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BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/1
Keywords: forensic, Comet assay, spermatozoa
Introduction
In recent years the techniques for DNA analysis have improved
in terms of sensitivity (4, 5). DNA analysis in the forensic
medicine, aiming to determine one’s identity has developed
with an extremely fast rate. The
advances of forensic DNA
testing methodologies led to requirement of extremely smaller
amounts of genetic material in order to produce a given prole
and it is relatively easy to nd identity in rape cases through
analysis of semen material. However, a
persistent problem
that has long plagued forensic pathologists has been the
determination of the time since the rape. The idea of using the
decay of macromolecules in forensic science is not new. DNA
is relatively stable molecule. Quite surprisingly, fragments
of DNA, suitable for analysis, could be preserved for up to
100 000 years (1, 3). Therefore, DNA may be preserved from
degradation at least in some cells for quite long periods of time.
It was shown that DNA degradation could be used in forensic
science for the assessment of the time of death (2). In sexual
assault cases dried semen uid, which usually can be found
on cloth, includes spermatozoa. One of the rst steps in the
investigative process in rape cases is to identify the presence
of spermatozoa. The process of proving the presence of semen
liquid in dried ndings, following the rules of forensics’
practice, is done most often by microscopic search for whole
spermatozoids or through looking for distinctive ingredients of
the sperm like the presence of the Y chromosome or Prostate
Specic Antigen (2). The next step is to carry out DNA
proling on the evidential material e.g. swab.
It is tempting to assume that there is a signicant association
between DNA degradation and the time since the intercourse.
Moreover, in the sperm DNA is compacted more tightly by
protamines in the spermatozoid heads. This tight compaction
presumably leads to higher resistance of DNA to the action of
nucleases and genotoxins. In addition, less enzymatic activities
have been found in the head of spermatozoa. Some degradation
of DNA in the spermatozoa does exist and has been assessed in
several studies. It was shown that degradation could be initiated
by stress conditions or by changes in chromatin structure (6, 7).
As DNA degradation in spermatozoa unavoidably takes place,
it is of interest to make a kinetic analysis of this degradation.
Such information could be very useful in the eld of forensic
science.
In order to understand better the process of sperm DNA
degradation along the time we designed our own conditions for
Comet assay. By using these conditions we were able to pursue
the process not only in the semen but also in dried on a piece
of cloth spermatozoa.
Materials and methods
All reagents were purchased from the Sigma-Aldrich company
unless stated otherwise.
Semen collection
Samples from sperm ejaculates of healthy, donors have been
collected in sterile tubes. Spermograms were performed
on each sample in order to conrm normozoospermia of all
individual semen liquids.
Sample preparation and manipulation
100 μl of cell sperm ejaculates were dropped on 3x3 cm clean
cotton cloths. The last were stored at room temperature for the
time-course studying.
DEVELOPMENT OF CONDITIONS FOR COMET ASSAY APPLICATION IN
FORENSIC INVESTIGATION OF RAPE AND OTHER SEXUAL ASSAULTS
R. Miteva
1
, M. Georgieva
2
, E. Peycheva
2
, T. Efremov
2
, G. Miloshev
2
Trakia University, Faculty of Medicine, Forensic Medicine and Deontology, Department of General and Clinical Pathoanatomy,
Stara Zagora, Bulgaria
1
Bulgarian Academy of Sciences, Institute of Molecular Biology “Roumen Tsanev”, Laboratory of Molecular Genetics, Soa,
Bulgaria
2
Correspondence to: George Miloshev
E-mail: miloshev@bio21.bas.bg
ABSTRACT
The problem of personal identity in rape cases is solved by DNA analysis as a standard procedure in forensic laboratories.
However, xing of the exact time of the sexual abuse is still an unsolved issue in the forensic science. Here, we present our
attempt to apply the method of Comet assay to measure the kinetics of sperm DNA degradation. Modication of the procedure
has enabled us to obtain a good correlation between time of ejaculation and the time of test performance. We see this work as
a proof of the principle that sperm DNA degradation could be used as a molecular clock for better estimation of the time when
rape has occurred. A vast database built from the work of many laboratories is required in order such a phenomenon to be used
in practice by forensic scientists.
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BIOTECHNOL. & BIOTECHNOL. EQ. 23/2009/1
Comet assay on spermatozoa
Liquid aliquots of 20 μl of the same ejaculates were preserved
in 1.5 μl eppendorf tubes as control probes for assessing the
DNA degradation in semen. Liquid probes were stored at 4
o
C until the time of the experiment. Spermatozoa cells were
extracted from dried cloths by soaking for 5 min at 37
o
C
and then subsequent washings with 200 μl of PBS buffer.
Spermatozoa cells were centrifuged at 3500 rpm/min and next,
resuspended in 1 ml of PBS.
The sperm suspesion (approximately 2 -10
5
sperm cells)
was mixed with 0.7% agarose and dropped on microscopic
slides, precoated with agarose. The slides were transferred
to two different solutions for enzyme treatment for specic
time periods, crucial steps for decondensing sperm chromatin
and allowing migration of broken DNA out of the nucleus.
Slides were electrophoresed under neutral conditions at 9
V and 130 mA for 10 minutes at 10
o
C. After that the slides
were air-dried. The comets in the gel were stained with SYBR
green I (Molecular Probes) and visualized under uorescent
microscope Leitz (Orthoplan, VARIO ORTHOMAT 2) using
450-490 nm bandpass lter. Pictures were taken with a build-
in microscope photo camera.
Results and Discussion
The estimation of the time since rape or other sexual assaults is
a problem in the forensic investigation. An intriguing question
is whether kinetics of DNA degradation in the spermatozoa
could be a “molecular clock” for an exact estimation of the
time since the ejaculation. We placed drops (100 μl each)
of semen taken from healthy donors on 5 pieces of cotton
cloths for each individual. In order to imitate a real situation
the pieces of cloths were kept in paper envelops at room
temperature without additional precautions for preservation of
the spermatozoa. As controls part of the semen uid was kept
in tubes in a fridge at 4
o
C. On the second, forth, seventh and
tenth day after sample collection spermatozoids were washed
from cloths in a tube (see Material and Methods) and were
subjected to Comet assay.
Fig. 1. Comet assay on spermatozoa cells: A – sopermatozoa cells; B –
Spermoplast; C – Comet from spermatozoid.
Examples of comets obtained by us from spermatozoa are
presented on Fig. 1. Wang et al. and Trisini et al. have been
studied the degradation of DNA in spermatozoa. These authors,
however, did not follow the degradation over a time period (6,
7). It has to be noted that by our modications of the method
we obtained comets from spermatozoa resembling those of
somatic cells (Fig. 1D). After the enzymatic disruption of the
heads of the spermatozoids (Fig.1 A) we obtained objects with
spread chromatin (Fig. 1B). By analogy with spheroplasts (yeast
cells without cell wall) we called such objects spermoplasts. In
these objects DNA has not been degraded. Spermatozoid with
partially degraded DNA (comet) is shown on Fig. 1C
.
On Fig. 2 an example of the kinetics of DNA degradation
in liquid probes and from dry probes is present. The number
of comets from spermatozoa is presented as a percentage from
the whole objects counted (spermoplasts and comets). As can
be seen from the gure DNA is subjected to degradation both
in the liquid and dried probes from the cloths. It has to be
noted that, although, for each individual the kinetics of DNA
degradation was different, there was always an increase of
DNA degradation with time.
Fig. 2. Kinetics of DNA degradation in spermatozoa cells revealed by Comet
assay.
These results, although promising, are preliminary and
represent only a proof-of-principle that DNA degradation
could be a “molecular clock” in crime investigations. Results
from many groups and laboratories obtaining huge database
are required in order such a phenomenon to be used in practice
by forensic scientists.
Conclusions
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