The role of DNA in criminal indictments in Israel.

In their investigations of criminal cases, law enforcement agencies rely heavily on forensic evidence. Numerous studies have examined the scientific and technological advancements of DNA testing, but little evidence exists on how the availability of DNA evidence influences prosecutors' decisions to move cases forward in the criminal justice system. We created a new database by juxtaposing data from the Forensics Division of the Israel Police, which recorded the presence (or not) of DNA profiles in criminal cases (n = 9862), and data on the indictment decision for each case (2008-2019). Rates of indictments are computed for each case, and trend lines are used to present variations in the rates of indictment decisions with and without DNA profiles. Approximately 15% of all criminal cases without DNA presented to the prosecutor's office are subsequently prosecuted, compared with nearly 55% of cases with DNA profiles. The presence of DNA evidence influences the prosecutor's decision to move a case forward in the criminal justice system. Utilizing a scientific approach to prosecute offenders is a welcome development; however, DNA evidence is not infallible, and caution must be exercised in regard to DNA's overuse in the legal system.

Application of a Forensic DNA Extraction System for Cannabis sativa Seed Identification.

Identification of Cannabis sativa seeds is crucial for law authorities fighting drug abuse and global trafficking. However, because they lack chemicals that are routinely used to pinpoint C. sativa plants, their identification becomes far more challenging. Germinating the seeds requires tremendous resources, is time consuming and is not effective when dealing with nonviable seeds. Botanical identification relies on well-trained experts. In recent years, laboratories have started to use specific DNA markers to achieve this goal. Real-time quantitative polymerase chain reaction (qPCR) was found to be a simple and efficient approach. However, seed DNA extraction is often labor intensive and involves many steps, which may also lead to DNA loss and contaminations. In the present study, we explored whether the PrepFiler™ Express Forensic DNA Extraction Kit, which is used in our DNA casework forensic laboratory, can reduce the work required for this critical step. We show that this kit has several advantages when compared with a dedicated plant extraction kit. In addition, we show that the combination of this extraction method and qPCR can enable high-throughput C. sativa seed identification.

Applying microbial biogeography in soil forensics.

The ubiquity, heterogeneity and transferability of soil makes it useful as evidence in criminal investigations, especially using new methods that survey the microbial DNA it contains. However, to be used effectively and reliably, more needs to be learned about the natural distribution patterns of microbial communities in soil. In this study we examine these patterns in detail, at local to regional scales (2 m-260 km), across an environmental gradient in three different soil types. Geographic location was found to be more important than soil type in determining the microbial community composition: communities from the same site but different soil types, although significantly different from each other, were still much more similar to each other than were communities from the same soil type but from different sites. At a local scale (25-1000 m), distance-decay relationships were observed in all soil types: the farther apart two soil communities were located, even in the same soil type, the more they differed. At regional-scale distances (1-260 km), differences between communities did not increase with increased geographic distance between them, and the dominant factor determining the community profile was the physico-chemical environment, most notably annual precipitation (R2 = 0.69), soil sodium (R2 = 0.49) and soil ammonium (R2 = 0.47) levels. We introduce a likelihood-ratio framework for quantitative evaluation of soil microbial DNA profile evidence in casework. In conclusion, these profiles, along with detailed knowledge of natural soil microbial biogeography, provide valuable forensic information on soil sample comparison and allow the determination of approximate source location on large (hundreds of km) spatial scales. Moreover, at small spatial scales it may enable pinpointing the source location of a sample to within at least 25 m, regardless of soil type and environmental conditions.

Acid phosphatase test on Phadebas® sheets - An optimized method for presumptive saliva and semen detection.

The precise and efficient detection of semen and saliva in sexual assault case-work items is a critical step in the forensic pipeline. The outcome of this stage may have a profound impact on identifying perpetrators as well as on the investigation process and the final outcome in court. Semen detection is usually based on the activity of acid phosphatase (AP), an enzyme found in high concentration in the seminal plasma. Amylase, an enzyme catalyzing starch hydrolysis is found in high concentrations in saliva and therefore is a useful target for its detection. To screen case-work items, both presumptive tests require transfer of biological material from the item to paper in a moisturized environment. Since semen and saliva may appear in the same item, it is required in some cases to perform the tests one after the other. This may reduce the chances of identifying all stains on the item and obtaining a DNA profile. In the present study, we applied the AP biochemical test on a Phadebas® sheet, a commercial starch containing paper used to detect saliva. This approach was found to be sensitive enough to detect diluted semen (1:50) after performing the Phadebas® press test. In addition, it enabled detection of adjacent saliva and semen stains and stains containing a semen-saliva mixture. Finally, a DNA profile was successfully obtained from the Phadebas® sheets after semen detection, a useful feature if the original item is lost or damaged. Taken together, this method provides a practical, reliable and convenient tool for screening sexual assault items of evidence.

Laundry in a washing machine as a mediator of secondary and tertiary DNA transfer.

The aim of this work was to investigate the possibility of secondary and tertiary DNA transfer during laundry. The modes of transfer tested were mixed and separate laundry of worn and unworn garments in household and public washing machines. In addition, the possibility of a background DNA carry-over from a washing machine's drum was investigated. In the mixed (worn and unworn garments washed together) laundry experiment, 22% of samples from new unworn socks with no traceable DNA prior to experiment produced DNA profiles post-laundry. In the tertiary DNA transfer experiment performed in a public washing machine (unworn garments only), no detectable DNA profiles were observed. Samples collected from the internal drum of 25 washing and drying machines did not produce detectable STR profiles. The implications of these results are discussed in the context of forensic DNA casework analysis. Graphical Abstract ᅟA real-life scenario of secondary DNA transfer between worn and unworn garments during machine washing has been evaluated. Experiments demonstrated this scenario is possible (22% of samples) and may in fact result in high quality DNA profiles. On the contrary, testing washing machine's interior for deposition of biological material between separate washing cycles to serve as a mediator of tertiary DNA transfer resulted in no DNA profiles.

Improved amplification results following episodes of failure to amplify at the Amelogenin Locus using PowerPlex® ESI 16 Fast System.

In 2012 the Israel Police DNA Casework laboratory adopted the 16 STR PowerPlex® ESI kit for routine use. The Promega Company updated this kit and developed the PowerPlex® ESI 16 Fast System in which all autosomal primer pairs remained identical to the original set, except at the amelogenin site. The master mix was improved and optimized which allowed for direct, faster and more robust amplification. Prior to implementing the PowerPlex® ESI 16 Fast System in our lab, we conducted a preliminary assay where 213 casework samples were amplified using the new kit. These samples had previously been extracted by one of two extraction kits employed by our lab. (the PrepFiler ExpressTM and PrepFiler BTATM Forensic DNA Extraction Kits). The amplification results from these samples were compared to samples amplified using the original PowerPlex® ESI 16 kit. Multiple incidents of failure to amplify at the amelogenin locus were noted using the new system with the recommended protocol at a rate of 13% (28 of 213 samples). Experiments were performed to understand whether these amplification failures could be a result of primer binding site mutations, extraction method reagents and/or inhibitors. The conclusions reached following these experiments, in conjunction with consultation with the manufacturer, led to the trial of a modified amplification protocol where the suggested annealing temperature was reduced by 2 degrees. To evaluate the efficiency of this altered protocol, a comparison study was undertaken where 88 additional casework samples were chosen and amplified using both the modified 58°C and the recommended 60°C annealing temperatures. We concluded that the most effective method in our laboratory for achieving a consistent and balanced amplification at the amelogenin locus was to reduce the annealing temperature from the manufacturer's recommended 60°C to 58°C. This modification resulted in a reduction of the failure to amplify at the amelogenin locus from 13% (28/213) to 1.1% (1/88) without any observed changes to the autosomal STR amplification results.

Acetone facilitated DNA sampling from electrical tapes improves DNA recovery and enables latent fingerprints development.

Electrical tapes (ETs) are a common component of improvised explosive devices (IEDs) used by terrorists or criminal organizations and represent a valuable forensic resource for DNA and latent fingerprints recovery. However, DNA recovery rates are typically low and usually below the minimal amount required for amplification. In addition, most DNA extraction methods are destructive and do not allow further latent fingerprints development. In the present study a cell culture based touch DNA model was used to demonstrate a two-step acetone-water DNA recovery protocol from ETs. This protocol involves only the adhesive side of the ET and increases DNA recovery rates by up to 70%. In addition, we demonstrated partially successful latent fingerprints development from the non-sticky side of the ETs. Taken together, this protocol maximizes the forensic examination of ETs and is recommended for routine casework processing.

Combination of prostate-specific antigen detection and micro-Raman spectroscopy for confirmatory semen detection.

Detecting prostate specific antigen (PSA) in a questioned body fluid stain or other sexual assault casework items strongly indicates the presence of semen and is extremely useful when no sperm cells are observed. However, it has several false-positives which may prevent confirmative semen classification. Namely, the presence of PSA and nucleated cells in male urine could lead a forensic examiner to an incorrect body fluid conclusion. Micro Raman spectroscopy is a molecular spectroscopy based on the inelastic scattering of monochromatic light and its potential to detect a wide range of body fluids has been demonstrated in recent years. In the present study, we show how the combination of PSA tests and micro Raman spectroscopy offers a simple, non-destructive and quick method for confirmatory semen detection. After positive PSA tests, micro Raman spectroscopy can easily corroborate semen presence and exclude the possibility of urine false positive detection. The sensitivity of this practice was demonstrated by measuring micro Raman spectra of diluted urine and semen (up to 1:100), as well as their spectra after extraction from cloth and swabs. These results strongly show the advantages of combining micro Raman spectroscopy and PSA tests when examining sexual assault casework items.

Soil characterisation by bacterial community analysis for forensic applications: A quantitative comparison of environmental technologies.

The ubiquity and transferability of soil makes it a resource for the forensic investigator, as it can provide a link between agents and scenes. However, the information contained in soils, such as chemical compounds, physical particles or biological entities, is seldom used in forensic investigations; due mainly to the associated costs, lack of available expertise, and the lack of soil databases. The microbial DNA in soil is relatively easy to access and analyse, having thus the potential to provide a powerful means for discriminating soil samples or linking them to a common origin. We compared the effectiveness and reliability of multiple methods and genes for bacterial characterisation in the differentiation of soil samples: ribosomal intergenic spacer analysis (RISA), terminal restriction fragment length polymorphism (TRFLP) of the rpoB gene, and five methods using the 16S rRNA gene: phylogenetic microarrays, TRFLP, and high throughput sequencing with Roche 454, Illumina MiSeq and IonTorrent PGM platforms. All these methods were also compared to long-chain hydrocarbons (n-alkanes) and fatty alcohol profiling of the same soil samples. RISA, 16S TRFLP and MiSeq performed best, reliably and significantly discriminating between adjacent, similar soil types. As TRFLP employs the same capillary electrophoresis equipment and procedures used to analyse human DNA, it is readily available for use in most forensic laboratories. TRFLP was optimized for forensic usage in five parameters: choice of primer pair, fluorescent tagging, concentrating DNA after digestion, number of PCR amplifications per sample and number of capillary electrophoresis runs per PCR amplification. This study shows that molecular microbial ecology methodologies are robust in discriminating between soil samples, illustrating their potential usage as an evaluative forensic tool.

A novel cell culture model as a tool for forensic biology experiments and validations.

To improve and advance DNA forensic casework investigation outcomes, extensive field and laboratory experiments are carried out in a broad range of relevant branches, such as touch and trace DNA, secondary DNA transfer and contamination confinement. Moreover, the development of new forensic tools, for example new sampling appliances, by commercial companies requires ongoing validation and assessment by forensic scientists. A frequent challenge in these kinds of experiments and validations is the lack of a stable, reproducible and flexible biological reference material. As a possible solution, we present here a cell culture model based on skin-derived human dermal fibroblasts. Cultured cells were harvested, quantified and dried on glass slides. These slides were used in adhesive tape-lifting experiments and tests of DNA crossover confinement by UV irradiation. The use of this model enabled a simple and concise comparison between four adhesive tapes, as well as a straightforward demonstration of the effect of UV irradiation intensities on DNA quantity and degradation. In conclusion, we believe this model has great potential to serve as an efficient research tool in forensic biology.

Wild gazelles of the southern Levant: genetic profiling defines new conservation priorities.

The mountain gazelle (Gazella gazelle), Dorcas gazelle (Gazella Dorcas) and acacia gazelle (Gazella arabica acacia) were historically abundant in the southern Levant, and more specifically in Israel. Anthropogenic and natural changes have caused a rapid decline in gazelle populations, raising concerns about their conservation status and future survival. The genetic profile of 111 wild gazelles from Israel was determined based on three regions of mitochondrial DNA (control region, Cytochrome b and 12S ribosomal RNA) and nine nuclear microsatellite markers. Genetic analysis of the mountain gazelle population, the largest known population of this rare species, revealed adequate diversity levels and gene flow between subpopulations. Nevertheless, ongoing habitat degradation and other human effects, such as poaching, suggest the need for drastic measures to prevent species extinction. Dorcas gazelles in Israel displayed inbreeding within subpopulations while still maintaining considerable genetic diversity overall. This stable population, represented by a distinctive genetic profile, is fragmented and isolated from its relatives in neighboring localities. Based on the genetic profile of a newly sampled subpopulation in Israel, we provide an alternative hypothesis for the historic dispersal of Dorcas gazelle, from the Southern Levant to northern Africa. The small acacia gazelle population was closest to gazelles from the Farasan Islands of Saudi Arabia, based on mitochondrial markers. The two populations did not share haplotypes, suggesting that these two populations may be the last remnant wild gazelles of this species worldwide. Only a dozen acacia gazelles survive in Israel, and urgent steps are needed to ensure the survival of this genetically distinctive lineage. The genetic assessments of our study recognize new conservation priorities for each gazelle species in the Southern Levant.

Spatial and temporal biogeography of soil microbial communities in arid and semiarid regions.

Microbial communities in soils may change in accordance with distance, season, climate, soil texture and other environmental parameters. Microbial diversity patterns have been extensively surveyed in temperate regions, but few such studies attempted to address them with respect to spatial and temporal scales and their correlations to environmental factors, especially in arid ecosystems. In order to fill this gap on a regional scale, the molecular fingerprints and abundance of three taxonomic groups--Bacteria, α-Proteobacteria and Actinobacteria--were sampled from soils 0.5-100 km apart in arid, semi-arid, dry Mediterranean and shoreline Mediterranean regions in Israel. Additionally, on a local scale, the molecular fingerprints of three taxonomic groups--Bacteria, Archaea and Fungi--were sampled from soils 1 cm-500 m apart in the semi-arid region, in both summer and winter. Fingerprints of the Bacteria differentiated between all regions (P<0.02), while those of the α-Proteobacteria differentiated between some of the regions (0.010.05). Locally, fingerprints of archaea and fungi did not display distance-decay relationships (P>0.13), that is, the dissimilarity between communities did not increase with geographic distance. Neither was this phenomenon evident in bacterial samples in summer (P>0.24); in winter, however, differences between bacterial communities significantly increased as the geographic distances between them grew (P<0.01). Microbial community structures, as well as microbial abundance, were both significantly correlated to precipitation and soil characteristics: texture, organic matter and water content (R(2)>0.60, P<0.01). We conclude that on the whole, microbial biogeography in arid and semi-arid soils in Israel is determined more by specific environmental factors than geographic distances and spatial distribution patterns.

Recognizing the advantage of employing the PowerPlex(®) ESI and PowerPlex(®) ESX complementary kits for purposes of clarification in routine casework.

In an effort to promote European cross-border cooperation in fighting crime and international terrorism the Treaty of Prüm was drafted and accepted within the European forensic community. This move led to the commercial development of new multiplex kits which introduced five new STR loci and promised better performance. Recently the Israel Police DNA Casework and Database laboratories adopted the PowerPlex(®) ESI kit for routine use in our laboratories. Presented in this paper are examples of three types of ambiguous results encountered during the implementation of the PowerPlex(®) ESI kit into routine work. These ambiguous products presented themselves in the form of (1) extreme variants outside of loci borders, (2) failure to amplify sister allele pairs or expression of null alleles and (3) episodes of loss of separation of adjacent microvariants primarily in mixture samples. The re-analysis of all these samples using the PowerPlex(®) ESX kit successfully and rapidly clarified all three categories of anomalies. Spotlighting such events to the forensic community, especially regarding the novel loci introduced in these next generation kits, can aid in raising the analyst's awareness to their future appearances and prevent possible erroneous conclusions. In addition, providing timely DNA results to investigating teams is of great importance and operational forensic laboratories do not have at their immediate disposal methods such as sequencing to elucidate such manifestations. We suggest that the complementary use of the PowerPlex(®) ESI and PowerPlex(®) ESX can provide a benefit for clarification purposes in routine casework.

Demonstration of DSI-semen--A novel DNA methylation-based forensic semen identification assay.

Determining whether the source tissue of biological material is semen is important in confirming sexual assaults, which account for a considerable percentage of crime cases. The gold standard for confirming the presence of semen is microscopic identification of sperm cells, however, this method is labor intensive and operator-dependent. Protein-based immunologic assays, such as PSA, are highly sensitive and relatively fast, but suffer from low specificity in some situations. In addition, proteins are less stable than DNA under most environmental insults. Recently, forensic tissue identification advanced with the development of several approaches based on mRNA and miRNA for identification of various body fluids. Herein is described DNA source identifier (DSI)-semen, a DNA-based assay that determines whether the source tissue of a sample is semen based on detection of semen-specific methylation patterns in five genomic loci. The assay is comprised of a simple single tube biochemical procedure, similar to DNA profiling, followed by automatic software analysis, yielding the identification (semen/non-semen) accompanied by a statistical confidence level. Three additional internal control loci are used to ascertain the reliability of the results. The assay, which aims to replace microscopic examination, can easily be integrated by forensic laboratories and is automatable. The kit was tested on 135 samples of semen, saliva, venous blood, menstrual blood, urine, and vaginal swabs and the identification of semen vs. non-semen was correct in all cases. In order to test the assay's applicability in "real-life" situations, 33 actual casework samples from the forensic biological lab of the Israeli police were analyzed, and the results were compared with microscopic examination performed by Israeli police personnel. There was complete concordance between both analyses except for one sample, in which the assay identified semen whereas no sperm was seen in the microscope. This sample likely represents true semen because sperm cells were detected from an adjacent sample from the same garment, therefore in this case the assay appears to be more sensitive than the microscopic examination. These results demonstrate that this assay is a bona fide confirmatory test for semen.

A search for obligatory paternal alleles in a DNA database to find an alleged rapist in a fatherless paternity case.

A sexual assault case resulted in a pregnancy, which was subsequently aborted. The alleged father of the fetus was unknown. Maternal and fetal types were obtained using the 11-locus AmpFℓSTR(®) SGM Plus(®) kit. The national DNA database was searched for the paternal obligatory alleles and detected two suspects who could not be excluded as father of the male fetus. Additional typing using the AmpFℓSTR(®) Minifiler(™) kit, containing three additional autosomal loci, was not sufficient to exclude either suspect. Subsequent typing using the PowerPlex(®) 16, containing four additional loci, and Y-Filer(™) kits resulted in excluding one suspect. Searching a database for paternal obligatory alleles can be fruitful, but is fraught with possible false positive results so that finding a match must be taken as only preliminary evidence.

Effect of benzyladenine on recovery of cryopreserved shoot tips of grapevine and citrus cultured in vitro.

The effect of N6-benzyladenine (BA) on the recovery of cryopreserved shoot tips of the LN33 hybrid (Vitis L.) and Troyer citrange [Poncirus trifoliata (L.) Raf. x Citrus sinensis [L.] Osbeck.] cultured in vitro was examined. For the LN33 hybrid, the presence of BA in the recovery medium was essential for survival of control and cryopreserved shoot tips, although the BA concentration did not influence the survival percentage. BA at 5, 2, and 5 microM or higher induced callus formation in control, and shoot tips cryopreserved by vitrification, and by encapsulation-dehydration, respectively. While a BA concentration of 4 microM was found optimal for recovery of control shoot tips, 1 and 2-4 microM produced the best recovery of shoot tips cryopreserved by vitrification and encapsulation-dehydration, respectively. A similar pattern of effect of BA on recovery was found for 'Troyer' citrange. Low survival of control and cryopreserved shoot tips was observed with a BA-free recovery medium. The addition of BA to the recovery medium significantly increased survival. The BA concentration that induced callus formation in shoot tips cryopreserved by encapsulation-vitrification was higher than that which induced it in those cryopreserved by encapsulation-dehydration. Recovery of control shoot tips was best with an addition of 6-10 microM BA to the medium. Optimal recovery of shoot tips cryopreserved by encapsulation-vitrification and encapsulation-dehydration was achieved with 3-4 and 2 microM BA, respectively. Results from the present study suggest that an optimal BA concentration for recovery of control shoot tips may be different from that for cryopreserved shoot tips; furthermore, the optimal BA concentration for recovery of cryopreserved shoot tips may also differ among different cryogenic procedures.

'The story of Abraham, Isaac and Jacob" or "am I my brother's keeper?".

Presented is a case report of a violent sexual assault where the DNA profile obtained from an item of evidence was compared to a suspect's profile. The profiles did not match, but the sharing of such a large number of alleles raised the suspicion that perhaps the real perpetrator was a blood relative of the suspect. The investigators requested a sample from the suspect's brother, and a match was defined. In an era of technological breakthroughs in the field of forensic DNA analysis, the importance of the scientist's attention to the evidence presented in each case is stressed.

Presentation of a three-banded allele pattern--analysis and interpretation.

The Israel police forensic biology laboratory received as an item of evidence in an attempted murder case, a pair of trousers belonging to a suspect. A bloodstain was observed on the trousers and analyzed by STR typing for nine loci using the Promega GenePrint STR silver stain detection kits. The genetic profile defined was found to be identical to that of the victim's at all nine loci. Within this profile a three-banded allele pattern was observed at the D16S539 locus, both in the bloodstain and in the victim's reference blood sample. Confirmation of this phenomenon was accomplished by amplifying the extracted DNA from both the trousers and the victim's blood sample using the PowerPlex 16 kit by Promega and the AmpFlSTR SGM Plus kit by Perkin Elmer, followed by analysis of the amplification products by capillary electrophoresis on the ABI prism 310 genetic analyzer. The same three-banded allele pattern was observed at the D16S539 locus in both specimen and reference DNA, using each of the three kits. Three additional loci located on chromosome 16 (D16S3407, D16S2617 and D16S3082), not employed for forensic identification, were also analyzed and did not show three-banded allele pattern.