Increased Male Evidence Detection Rates by Utilization of DNA Markers in Forensic Rape Cases of Espírito Santo, Brazil

Abstract

Sexual assault ignores cultural barriers, social class, socioeconomic and personal limitations. Cases of rape, murder and other forms of sexual abuse have been recognized as a health problem. These cases have been targeted by forensic sciences, with the goal of developing more effective strategies for the identification and punishment of aggressors. Seminal biomarkers have already being used for detection of sperm in vaginal samples in cases of sexual violence, but in some cases biological fluids represent a mixture of serological types, making it impossible to identify the perpetrator. Since 1985, DNA has been used as a tool of genetic identification, bringing progress and helping solve many criminal and civil cases. In rape forensic investigations, the conclusive identification of semen is required to corroborate the alleged sexual crime. However, even when sperm cells are absent, various types of non-sperm cells are left by the perpetrator (epithelial cells, leukocytes, immature germ cells), representing viable sources of DNA for analysis. Aiming to identify the presence of male DNA in samples of women victims of sexual violence, we analyzed 132 sexual abuse samples. Using five specific molecular markers for the Y chromosome, it was possible to identify 19 positive samples, which were negative for SC and PSA. Primers AMEL Y, SRY D&E and DYS270 showed high score rates, sensitivity, specificity and accuracy, suggesting that this new method presents good reliability and gives new perspectives for the sample screening process, providing more information about forensic samples involving sexual crimes in the Espírito Santo State, Brazil.

Full text

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Annals of Forensic Research and Analysis
Cite this article: Stange V, Pelição FS, Mendonça JB, Pissinate JF, Barbirato C, et al. (2014) Increased Male Evidence Detection Rates by Utilization of DNA
Markers in Forensic Rape Cases of Espírito Santo, Brazil. Ann Forensic Res Anal 1(2): 1008.
*Corresponding author
Iuri Drumond Louro*, Rua Horácio Andrade de
Carvalho, 210, Ilha do Boi, Vitória, Espírito Santo, Brasil,
CEP: 29052-620; Tel: 55 27-3335-7251; E-mail:
Submitted: 22 July 2014
Accepted: 28 September 2014
Published: 30 September 2014
Copyright
© 2014 Louro et al.
OPEN ACCESS
Keywords
•Sexual crime
•Y-chromosome molecular markers
•Foreign DNA
•Rape cases
Research Article
Increased Male Evidence
Detection Rates by Utilization
of DNA Markers in Forensic
Rape Cases of Espírito Santo,
Brazil
Victor Stange1, Fabrício Souza Pelição2, Josidéia Barreto
Mendonça2, Jauber Fornaciari Pissinate2, Clara Barbirato1,
Fernando Colnago Gonçalves1, Quezia Silva Anders1, Raquel Silva
dos Reis1 and Iúri Drumond Louro1*
1Department of Núcleo de Genética Humana e Molecular, Universidade Federal do
Espírito Santo, Brazil
2Department of Laboratório de Toxicologia, Universidade Polícia Civil do Espírito
Santo, Brazil
Abstract
Sexual assault ignores cultural barriers, social class, socioeconomic and personal
limitations. Cases of rape, murder and other forms of sexual abuse have been
recognized as a health problem. These cases have been targeted by forensic sciences,
with the goal of developing more effective strategies for the identication and
punishment of aggressors. Seminal biomarkers have already being used for detection
of sperm in vaginal samples in cases of sexual violence, but in some cases biological
uids represent a mixture of serological types, making it impossible to identify the
perpetrator. Since 1985, DNA has been used as a tool of genetic identication, bringing
progress and helping solve many criminal and civil cases. In rape forensic investigations,
the conclusive identication of semen is required to corroborate the alleged sexual
crime. However, even when sperm cells are absent, various types of non-sperm cells are
left by the perpetrator (epithelial cells, leukocytes, immature germ cells), representing
viable sources of DNA for analysis. Aiming to identify the presence of male DNA in
samples of women victims of sexual violence, we analyzed 132 sexual abuse samples.
Using ve specic molecular markers for the Y chromosome, it was possible to identify
19 positive samples, which were negative for SC and PSA. Primers AMEL Y, SRY D&E
and DYS270 showed high score rates, sensitivity, specicity and accuracy, suggesting
that this new method presents good reliability and gives new perspectives for the
sample screening process, providing more information about forensic samples involving
sexual crimes in the Espírito Santo State, Brazil.
ABBREVIATIONS
AMEL : Amelogenin; APA: acid phosphatase activity; APS:
Amonium persulfate; Art.: article; AS: Autosomal; BP: base pairs;
DNA: Desoxirribonucleic acid; dNTP: Desoxirribonucleotides
trifosfate; EDTA: ethylenediaminetetracetic acid; PCR: polymerase
chain reaction; PSA:  SC: spermatoscopy;
NGHM: Núcleo de Genética Humana e Molecular (Center of
Human and Molecular Genetics); LoTE: tris/EDTA pH 8 buffer;
SDS: sodium dodecyl sulfate; STS:   Y-STR:
Y Short Tandem Repeats; STR: Short Tandem Repeats; SRY: Sex-

INTRODUCTION
         
        
assault. Victim’s medical examination and laboratory analyses
of biological samples aim to detect the perpetrator’s semen
and supply legal evidences for the criminal process. Vaginal

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Louro et al. (2014)
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Ann Forensic Res Anal 1(1): 1008 (2014) 2/4
swabs are, in these cases, the most reliable source of biological
evidence, despite the enormous variation of samples condition
and degradation degree.
     
forensic rape analyzes include sperm cytology (SC), acid
      

is a presumption test [2], whereas PSA detection represents a

investigations of rape in 1971 [4] and it has been accepted as a
       
because detectable levels may persist in the vagina up to 48
        
         
misinterpretation of results.
         
violence [6,7], however, in approximately two thirds of cases,
it is not possible to identify the semen’s donor, because mixed

Even when sperm is absent, several types of non-sperm
   
germ cells) and represent a viable source of DNA for analysis.
These cells are normally found in approximately 15% of sperm
materials [8] provided by the prostate gland and seminal vesicles
as major sources.
More recent methodologies based on detection of DNA
(autosomal and Y-chromosome short tandem repeats [AS- and
Y-STRs]) have been described for forensic purposes [9] and
         
  
       
        
world. Its utilization more than tripled from 2001 to 2010, with
       
        
worldwide [10].
With the purpose to increase perpetrator DNA detection

PSA and SC tests, when compared to DNA based tests in forensic
analysis of rape.
MATERIALS AND METHODS
PSA test and Sperm cytology
PSA and SC tests were performed by experts in the Toxicology
Laboratory of the Civil Police of Espírito Santo State. PSA scores
were determined by immunochromatographic membrane test
assay performed by One Step ABA card Imuno-Rapido PSA
       
to manufacturer’s recommendations. SC test was performed
by experienced professionals using the smear slide technic
       
were microscopically screened for spermatozoa under 100x
 
was considered positive when at least 1 sperm (usually head)

Vaginal swabs
Samples were provided by the Toxicology Laboratory of the
          
after tested by routine PSA and SC, at which point samples would
be discarded. Vaginal swabs were collected using cotton swabs,
by Forensic examiners at the Police Legal Medicine Department

       
stored at -20oC until analyzed.
DNA extraction and evaluation
       
Proteinase K and SDS treatment followed by organic phenol/
chloroform extraction and ethanol precipitation [12]. DNA from
          
buffer, 5µl was used per reaction. The Y chromosome regions
        
Personal Eppendorf) in simplex reactions, using primers
         
described by Carvalho et al., 2007[14] (table 1).
Reactions were performed using a male DNA positive control

dilutions of 1:100. Contamination controls were represented by
ultrapure water pipetted simultaneously with the test samples
        

was expected.
L of PCR product were applied to a 7%
        
at 240V for 60 to 90 minutes, silver nitrate stained according to
Sanguinnetti et al. 1994 [15] and photo documented.
Results interpretation, sensitivity and specificity

the presence of male DNA in the swab wash. Positive scores were
    
PCR. Presence of male DNA was compared with results from SC
(gold standard test) and PSA tests.
       
validate the methodology [16].
RESULTS AND DISCUSSION
Male DNA detection by PCR
When compared with sperm cytological analysis, PCR
       
corresponding to a sensitivity of 82%. This difference can be

samples, which may result in degradation of male genetic material
by human or bacterial nucleases [17]. Cytological analysis errors

Among 115 cytologically negative samples for spermatozoa,
29 were positive for male DNA, of which 10 also showed positive
in the PSA test, increasing the reliability of DNA results.

(56% sensitivity). The lower sensitivity may be explained by the

Central
Louro et al. (2014)
Email:
Ann Forensic Res Anal 1(1): 1008 (2014) 3/4
fact PSA is a protein based test, which may be positive even in the
absence of male cells.
We attributed particular importance to the 19 positive
samples by PCR which were among the 89 negative samples
for PSA. This represents a 20% fraction of false negatives by
PSA analysis and strongly supports the use of DNA based tests
        
affordable and simple genetic testing in Police routine cases of
sexual violence.
       
satisfactory results, presenting values higher than 75% of
concordance with cytological results (gold standard). When
      
           
 
75%), and amelogenin (89%).
As Sartori in 2008 [19], best results in detecting portions
of the Y chromosome in low representation mixtures was
using primers SRY D & E and AMEL Y, which we recommend as

was also satisfactory.
CONCLUSION
Our results show that the sole utilization of cytological and
         
detection of male evidence in female rape cases, and therefore
   
is highly recommended. By doing so we were able to increase
          
Furthermore, we suggest that a further analysis with autosomal
        

and DYS270 showed the best results overall, being considered
 
recommend the combination of DNA based tests and regularly
used PSA and SC tests to increase male evidence detection rates

ACKNOWLEDGEMENTS

          
Espírito Santo, Brazil for providing the samples.
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
PRIMER REGION BP SEQUENCE
DXYS96 Y STS  

SRY D&E SRY 195 

SRY F&G SRY 212 

DYS270 Y STS 216 

AMEL -Y AMELOGENIN 100 

Table 1: Primers used to amplify fragments.
Abbreviations:       SRY: Sex-Determining Region of the Y chromosome; BP: base pairs; F:
Forward; R: Reverse.

Central
Louro et al. (2014)
Email:
Ann Forensic Res Anal 1(1): 1008 (2014) 4/4
Stange V, Pelição FS, Mendonça JB, Pissinate JF, Barbirato C, et al. (2014) Increased Male Evidence Detection Rates by Utilization of DNA Markers in Forensic
Rape Cases of Espírito Santo, Brazil. Ann Forensic Res Anal 1(2): 1008.
Cite this article
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