Isolation and Identification of Male and Female DNA on a Postcoital Condom.
It follows that if a condom is worn by the assailant during a sexual assault, cells should be deposited on the latex surfaces, which can be used to link both the suspect and victim to the crime. Multicolor fluorescence in situ hybridization (FISH) employing X- and Y-chromosome probes has been used to identify male epithelial cells on cervicovaginal smears following sexual intercourse.[1,2] Theoretically, this technique can be used to screen for the presence of male epithelial cells and spermatozoa on the internal surface of a used condom. Furthermore, a population of epithelial and inflammatory cells of female origin must be deposited on the external surface of the condom. It should be possible to screen for the presence of female cells on a used condom using multicolor FISH.
The polymerase chain reaction (PCR) has revolutionized DNA analysis.[3-7] Using PCR-based techniques, positive identification can be based on the molecules recovered from a single cell or hair. This study proves that male and female cells retrieved from a used condom can be identified using FISH and that positive identification of both sexual participants can be achieved using PCR-based techniques.
MATERIALS AND METHODS
A male and female volunteer couple consented to participate in this study. Eight hours following sexual intercourse, the male partner presented to one of the investigators with a used condom. Four sterile cotton-tipped applicators were slightly moistened in sterile saline and swabbed along both the internal and external surfaces of the condom. Three air-dried slides were prepared from each set of swabs and stored for subsequent FISH. The remaining swabs were air-dried and submitted for DNA analysis. Buccal swabs were taken of both sex partners for preparation of controls.
Polymerase chain reaction-based DNA identification was performed on the swabs at the DNA Laboratory of the South Carolina Law Enforcement Division (Columbia, SC). Total DNA was extracted from each swab by standard organic extraction. Spermatozoa were not lysed due to an expectation of a preponderance of male DNA in the samples. DNA was quantitated using a fluorescence-based DNA quantitation kit (PanVera, Madison, Wis) and a fluorescent microplate reader (BMG Technologies, Offenburg, Germany). Extracted DNA was amplified for 8 different genetic loci using the Promega PowerPlex (Promega, Madison, Wis) kit and for the amelogenin sex marker. One nanogram of DNA was amplified under the following conditions: 94 [degrees] C for 10 minutes to activate amplitaq gold, then 94 [degrees] C for 1 minute, 60 [degrees] C for 1 minute, and 72 [degrees] C for 1.5 minutes for 30 cycles, followed by 15 minutes at 72 [degrees] C. Amplified samples were electrophoresed on precast sequencing gels (Hitachi/Boehringer Mannheim Corp, Indianapolis, Ind) and analyzed fluorescently using a Hitachi FMBIO 2 fluorescent scanner and software. Each sample obtained from a condom was compared to male and female buccal controls.
Multicolor FISH was performed using X and Y chromosome probes with 4'-6-diamidino-2-phenylindole (DAPI) counterstain. The probes used for this study were the Spectrum CEP Direct Chromosome Enumeration System (VYSIS, Downers Grove, Ill). The X-chromosome probe (alpha satellite DNA) is labeled with Spectrum green and the Y-chromosome-specific probe (satellite III DNA) is labeled with Spectrum orange. The Y-chromosome probe is highly specific for the repeated alphoid DNA located at the centromere of the human chromosome Y, while the X DNA probe hybridizes to the short repeats related to the AATGG in the pericentric heterochromatin of the X chromosome. In our experience, the hybridization efficiency of the Y probe using FISH on uncultured normal tissue samples is 99.97% in males and 0.001% in females.
Slides were pretreated with pepsin (0.002%) for 10 minutes at 37 [degrees] C then rinsed in 2x standard saline citrate (SSC) at room temperature for 2 minutes. Slides were dehydrated through a series of ethanol dilutions then denatured in 70% formamide/2x SSC at 75 [degrees] C for 5 minutes, followed by dehydration through an ethanol series and subsequently air dried. The DNA probes were denatured at 75 [degrees] C for 5 minutes. Slides were hybridized simultaneously with the chromosome X and Y DNA hybridization mixture being added to each slide, then covered with a glass coverslip and sealed with rubber cement. Slides were incubated at 42 [degrees] C for 1 hour in a humidified chamber. Postwashing was done in 2x SSC at 75 [degrees] C for 2 minutes followed by a washing in 2x SSC/0.1% NP40 at room temperature for 1 minute.
Cells were counterstained with DAPI and covered with a glass coverslip prior to microscopic analysis using a fluorescent microscope equipped with a triple band pass (Omega Optical Inc, Brattleboro, Vt). Cells were easily visualized and only nonoverlapping cells were counted. All slides were examined, and all cells were scored for the presence of either 2 orange signals (female) or an orange and a green signal (male). Without exception, cells were scored as positive and of male origin if a green fluorescent signal indicating the presence of the Y chromosome was identified within a nucleus.
A pure sample of female DNA was isolated from the external surface of the condom as determined by exclusive amplification of the X-chromosome-specific 212-base pair amelogenin marker. Only male DNA was extracted from the internal surface of the condom. In both samples, there was conclusive identification at 8 of 8 genetic loci compared to controls (Figure 1, A and B; Table). Fluorescence in situ hybridization identified male epithelial cells and spermatozoa on slides prepared from the internal surface of the condom (Figure 2). Female epithelial cells only were seen on slides generated from the external surface of the condom (Figure 3). The matching probability calculated for the female was 1 in 643 billion based on recently published allelic distribution data.
[Figures 1-3 ILLUSTRATION OMITTED]
Short Tandem Repeat Genotyping Results for Postcoital Condom Swabs(*)
Condom Condom Male Female Inside Outside D16S539 11, 11 10, 10 11 11 10, 10 D7S820 11, 11 10, 11 11 11 10, 11 D13S317 10, 10 11, 11 10 10 11, 11 D5S818 10, 13 11, 11 10 13 11, 11 CSF1PO 10, 11 9, 10 10 11 9, 10 TPOX 8, 11 9, 11 8 11 9, 11 THO1 9, 10 6, 7 9 10 6, 7 VWA 14, 19 16, 16 14 19 16, 16 Amelogenin X, Y X X Y X
(*) Conclusive identification was made at 8 of 8 genetic loci and with the amelogenin sex marker.
Our results show that female cells can be identified by FISH from the external surface of a condom following sexual intercourse. These cells can be recovered in adequate numbers to allow for extraction of adequate amounts of DNA for PCR-based conclusive identification of the female participant. As expected, positive identification of the male can be made from cells and semen recovered from the internal surface of the condom.
In some cases of sexual assault, a condom is worn by the assailant. As a result, the amount of trace evidence (eg, semen, male epithelial cells) deposited on the victim or within the vaginal vault may be minimal. A condom recovered at or near the scene of a sexual assault may yield the only evidence linking a suspect to a crime. The presence of DNA originating from a sexual assault victim on a recovered condom can strengthen the link between assailant and victim.
This work was supported in part by federal formula grant 98-DB-MU-0045, awarded by the Bureau of Justice Assistance, US Department of Justice, Washington, DC, through the South Carolina Department of Public Safety, Columbia, SC. The Assistant Attorney General, Office of Justice Programs, Washington, DC, coordinates the activities of the following program offices and bureaus: Bureau of Justice Assistance, Bureau of Justice Statistics, National Institute of Justice, Office of Juvenile Justice and Delinquency Prevention, and the Office for Victims of Crime.
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[2.] Collins KA, Rao PN, Hayworth R, et al. Identification of sperm and nonsperm male cells in cervicovaginal smears using fluorescence in situ hybridization: applications in alleged sexual assault cases. J Forensic Sci. 1994;39:1347-1355.
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Accepted for publication January 12, 1999.
From the Office of the Armed Forces Medical Examiner, Department of Pathology, Wilford Hall Medical Center, Lackland AFB, Tex (Maj Cina); Department of Pathology and Laboratory Medicine, Medical University of South Carolina, Charleston, SC (Dr Collins); Forensic DNA/Serology, South Carolina Law Enforcement Division, Columbia, SC (Dr Fitts); and the Department of Pediatrics, Cytogenetics Laboratory, Wake Forest University School of Medicine, Winston-Salem, NC (Dr Pettenati).
Points of view or opinions contained within this document are those of the authors and do not necessarily represent the official position or policy of the US Department of Justice, the Department of the Defense, or the United States Air Force. One author (S.J.C.) is a full-time federal employee and this work is in the public domain.
Reprints: Maj Stephen J. Cina, USAF, MC, FS, Office of the Armed Forces Medical Examiner, Department of Pathology, Wilford Hall Medical Center, 59th MDW/MTLP, Lackland AFB, TX 78236.
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|Author:||Cina, Stephen J.; Collins, Kim A.; Fitts, Matthew; Pettenati, Mark J.|
|Publication:||Archives of Pathology & Laboratory Medicine|
|Article Type:||Statistical Data Included|
|Date:||Jul 1, 2000|
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