Krokan H. 2004, Alkylation Damage in DNA and RNA – Repair
Mechanisms and Medical Significance, DNA Repair, vol. 3, pp. 1389-1407.
Diculescu V., Paquim A., Brett A. 2005, Electrochemical DNA Sensors for Detection
of DNA Damage, Sensors, vol. 5, pp. 377-393.
Farkas D. H. 1993, Specimen Procurement, Processing, Tracking, and Testing
by the Southern Blot, in: Farkas D. H. (red.) Molecular Biology and Pathology:
A Guidebook for Quality Control, Academic Press, San Diego, California,
pp. 51-75.
Hoeijmakers J. H. 2001, Genome Maintenance Mechanisms for Preventing Cancer,
Nature, vol. 411, pp. 366-374.
Jun S. 2010, DNA Chemical Damage and Its Detected, International Journal
of Chemistry, vol. 2, issue 2, pp. 261-265.
Keer J., Brich L. 2008, Essentials of Nucleic Acid Analysis. A Robust Approach,
RSC Publishing, Teddington.
Kumari S., Rastogi R. P., Singh K. L., Singh S. P., Sinha R. P. 2008, DNA Damage:
detection strategies, EXCLI Journal, vol. 7, pp. 44-62.
Ligaj M., Dudczak J., Mikołajczak A., Filipiak M. 2014, Electrochemical Detection
of Heat-Induced Nucleobases Damage and DNA Samples Aging, in: Chemistry
244
of Nucleic Acid Components, Institute of Organic Chemistry and Biochemistry
Academy of Sciences of the Czech Republic, Prague, pp. 314-317.
Liu D., Yu S. 2006, DNA Damage Checkpoint, Damage Repair and Genome Stability,
Journal of Genetics and Genomics, vol. 5, pp. 3-12.
Miller S. A., Dykes D. D., Polesky H. F. 1988, A Simple Salting out Procedure
for Extracting DNA from Human Nucleated Cells, Nucleic Acids Research,
vol. 16, pp. 1215.
Palecek E., Jelen F. 2005, Eletrochemistry of Nucleic Acids, in: Palacek E. (red.),
Electrochemistry of Nucleic Acids and Proteins, Elsevier, pp. 86-104.
Qiu Y., Qu X. 2011, Electrochemical Detection of DNA Damage Induced
by Acrylamide and its Metabolite AT the Graphene-ionic Liquid-Nafion Modified
Pyrolytic Graphite Electrode, Journal of Hazardous Materials, vol. 190,
pp. 480-485.
Sun S., Li I., Li Z. 2005, Radical-mediated Cytosine and 5-methylcytosine Hydrolytic
Deamination Reaction, International Journal of Quantum Chemistry, vol. 106,
pp. 1878-1884.
Słomski R. 2008, Analysis of DNA. Theory and Practise (in Polish: Analiza DNA.
Teoria i praktyka), Wydawnictwo UP, Poznań.
Szczepaniak W. 2002, Instrumental Methods in Chemical Analysis (in Polish: Metody
instrumentalne w analizie chemicznej), Wydawnictwo PWN.
Tuteja N., Ahmad P., Panda B., Tuteja R. 2009, Genotoxic Stress in Plants: Hedding
Light on DNA Damage, Repair and DNA Repair Helicases, Mutation Research,
vol. 681, pp. 134-149.
Vongchampa V., Dong M., Gingipalli L., Dedon P. 2003, Stability of 2’-deoxy
xanthosine in DNA, Nucleic Acids Research, vol. 31, pp. 1045-1051.
Wang G., Huang D. 2005, Denaturing High-performance Liquid Chromatography
and its Application in Forensic DNA Analysis, Forensic Science International,
vol. 3, pp. 34-37.
Wang Y., Li J., Lin Z. 2003, Studies on the Application of Cre/LoxP Site-specific
Recombination System on Plant Male Sterility and Hybrid Heterosis, Molecular
Plant Breeding, vol. 1, issue 4, pp. 557-558.
245
Wolnicka-Głubisz A., Płonka P. 2007, The Role of the UV Radiation
in the Pathogenesis of Melanoma (in Polish: Rola promieniowania UV w etiopatogenezie czerniaka skóry), Współczesna onkologia, vol. 11, issue 9, pp. 419-429.
Wyatt M., Pittman D. 2006, Methylating Agents and DNA Repair Responses:
Methylated Bases and Sources of Strand Breaks, Chemical Research
in Toxicology, vol. 19, pp. 1580-1594.
Yan L., Iwasaki H. 2004, Fractal Aggregation of DNA after Thermal Denaturation,
Chaos, Solitons & Fractals, vol. 20, pp. 877-881.
Yang C., Garcia K., He C. 2009, Damage Detection and Base Flipping in Direct DNA
Alkylation Repair, ChemBioChem., vol. 695, pp. 29-34.
Zhang A., Yang B., Li Z. 2007, Theoretical Study on the Hydrolytic Deamination
Reaction Mechanism of Adenine = (H2On) (n = 1-4), Journal of Molecular
Structure: THEOCHEM, vol. 819, pp. 95-101.
STRESZCZENIE
Materiał genetyczny jest podstawą funkcjonowania oraz rozwoju wszystkich
organizmów żywych. Pomimo ogólnej stabilności struktury DNA, jest wrażliwy
na działanie czynników zewnętrznych. Mogą one powodować nieodwracalne zmiany,
niebezpieczne dla organizmu. Monitorowanie tych zmian jest trudne, ale zarazem
bardzo istotne, ponieważ pozwala na sprawdzenie wpływu określonych czynników
na DNA oraz ocenę jakości kwasów nukleinowych przed dalszą analizą.
Właśnie ta kwestia jest przedmiotem poniższego opracowania. Mimo, że metod
kontroli oraz izolacji materiału genetycznego jest wiele nie ma dowodów na to,
że są one odpowiednie. Dobre wyniki oceny jakości próbek DNA można otrzymać
stosując metody, które nie powodują uszkodzenia materiału. W związku z czym
ważne jest, aby wykorzystywać technikę, którą można w łatwy i szybki sposób
kontrolować poprzez ocenę stanu próbki.
W badaniach stosowano równocześnie trzy metody pomiaru jakości próbek DNA.
Aby porównać skuteczność w ocenie jakości próbek DNA zastosowano metodę
spektrofotometryczną, elektro-foretyczną oraz elektrochemiczną. Na podstawie
246
otrzymanych wyników stwierdzono, że najbardziej efektywną i czułą techniką
stosowaną do sprawdzania uszkodzeń struktury DNA jest SWV (woltamperometria
fali prostokątnej). Wszystkie próbki, które zostały nieprawidłowo przechowywane
lub
poddane
działaniu
podwyższonej
temperatury
wykazały
widoczne
nieprawidłowości. Metody elektroforetyczne wykazały jedynie stopień fragmentacji
i mogą stanowić tylko uzupełnienie do metod SWV. Natomiast metody
elektroforetyczne mogą być wykorzystywane do oceny czystości i stężenia próbek
kwasów nukleinowych, lecz nie są przydatne do oceny uszkodzeń.
Słowa kluczowe: jakość preparatów DNA, metody wykrywania uszkodzeń DNA,
SWV
247