Background levels of body fluids and DNA on the shaft of the penis and associated underpants in the absence of sexual activity

This study examines the background of blood, saliva, semen and autosomal DNA on penile swabs and underpants from males in the absence of recent sexual activity. Based on the data collected by the AFSP Body Fluid Forum, the results of this study show that; there is a very low expectation of detecting blood on penile swabs and male underpants; a low expectation of detecting saliva on penile swabs and male underpants; and spermatozoa would be expected in less than a quarter of penile swabs and three quarters of male underpants. As none of the samples had detectable levels of DNA which were suitable for meaningful comparison that did not match the donor or their partner, the expectation of detecting a DNA profile from the cellular background on penile swabs or underpants from a male who has not been involved in recent sexual intercourse is very low. The results of this study are extremely informative when evaluating the significance of blood, saliva, semen and DNA detected on the penile swabs and underpants of males in cases of alleged sexual assault.     

Evaluation of usefulness of further Y-STR analysis in sexual assault cases on PSA positive samples resulting in female autosomal STR profiling

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.     

Y-STR profiling in extended interval (> or = 3 days) postcoital cervicovaginal samples

Depending upon specific situations, some victims of sexual assault provide vaginal samples more than 36-48 h after the incident. We have tested the ability of commercial and in-house Y-STR systems to provide DNA profiles from extended interval (> or =3 days) postcoital samples. The commercial Y-STR systems tested included the AmpFlSTR Yfiler (Applied Biosystems), PowerPlex Y (Promega) and Y-PLEX 12 (Reliagene) products whereas the in-house systems comprised Multiplex I (MPI) and Multiplex B (MPB). Three donor couples were recruited for the study. Postcoital cervicovaginal swabs (x2) were recovered by each of the three females at specified intervals after sexual intercourse (3-7 days). Each time point sample was collected after a separate act of sexual intercourse and was preceded by a 7-day abstention period. As a negative control, a precoital swab was also recovered prior to coitus for each sampling and only data from postcoital samples that demonstrated a lack of male DNA in the associated precoital sample was used. A number of DNA profile enhancement strategies were employed including sampling by cervical brushing, nondifferential DNA extraction methodology, and post-PCR purification. Full Y-STR profiles from cervicovaginal samples recovered 3-4 days after intercourse were routinely obtained. Profiles were also obtainable 5-6 days postcoitus although by this stage partial profiles rather than full profiles were a more likely outcome. The DNA profiles from the sperm fraction of a differential lysis were superior to that obtained when a nondifferential method was employed in that the allelic signal intensities were generally higher and more balanced and exhibited less baseline noise. The incorporation of a simple post-PCR purification process significantly increased the ability to obtain Y-STR profiles, particularly from 5- to 6-day postcoital samples. Remarkably an 8 locus Y-STR profile was obtained from a 7-day postcoital sample, which is approaching the reported time limit for sperm detection in the cervix.     

Analysis of non-suspect samples lacking visually identifiable sperm using a Y-STR 10-plex

Y-STRs are valuable in the investigation of sexual assaults in which autosomal STR genotype interpretation is challenging. To detect male DNA from compromised sexual assault evidence, 45 non-suspect samples were differentially extracted and analyzed with 10 Y-STRs. These samples were positive for the presence of human seminal fluid, but were negative for spermatozoa by microscopic examination. Y-STR data were obtained in approximately 86.2% of the epithelial or sperm fractions. On samples yielding incomplete profiles, results were obtained on an average of 5 loci per sample. The inability to obtain results may be due to insufficient amplifiable male DNA, PCR inhibition, or unfounded accusations of sexual assault. This study indicates that it is possible to obtain a male STR profile even in the absence of visually identifiable spermatozoa. Furthermore, Y-STR loci should become components of CODIS if they are to be used in solving non-suspect sexual assaults.     

The efficiency of Y-chromosome markers in forensic trace analysis and their inclusion in the Austrian National DNA Database

Y-STR markers are a valuable tool in the analysis of biological traces in which a mixture of male and female trace material is to be expected. It is possible to generate a Y-chromosome DNA profile, even if all the prior sperm tests are negative and no sign of any male component is found in amelogenin. In 38 of a total of 239 sexual offences a perpetrator trace was identified solely using Y-STR analysis. Based on these findings, the Austrian National DNA Database was expanded to include Y-STRs in 2012 with the primary objective to identify serial sexual offences.     

Examining the transfer of male DNA onto female underwear in simulated non sexual social interaction versus digital penetration to assist in the evaluation of results in casework scenarios

Experiments have been carried out by the UK and Ireland Association of Forensic Science Providers Body Fluid Forum (AFSP BFF) to determine the levels of male DNA, detected during Y-STR analysis, that may be expected on female underwear from non-sexual social interaction and digital penetration, versus non-sexual social interaction only. The data obtained strongly supports the existing assumptions made: whilst low levels of DNA may be inadvertently transferred to the inside surface of a female’s underwear during social interaction with a male, there is a low expectation of detecting a matching Y-STR profile to that male, which is suitable for statistical evaluation, unless he is a co-habitant of that female.     

DNA profiling of trace DNA recovered from bedding

Trace DNA is often detected on handled items and worn clothing examined in forensic laboratories. In this study, the potential transfer of trace DNA to bedding by normal contact, when an individual sleeps in a bed, is examined. Volunteers slept one night on a new, lower bed sheet in their own bed and one night in a bed foreign to them. Samples from the sheets were collected and analysed by DNA profiling. The results indicate that the DNA profile of an individual can be obtained from bedding after one night of sleeping in a bed. The DNA profile of the owner of the bed could also be detected in the foreign bed experiments. Since mixed DNA profiles can be obtained from trace DNA on bedding, caution should be exercised when drawing conclusions from DNA profiling results obtained from such samples. This transfer may have important repercussions in sexual assault investigations.     

Performance characteristics of commercial Y-STR multiplex systems

In this work, a number of performance checks were carried out to evaluate the efficacy of commercial Y-short tandem repeats (Y-STR) kits for casework applications. The study evaluated the sensitivity, specificity and stability of the Y-STR markers used and the ability to obtain a male profile from postcoital samples taken at various time points after intercourse. All systems performed well with 1-3 ng of male DNA as recommended by the manufacturers. All systems gave full profiles at 100 pg of input DNA, which is within the realm of low copy number DNA analysis. Moreover all, except Y-Plex12, gave full profiles with 30-50 pg of male DNA. No increased performance was obtained with any of the systems by increasing the cycle number beyond that recommended by the various manufacturers. When up to 1 microg of female DNA was used (in the absence of male DNA) no female DNA cross reactivity was observed with the Y-Plex 12 and Y-Filer systems. PowerPlex Y produced female DNA derived products near the DYS438 and within the DYS392 loci at a rare allele position with high input DNA levels (300 ng and 1 microg, respectively). Male/female DNA admixture experiments indicated the particularly high specificity of the Y-Filer and PowerPlex Y systems under conditions of several thousand fold female DNA excess. All systems were able to detect the minor alleles in male/male DNA admixtures at a 1:5 dilution with the PowerPlex Y and Y-Filer being able to detect some minor alleles at 1:20. Species testing indicated some limited, minor cross reactivity of the commercial systems with some domestic male mammals although it is easily recognizable and would not pose any problems in casework analysis. As expected a significant number of cross-reacting products were obtained with nonhuman primate species. All Y-STR multiplex systems tested were able to produce complete Y-STR profiles from bloodstains and semen stains exposed up to 6 weeks when the samples were protected against precipitation and sunlight. However, exposure of the samples to precipitation either in the presence or absence of sunlight resulted in Y-STR profile loss over time, with total profile loss occurring with all systems after 3 weeks or more. Complete Y-STR profiles of the male donors up to 72 h postcoitus were obtained with all of the multiplex systems tested, except for Y-Plex12, which gave partial profiles.     

Novel Y-STR typing strategies reveal the genetic profile of the semen donor in extended interval post-coital cervicovaginal samples

For a variety of reasons, some victims of sexual assault provide vaginal samples more than 24-36 h after the incident. In these cases, the ability to obtain an autosomal STR profile of the semen donor from the living victim diminishes rapidly as the post-coital interval is extended. We have used a number of carefully selected Y-STR loci in a variety of multiplex or monoplex formats to extend the post-coital interval from which a genetic profile of the semen donor can be obtained. The proposed Y-STR typing strategies enable the routine detection of the male donor Y-STR haplotype in cervicovaginal samples recovered up to 4 days post-coitus. We attribute our success to a number of factors that significantly improve the sensitivity and specificity of the analysis. Firstly, we utilize a subset of Y-STR loci that have been carefully selected for their superior performance under stressed conditions in both multiplex and monoplex formats. Specifically these loci function with low copy number templates in the presence of a vast excess of potentially confounding female DNA. Secondly, sperm and non-sperm DNA is co-extracted without a differential extraction process to prevent the unnecessary loss of the small number of structurally fragile sperm remaining in the cervicovaginal tract several days after intercourse. Thirdly, low copy number detection is facilitated by increasing the cycle number to 34-35 cycles and by the ability to input up to 450 ng of co-extracted sperm/non-sperm DNA into the PCR reaction without the appearance of confounding female artifacts. Lastly, the proper collection of post-coital cervicovaginal samples, instead of the lower or mid-vaginal tract samples often taken, is required for optimal recovery of sperm for analysis. In this report we demonstrate that our previously described 19 Y-STR loci systems (MPI and MPII) permit a reliable high resolution haplotype determination of the semen donor in cervicovaginal samples taken up to 48 h after intercourse. However, as the post-coital interval is extended further, dramatic loss of signal is observed and haplotype determination of the male donor is no longer possible with MPI and MPII. Nonetheless, subsets of these 19 loci (MPA and MPB) have been developed specifically to detect the male haplotype in samples recovered 4 days after intercourse. Thus, it is possible to derive an 11-19 locus Y-STR profile of the semen donor in cervicovaginal samples recovered 2-4 days after intercourse.     

SWGDAM developmental validation of a 19-locus Y-STR system for forensic casework

A Scientific Working Group on DNA Analysis Methods (SWGDAM) developmental validation study was carried out on two Y-STR multiplex systems (MPI and MPII) that collectively permit the co-amplification of 19 Y-STR markers, including DYS393, DYS392, DYS391, DYS389I, DYS389II, Y-GATA-A7.2 (DYS461), DYS438, DYS385a and DYS385b (MPI); DYS425, DYS388, DYS390, DYS439, DYS434, DYS437, Y-GATA-C.4, Y-GATA-A7.1 (DYS460), Y-GATA-H.4, and DYS 19 (MPII). Performance checks subsequent to PCR parameter optimization indicated that MPI and MPII were suitably reproducible, precise and accurate for forensic use. The sensitivity of the systems was such that a full 19-locus Y-STR profile was obtainable with 150-200 pg of male DNA, and some loci were detectable even with as little as 20-30 pg of input DNA. Primate specificity was demonstrated by the lack of cross-reactivity with a variety of commonly encountered bacterial and animal species, with the single exception of a monomorphic canine product that was outside of the size range of human alleles from any of the 19 loci. Not surprisingly, cross-reactivity was observed with a number of male and female nonhuman primates. Environmentally compromised samples produced full or partial Y-STR profiles. For example, a semen stain exposed to the outdoor elements for six months still gave a 13-locus Y-STR profile. Although a limited number of female DNA artifacts were observed in mixed stains in which the male DNA comprised 1/300 of the total, the full 19-locus male profile was easily discernible. Even at a 1500-to-2000-fold dilution of male DNA with female DNA partial Y-STR profiles were obtained. Furthermore, the potential utility of MPI and MPII for forensic casework is exemplified by their ability to dissect out the male haplotype in a variety of case-type samples, including, inter alia, post-coital vaginal swabs, admixed male and female bloodstains, the nonsperm fraction from a differentially extracted semen stain, and determination of the number of male donors in mixed semen stains.     

Y-STR基因座应用于刑事案件的独特作用

目的探讨Y-STR基因座在刑事案件中的应用价值。方法采用Y-STR荧光标记复合扩增技术,结合案例应用。结果Y-STR基因座对于涉及男女混合、多名男性混合样本、性别鉴定、父权鉴定等案例中具有特有应用价值。结论Y-STR基因座可应用于法庭科学中的个体识别与同一认定,但在应用中要注意各种特例的发生。     

Assessment of the Yfiler® Plus PCR amplification kit for the detection of male DNA in semen-negative sexual assault cases

The ability to detect male epithelial cells deposited during digital penetration or penile penetration without ejaculation is limited by the sensitivity of the Y-STR profiling kit. In this study, the relative profiling success of the Thermofisher Yfiler® Plus kit was compared to its predecessor, AmpFlSTR Yfiler®, for 104 semen-negative sexual assault samples from casework at Forensic Science SA, Adelaide, South Australia. Yfiler Plus generated allele information in 25% more samples than Yfiler and gave a higher recovery of informative alleles in all but two samples where detectable male DNA was present. Where a profile was obtained in both kits, 92% of samples gave a higher percentage of informative loci with Yfiler Plus compared to Yfiler. Yfiler Plus also resolved DNA mixtures in 15 samples as compared to 1 sample with Yfiler. Detection of male DNA with the Quantifiler™ Trio DNA Quantification kit was shown to correlate with a successful profiling outcome with Yfiler Plus. The success of profiling with Yfiler Plus was independent of the time elapsed between the alleged offence and the sample being collected, the type of sexual penetration which occurred, and the anatomical origin of the sample.     

Y-STR analysis on DNA mixture samples—Results of a collaborative project of the ENFSI DNA Working Group

The ENFSI (European Network of Forensic Science Institutes) DNA Working Group undertook a collaborative project on Y-STR typing of DNA mixture samples that were centrally prepared and thoroughly tested prior to the shipment. Four commercial Y-STR typing kits (Y-Filer, Applied Biosystems, Foster City, CA, USA; Argus Y Nonaplex, Biotype, Dresden, Germany; Powerplex Y, Promega, Madison, WI, USA; and DYSplex-3, SERAC, Bad Homburg, Germany) were used for the amplification of the mixture samples. The results of the study showed a striking inter-laboratory difference of kit performance as determined from the peak heights of the obtained Y-STR genotypes. Variation in quantity and quality of the shipped DNA can be excluded as reason for the observed differences because both samples and shipping conditions were found to be reproducible in an earlier study. The results suggest that in some cases a laboratory-specific optimization process is indicated to reach a comparable sensitivity for the analysis of minute amounts of DNA.     

DNA recovery from different evidences in 300 cases of sexual assault

Almost 60% of the DNA evidences analyzed in our laboratory correspond to sexual assault cases. With the aim to assess the efficiency of the DNA IQ System (Promega) in recovering the perpetrator DNA profile, the statistical analysis of results obtained in 300 casework was performed. In such cases, 850 evidence samples were processed. In 71% of the cases the perpetrator DNA profile was detected in at least one of the submitted casework samples, with a minimum of 13 STRs markers typed using the AmpFlSTR Identifiler PCR amplification kit (Applied Biosystem). When the suspect DNA profile was available, 67% matched with the evidence.With regard to the type of evidence, the best performance corresponded to panties, with more than 70% of success in recovering male profile, whereas the efficiency of vaginal swabs was almost 60%, with a higher incidence of victim/perpetrator mixed profiles.     

Y-chromosome STR system, Y-PLEX 12, for forensic casework: development and validation

The Y-PLEX 12 system, developed for use in human identification, enables simultaneous amplification of eleven polymorphic short tandem repeat (STR) loci, namely DYS392, DYS390, DYS385 a/b, DYS393, DYS389I, DYS391, DYS389II, DYS 19, DYS439 and DYS438, residing on the Y chromosome and Amelogenin. Amelogenin provides results for gender identification and serves as internal control for PCR. The validation studies were performed according to the DNA Advisory Board's (DAB) Quality Assurance Standards. The minimal sensitivity of the Y-PLEX 12 system was 0.1 ng of male DNA. The mean stutter values ranged between 3.76-15.72%. A full male profile was observed in mixture samples containing 0.5 ng of male DNA and up to 400 ng of female DNA. Amelogenin did not adversely affect the amplification of Y-STRs in mixture samples containing male and female DNA. The primers for the Y-STR loci present in Y-PLEX 12 are specific for human DNA and some higher primates. None of the primate samples tested provided a complete profile at all 11 Y-STR loci amplified with the Y-PLEX 12 system. Y-PLEX 12 is a sensitive, valid, reliable, and robust multiplex system for forensic analysis, and it can be used in human forensic and male lineage identification cases.     

Generating DNA profiles from immunochromatographic cards using LCN methodology

The aim of this research was to obtain DNA profiles from immunochromatographic test devices which have already yielded positive results with body fluids obtained from fourteen volunteers. Three different immunochromatographic cards for the identification of human blood and one for the identification of human saliva were used for this research. Each body fluid was detected using the appropriate immunochromatographic card. The used cards were kept at room temperature for various lengths of time. The membranes were removed at the end of the designated times and the entire strip was extracted using low copy number (LCN) extraction procedure. The extracted DNA was amplified using reduced amplification volume and higher PCR cycle numbers. Autosomal STR profiles were detected using AmpFℓSTR^® Identifiler™ PCR Amplification Kit from Applied Biosystems (AB). Additionally, DNA extracted from the male volunteers was amplified using the AB AmpFℓSTR^® Yfiler™ PCR Amplification Kit. Analysis of the amplified products was carried out by capillary electrophoresis injection on the AB 3130xl Genetic Analyzer. The generated DNA data was analyzed using the SoftGenetics GeneMarker^® HID Version 1.7 software.Autosomal and Y-STR DNA profiles were obtained from most of the cards which were stored at room temperature for up to three months. DNA profile was obtained from all four types of the immunochromatographic cards used in this study. These profiles were concordant with the profiles obtained from the donors’ reference samples.     

Chromosomal duplications along the Y-chromosome and their potential impact on Y-STR interpretation

Y-chromosome short tandem repeat (Y-STR) markers are being used as potential tools for distinguishing low levels of male DNA in the presence of excess female DNA as is present in many sexual assault samples. Usually single copy Y-STR loci produce a single amplicon in single source samples, and thus the observation of multiple peaks at such a locus could suggest to an analyst that a mixture of more than one male contributor is present in the tested sample. However, many regions of the Y-chromosome are duplicated or even triplicated in some individuals and this fact can thus complicate potential mixture interpretation. Reasons for the presence of duplications at multiple loci within a single sample are explored in the context of Y-STR marker location along the chromosome. True male-male mixtures commonly exhibit more than one locus-specific PCR product across multiple Y-STR loci that are not adjacent to one another on the Y-chromosome. In addition, duplicated loci typically possess alleles that differ by only a single repeat unit and possess similar peak heights.     

The prevalence of mixed DNA profiles on fingernail swabs

There is a general acceptance that cellular material will transfer from one person to another person's fingernails through everyday contact. However, the level or degree of contact required to transfer sufficient cellular material in order to obtain a DNA profile is not known. This study examined swabs from the fingernails of 40 volunteers and compared the DNA profiles obtained to the daily activities of that individual. The majority (78%) of high level profiles obtained were associated with recent intimate contact. However, high level profiles were also obtained from the fingernails of individuals who shared accommodation with their partner, flatmates and/or children. Low level profiles and single profiles were associated with all levels of contact.     

Identification and isolation of male cells using fluorescence in situ hybridisation and laser microdissection, for use in the investigation of sexual assault

In cases of sexual assault involving an azoospermic assailant, vaginal swabs taken from the victim may fail to provide an autosomal DNA profile with which to search a suspect database, as the signal from any male cells present would be masked by that from the overwhelming number of female cells collected on the swab. Here, we describe a method of visually identifying diploid male cells in such samples using fluorescence in situ hybridisation, and selectively harvesting them by means of laser microdissection. This combination of techniques was tested on 26 post-coital vaginal swabs taken at a range of times after intercourse; the collected cells were then subjected to a simple lysis procedure and DNA was amplified using the AmpFlSTR^® SGMPlus^® multiplex under low copy number conditions. Useful DNA profiles were generated from samples taken up to 24 h after intercourse.