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DNA isolation in sexual assault cases is complicated by the presence of large numbers of female epithelial cells, which are often in vast excess when compared to spermatozoa. The two-step differential extraction has been a standard method for isolating the spermatozoa; however, new techniques in forensic science allow the use of precision techniques for capturing spermatozoa. We have used an immuno-magnetic bead-based technique for sperm cell separation. We identified two antibodies that were specific to spermatozoa: SP17 polyclonal antibody and SP10 Intra Acrosomal Protein monoclonal antibody. These were conjugated to Dynabeads® M-450 Epoxy beads and used to isolate the spermatozoa in samples that exhibited the characteristic of sexual assault samples. Using these antibodies, we achieved separation of spermatozoa in the samples with sperm concentration 104/ml and 103/ml; STR analysis produced full male profiles. Mixed profiles were obtained with reduced levels of spermatozoa. Our preliminary data illustrate the potential to apply antibody-based capture to sexual assault cases. [ABSTRACT FROM AUTHOR]

In the investigation of sexual-assault cases different analysis of collected biological samples from the victim and from the crime scene are applicable, depending on the results of the presumptive tests for sample characterization. DNA testing of semen stains can be performed in different ways. Differential extraction method is a golden standard for analysis of sexual assault mixture samples of sperm cells and victim epithelial cells, allowing straight forward interpretation of perpetrator's DNA profile. We have to keep in mind that it is not applicable in about 1.9% of semen stains, found in the casework that shows condition of azoospermia. We present two cases where differential extraction method was decisive approach in analysis of sexual assault evidence in the trial process. [ABSTRACT FROM AUTHOR]

Samples from male to female sexual assault cases that are positive for the presumptive prostate specific antigen (PSA) test often do not result in a male autosomal STR profile. Due to highly unequal proportions of female and male DNA in typical sexual assault samples, routine autosomal STR analysis often fails to detect the DNA of the assailant, even after differential extraction of the samples. Previous studies have already shown the value of Y-STR analysis in such cases [ 1 ]. In Belgium, forensic DNA laboratories are only allowed to perform Y-STR profiling on sexual assault samples by a specific requisition, after routine autosomal STR analysis has been performed. However, a request for additional Y-STR analysis is rather exceptional. In this study, we evaluated the usefulness of further Y-STR analysis. For 100 PSA positive rinses and swabs from male to female sexual assault cases resulting in female autosomal STR profiling, 7% resulted in a full or partial Y-STR profile useful for comparison, using the 23-loci Y-STR PowerPlex Y23 System (Promega). The success rate raised to 12.5% with a higher DNA input in the PCR mix. In conclusion, these results support the usefulness of performing Y-STRs analysis on the sperm DNA extracts to identify the alleged assailant in sexual assault cases. [ABSTRACT FROM AUTHOR]

In forensic investigations, especially in sexual assault cases, biological samples are often mixed stains of different types of body fluids. Highly uneven proportions DNA of male and female makes the detection for stains more difficult. Recently, DNA methylation was proposed to be a promising maker for body fluid identification, and successfully applied to mixed DNA samples. In this study, 7 semen-specific sites were selected from the dataset GSE59509. Methylation-specific polymerase chain reaction and SNaPshot technology were then used to detect the genotype of SNPs in these sites. The results showed that 7-plex SNP typing system was specific in semen samples. In mixed stains, when semen and vaginal fluid in volume ratios of 1:25, the analyses for 7-plex SNP showed that the semen derived genotype could be determined correctly. Meanwhile, unambiguous profiles can be obtained when the input DNA was 0.25 ng. In addition, the 7-plex assay may be useful in the identification of semen even in the degraded samples and complex mixtures. [ABSTRACT FROM AUTHOR]

Sexual assault samples are among the most difficult sample types encountered by forensic laboratories. Typically, a sexual assault sample poses multiple challenges including small quantity of male DNA and a relatively high quantity of female DNA. Differential extraction procedures are time-consuming and labor-intensive, particularly with microscopic sperm identification. A rapid upfront screening or triage assay prior to use of differential extraction procedures could ensure that male DNA is present prior to the use of full DNA workflows. Previously reported rapid male screening assays do not provide a confirmation of whether sperm are present and therefore still require the use of microscopic sperm identification. This work was focused on an improved rapid male DNA screening assay with an upfront sperm identification using mRNA profiling. The rapid male screening assay consists of a brief lysis using only a small tip portion of a swab, to obtain both RNA (eluted) and DNA (extraction 'waste') followed by a one-step reverse transcription-high resolution melt assay for PRM2, a sperm-specific mRNA. Without additional purification, the DNA 'waste' fraction can be quantitated and used to obtain an upfront Y-STR profile of the sperm donor. The assay takes 30 min for lysis followed by ∼2 h for quantitation and sperm identification. Here, we demonstrate the specificity of the assay to detect male DNA in semen samples with no cross reactivity with other body fluids and no PRM2 detection in vasectomized males. RNA and DNA profiling results were obtained with as little as ∼0.15 μl of semen in vaginal-semen admixtures. In all samples tested, Y-STR profiling results were obtained when male DNA was indicated based on quantitation results. [ABSTRACT FROM AUTHOR]