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Development of Conditions for Comet Assay Application in Forensic Investigation of Rape and Other Sexual Assaults

  • Institute of Molecular Biology Bulgarian Academy of Sceinces

Abstract and Figures

The problem of personal identity in rape cases is solved by DNA analysis as a standard procedure in forensic laboratories. However, fixing 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. Modification 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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Keywords: forensic, Comet assay, spermatozoa
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 prole
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
Specic Antigen (2). The next step is to carry out DNA
proling on the evidential material e.g. swab.
It is tempting to assume that there is a signicant 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
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 conrm 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.
R. Miteva
, M. Georgieva
, E. Peycheva
, T. Efremov
, G. Miloshev
Trakia University, Faculty of Medicine, Forensic Medicine and Deontology, Department of General and Clinical Pathoanatomy,
Stara Zagora, Bulgaria
Bulgarian Academy of Sciences, Institute of Molecular Biology “Roumen Tsanev”, Laboratory of Molecular Genetics, Soa,
Correspondence to: George Miloshev
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. Modication 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.
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
C until the time of the experiment. Spermatozoa cells were
extracted from dried cloths by soaking for 5 min at 37
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
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 specic
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
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
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 modications 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
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.
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... Furthermore, Miteva et al. (2009) exhibited the forensic application of SCGE technique in sexual assault cases. This study revealed that SCGE help to measure the kinetics of sperm DNA degradation which help to estimate the time since deposition of sperm or time of ejaculation of sperm. ...
... This study revealed that SCGE help to measure the kinetics of sperm DNA degradation which help to estimate the time since deposition of sperm or time of ejaculation of sperm. With the help of this study, Miteva et al. (2009) elucidated that SCGE could be a valuable technique which may aid to the forensic investigation especially in the sexual assault cases. This study accurately measured the time since deposition/ejaculation of sperm qualitatively and quantitatively both. ...
... Existing literatures have demonstrated that Single Cell Gel Electrophoresis assay play an important role in estimation of time of sperm ejaculation in rape and other sexual assaults cases by analyzing degradation of DNA content in sperm cells (Miteva et al. 2009). In this study, results revealed that DNA degradation rate was increased with time since deposition of stain. ...
Full-text available
Time since death (TSD) estimation is a crucial issue in death investigation. In majority of homicide/suicide cases deceased body recovered within the first 48 h, therefore, it is critically important to determine the time of death quickly and precisely. In the present scenario, TSD estimation still remains difficult even for experienced pathologists undertaken with extreme caution. After death numerous factors may be of cadaveric or environmental origin can influence the ‘normal’ rate of postmortem changes. Therefore, it could be more difficult to estimate longer TSD. However, these environmental influencing factors can also effect on DNA in the form of degradation, which start after 1–2 h of death. This DNA degradation could be work as molecular clock which help to estimate early TSD. Single-cell gel electrophoresis (SCGE), also known as the Comet assay, is the technique by which DNA degradation can be visualized and measured qualitatively as well as quantitatively. Using this assay not only early TSD estimated but many other challenges such as time since deposition of biological fluids, repair of genetic material from degraded biological sample could also be resolved. With the help of this paper an attempt was made to introduce a well-known cytogenetic technique that is SCGE assay, which could be a versatile technique for forensic science.
... In this sequence, time since estimation of a rape/ sexual assault case has been a persistent problem [17]. In sexual assault cases, dried seminal fluid, which contains spermatozoa, is usually found on victim's cloth. ...
... In addition, the idea of DNA fragmentation correlated with PMI estimation is now new in forensic investigation. With this hypothesis, Miteva et al. [17] have shown the fragmentation pattern of sperm DNA using comet assay at different time points of deposition. The results revealed that fragmentation of sperm DNA induced with prolonged time since deposition of stain as evident by comet assay parameter. ...
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Postmortem interval (PMI) estimation is a recurring problem in the field of forensic medicine. Conventional methods are effective but are insufficient to estimate accurate and precise time of death or PMI. In addition, degradation of biological samples is another major problem in forensic science which affects the investigation process and misleads the result. Some previous studies reported that DNA fragmentation has strong correlation with PMI. DNA fragmentation increased with prolonged PMI. Comet assay is a rapid sensitive, versatile, reliable and cost effective technique that is specifically used for qualitative and quantitative estimation of nuclear DNA fragmentation. Due to this attribute, comet assay can help to estimate accurate and precise time of death for some extent that is for early PMI estimation. In addition, two confounding factors are responsible for DNA fragmentation: (1) micro-organism; (2) environmental condition. Here, comet assay plays a dual role: (1) partially degraded samples get repaired using repair enzyme; (2) accurate time since deposition can be measured without using repair enzyme. Furthermore, this assay can also help to identify potential exposures of environmental-released chemicals/toxicants and its deleterious effects on human population. In this way, comet assay shows its versatile applications that could be useful for forensic investigation. Therefore, with the help of this review, an attempt was made to explore the versatility of comet assay technique for forensic applications and its future perspective.
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About 20 years ago, DNA sequences were separately described from the quagga (a type of zebra) and an ancient Egyptian individual. What made these DNA sequences exceptional was that they were derived from 140- and 2400-year-old specimens. However, ancient DNA research, defined broadly as the retrieval of DNA sequences from museum specimens, archaeological finds, fossil remains, and other unusual sources of DNA, only really became feasible with the advent of techniques for the enzymatic amplification of specific DNA sequences. Today, reports of analyses of specimens hundreds, thousands, and even millions of years old are almost commonplace. But can all these results be believed? In this paper, we critically assess the state of ancient DNA research. In particular, we discuss the precautions and criteria necessary to ascertain to the greatest extent possible that results represent authentic ancient DNA sequences. We also highlight some significant results and areas of promising future research.
Objective: The objective of this review was to summarize application of DNA polymorphisms to individual identification. Design: A review of forensic medicine. Materials and Methods: As a powerful evidence, biological markers are not only widely used in criminal investigation, but also in clinical tissues identification such as pathological specimen mix-up as well as mass disasters and accidents such as airline crashes, train crashes, floods and building fires. While several methods are being used for human individual identification, the biological method is more important because the identification tests are not restricted to any particular or one body landmark. Furthermore, comparison of biological markers can be used to associate separated remains or body parts. Results: Polymerase chain reaction (PCR) commonly used in DNA polymorphisms has been developed to realize genetic analysis of multiplex loci employing fluorescent dye labeling technology. The genetic evidence provided by DNA polymorphic markers from the analyses of restriction fragment length polymorphisms (RFLPs), minisatellite (VNTR) polymorphisms, microsatellite (STR) polymorphisms, single nucleotide polymorphisms (SNPs) and mitochondrial DNA (mtDNA) has become the most powerful tool for individual identification in natural disasters and non-natural catastrophes, or criminal cases. Conclusion: DNA analysis is a highly discriminating tool that permits identification of remains, provided that a corresponding reference sample is available, either from biologically related family members or from the missing and unidentified person themselves. DNA markers have advantages over the traditional biological markers including the red blood cell group, serum protein types, isoenzyme types, HLA types and salivary protein types in several aspects: DNA markers are simply to analysis, require small amount of template for PCR amplification, can be used for the analysis of some degraded samples and have high power of discrimination. STR polymorphisms have become the major and widely used method for human identification tests both in criminal investigation and mass disaster victim identification.
To explore the association between semen parameters and sperm DNA damage. Cross-sectional. Andrology clinic. Two hundred fifty-seven men undergoing infertility assessment. None. Sperm concentration and motility were measured using computer-assisted sperm analysis; morphology was scored using the strict criteria. The neutral comet assay was used to measure sperm DNA damage. Comet assay parameters included comet extent, percent DNA in the comet tail, and tail distributed moment, an integrated measure of length and intensity. We also scored cells that were too long to measure (>300 microm), which we referred to as cells with high DNA damage. Men older than 35 years had a statistically significant increase in the number of cells with high DNA damage as compared with younger men. In age-adjusted regression analyses, the most consistent associations were found between semen parameters and the number of cells with high DNA damage. For an interquartile range change in the number of cells with high DNA damage, sperm concentration declined 14.2 x 10(6)/mL, motility declined 4.3%, and morphology declined 0.5%. Comet extent and percent DNA in the comet tail were also associated with a decline in sperm concentration and motility, respectively. Although there were associations between semen and comet assay parameters, their magnitudes were weak, suggesting that the comet assay provides additional independent information on sperm function.
  • J Tie
  • S Uchigasaki
  • S Oshida
Tie J., Uchigasaki S., Oshida S. (2008) International Medical J., 5, 307-314.
  • M J Saks
  • J J Koehler
Saks M.J, Koehler J.J. (2005) Science, 09, 893-895.