Search

There are situations in which it would be very valuable to have a DNA profile within a short time; for example, in mass disasters or airport security. In previous work, we have promoted reduced size STR amplicons for the analysis of degraded DNA. We also noticed that shorter amplicons are more robust during amplification, making them inhibition resistant, and potentially applicable to high-speed direct PCR. Here, we describe a set of miniSTRs capable of rapid direct PCR amplification. The selected markers are a subset of the Combined DNA Index System (CODIS) loci modified to permit high-speed amplification. Using the proposed protocol, the amplification of eight loci plus amelogenin directly from a saliva sample can be completed in 7 min and 38 s using a two-step PCR with 30 cycles of 98°C for 2 s and 62°C for 7 s on a Streck Philisa thermocycler. Selection of DNA polymerase, optimization of the two-step PCR cycling conditions, the primer concentrations, and the dilution of saliva is described. This method shows great potential as a quick screening method to obtain a presumptive DNA profile when time is limited, particularly when combined with high-speed separation and detection methods., (© 2021 Wiley-VCH GmbH.)

Cellular material derived from contact traces can be transferred via many direct and indirect routes, with the manner of contact and the time of transfer (in relation to the alleged crime-event) having an impact on whether DNA is recovered from the surface and a reportable profile generated. In an effort to acquire information on the transfer and recovery of DNA traces from clothing items worn during scenarios commonly encountered in casework, upper garments were worn during a normal working day before individuals were paired to embrace one another ('contact'), go on an outing together ('close proximity'), or individually asked to spend a day in another person's environment ('physical absence'). Each prescribed activity was repeated by sixteen individuals across four countries, and was the last activity performed before the garment was removed. Samples were collected from several areas of the upper garments and processed from DNA extraction through to profiling within the laboratory of the country in which the individual resided. Activities relating to the garment prior to and during wearing, including the prescribed activity, were recorded by the participant and considered during the interpretation of results. In addition to obtaining reference profiles from the wearer and their activity partner, DNA profiles from the wearers' close associates identified in the questionnaire were obtained to assess the impact of background DNA transferred prior to the prescribed activity. The wearer was typically, but not always, observed as the major contributor to the profiles obtained. DNA from the activity partner was observed on several areas of the garment following the embrace and after temporarily occupying another person's space. Particular areas of the garment were more prone to acquiring the hugging partner or office owner's DNA than others, and whether they were observed as the major or minor component was activity dependent. For each of the pairs, no DNA from the activity partner was acquired by the garments during the outing, even though both participants were in close proximity. This study provides empirical data on the transfer, persistence, prevalence and recovery of DNA from clothing items, and enables a better understanding of the mechanisms which lead to the transfer and detectability of DNA traces in different scenarios., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Among the various items recovered from crime scenes or persons involved in a crime event, clothing items are commonly encountered and submitted for forensic DNA sampling. Depending on the case circumstances and the activity-of-interest, sampling of the garment may concentrate on collecting DNA from the wearer, or from one or more offenders who have allegedly contacted the item and/or wearer. Relative to the targeted DNA, background DNA already residing on the item from previous contacts, or transferred during or after the crime event, may also be collected during sampling and observed in the resultant DNA profile. Given our limited understanding of how, and from where, background DNA is derived on clothing, research on the transfer, persistence, prevalence, and recovery (TPPR) of DNA traces from upper garments was conducted by four laboratories. Samples were collected from several areas of two garments, each worn on separate working or non-working days and individually owned by four individuals from each of the four laboratories, and processed from DNA extraction through to profiling. Questionnaires documented activities relating to the garment prior to and during wearing, and reference profiles were obtained from the wearer and their close associates identified in the questionnaire. Among the 448 profiles generated, variation in the DNA quantity, composition of the profiles, and inclusion/exclusion of the wearer and their close associates was observed among the collaborating laboratories, participants, garments worn on different occasions, and garment areas sampled., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Sharing data between forensic scientists on DNA transfer, persistence, prevalence and recovery (TPPR) is crucial to advance the understanding of these issues in the criminal justice community. We present the results of a collaborative exercise on reporting forensic genetics findings given activity level propositions. This exercise outlined differences in the methodology that was applied by the participating laboratories, as well as limitations to the use of published data on DNA TPPR. We demonstrate how publication of experimental results in scientific journals can be further improved to allow for an adequate use of these data. Steps that can be taken to share and use these data for research and casework purposes are outlined, and the prospects for future sharing of data through publicly accessible databases are discussed. This paper also explores potential avenues to proceed with implementation and is intended to fuel the discussion on sharing data pertaining to DNA TPPR issues. It is further suggested that international standardization and harmonization on these topics will benefit the forensic DNA community as it has been achieved in the past with the harmonization of STR typing systems., (Crown Copyright © 2018. Published by Elsevier B.V. All rights reserved.)

In forensic investigations involving human biological traces, cell type identification is often required. Identifying the cell type from which a human STR profile has originated can assist in verifying scenarios. Several techniques have been developed for this purpose, most of which focus on molecular characteristics of human cells. Here we present a microarray method focusing on the microbial populations that are associated with human cell material. A microarray with 863 probes targeting (sets of) species, specific genera, groups of genera or families was designed for this study and evaluated with samples from different body sites: hand, foot, groin, penis, vagina, mouth and faeces. In total 175 samples from healthy individuals were analysed. Next to human faeces, 15 feline and 15 canine faeces samples were also included. Both clustering and classification analysis were used for data analysis. Faecal and oral samples could clearly be distinguished from vaginal and skin samples, and also canine and feline faeces could be differentiated from human faeces. Some penis samples showed high similarity to vaginal samples, others to skin samples. Discriminating between skin samples from different skin sites proved to be challenging. As a proof of principle, twenty-one mock case samples were analysed with the microarray method. All mock case samples were clustered or classified within the correct main cluster/group. Only two of the mock case samples were assigned to the wrong sub-cluster/class; with classification one additional sample was classified within the wrong sub-class. Overall, the microarray method is a valuable addition to already existing cell typing techniques. Combining the results of microbial population analysis with for instance mRNA typing can increase the evidential value of a trace, since both techniques focus on independent targets within a sample., (Copyright © 2018 Elsevier B.V. All rights reserved.)

The use of DNA methylation (DNAm) for chronological age determination has been widely investigated within the last few years for its application within the field of forensic genetics. The majority of forensic studies are based on blood, saliva, and buccal cell samples, respectively. Although these types of samples represent an extensive amount of traces found at a crime scene or are readily available from individuals, samples from other tissues can be relevant for forensic investigations. Age determination could be important for cases involving unidentifiable bodies and based on remaining soft tissue e.g. brain and muscle, or completely depend on hard tissue such as bone. However, due to the cell type specificity of DNAm, it is not evident whether cell type specific age-dependent CpG positions are also applicable for age determination in other cell types. Within this pilot study, we investigated whether 13 previously selected age-dependent loci based on whole blood analysis including amongst others ELOVL2, TRIM59, F5, and KLF14 also have predictive value in other forensically relevant tissues. Samples of brain, bone, muscle, buccal swabs, and whole blood of 29 deceased individuals (age range 0-87 years) were analyzed for these 13 age-dependent markers using massive parallel sequencing. Seven of these loci did show age-dependency in all five tissues. The change of DNAm during lifetime was different in the set of tissues analyzed, and sometimes other CpG sites within the loci showed a higher age-dependency. This pilot study shows the potential of existing blood DNAm markers for age-determination to analyze other tissues than blood. We identified seven known blood-based DNAm markers for use in muscle, brain, bone, buccal swabs, and blood. Nevertheless, a different reference set for each tissue is needed to adapt for tissue-specific changes of the DNAm over time., (Copyright © 2018 Elsevier B.V. All rights reserved.)

In disaster victim identification (DVI), DNA profiling is considered to be one of the most reliable and efficient means to identify bodies or separated body parts. This requires a post mortem DNA sample, and an ante mortem DNA sample of the presumed victim or their biological relative(s). Usually the collection of an adequate ante mortem sample is technically simple, but the acquisition of a good quality post mortem sample under unfavourable DVI circumstances is complicated due to the variable degree of preservation of the human remains and the high risk of DNA (cross) contamination. This paper provides the community with an efficient method to collect post-mortem DNA samples from muscle, bone, bone marrow and teeth, with a minimal risk of contamination. Our method has been applied in a recent, challenging DVI operation (i.e. the identification of the 298 victims of the MH17 airplane crash in 2014). 98,2% of the collected PM samples provided the DVI team with highly informative DNA genotyping results without the risk of contamination and consequent mistyping the victim's DNA. Moreover, the method is easy, cheap and quick. This paper provides the DVI community with a step-wise instructions with recommendations for the type of tissue to be sampled and the site of excision (preferably the upper leg). Although initially designed for DVI purposes, the method is also suited for the identification of individual victims., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Analysis of human DNA methylation (DNAm) can provide additional investigative leads in crime cases, e.g. the type of tissue or body fluid, the chronological age of an individual, and differentiation between identical twins. In contrast to the genetic profile, the DNAm level is not the same in every cell. At the single cell level, DNAm represents a binary event at a defined CpG site (methylated versus non-methylated). The DNAm level from a DNA extract however represents the average level of methylation of the CpG of interest of all molecules in the forensic sample. The variance of DNAm levels between replicates is often attributed to technological issues, i.e. degradation of DNA due to bisulfite treatment, preferential amplification of DNA, and amplification failure. On the other hand, we show that stochastic variations can lead to gross fluctuation in the analysis of methylation levels in samples with low DNA levels. This stochasticity in DNAm results is relevant since low DNA amounts (1pg - 1ng) is rather the norm than the exception when analyzing forensic DNA samples. This study describes a conceptual analysis of DNAm profiling and its dependence on the amount of input DNA. We took a close look at the variation of DNAm analysis due to DNA input and its consequences for different DNAm-based forensic applications. As can be expected, the 95%-confidence interval of measured DNAm becomes narrower with increasing amounts of DNA. We compared this aspect for two different DNAm-based forensic applications: body fluid identification and chronological age determination. Our study shows that DNA amount should be well considered when using DNAm for forensic applications., (Copyright © 2017 Elsevier B.V. All rights reserved.)

To address questions on the activity that led to the deposition of biological traces in a particular case, general information on the probabilities of transfer, persistence and recovery of cellular material in relevant scenarios is necessary. These figures may be derived from experimental data described in forensic literature when conditions relevant to the case were included. The experimental methodology regarding sampling, DNA extraction, DNA typing and profile interpretation that were used to generate these published data may differ from those applied in the case and thus the applicability of the literature data may be questioned. To assess the level of variability that different laboratories obtain when similar exhibits are analysed, we performed an inter-laboratory study between four partner laboratories. Five sets of 20 cable ties bound by different volunteers were distributed to the participating laboratories and sampled and processed according to the in-house protocols. Differences were found for the amount of retrieved DNA, as well as for the reportability and composition of the DNA profiles. These differences also resulted in different probabilities of transfer, persistence and recovery for each laboratory. Nevertheless, when applied to a case example, these differences resulted in similar assignments of weight of evidence given activity-level propositions., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The use of DNA methylation (DNAm) to obtain additional information in forensic investigations showed to be a promising and increasing field of interest. Prediction of the chronological age based on age-dependent changes in the DNAm of specific CpG sites within the genome is one such potential application. Here we present an age-prediction tool for whole blood based on massive parallel sequencing (MPS) and a random forest machine learning algorithm. MPS allows accurate DNAm determination of pre-selected markers and neighboring CpG-sites to identify the best age-predictive markers for the age-prediction tool. 15 age-dependent markers of different loci were initially chosen based on publicly available 450K microarray data, and 13 finally selected for the age tool based on MPS (DDO, ELOVL2, F5, GRM2, HOXC4, KLF14, LDB2, MEIS1-AS3, NKIRAS2, RPA2, SAMD10, TRIM59, ZYG11A). Whole blood samples of 208 individuals were used for training of the algorithm and a further 104 individuals were used for model evaluation (age 18-69). In the case of KLF14, LDB2, SAMD10, and GRM2, neighboring CpG sites and not the initial 450K sites were chosen for the final model. Cross-validation of the training set leads to a mean absolute deviation (MAD) of 3.21 years and a root-mean square error (RMSE) of 3.97 years. Evaluation of model performance using the test set showed a comparable result (MAD 3.16 years, RMSE 3.93 years). A reduced model based on only the top 4 markers (ELOVL2, F5, KLF14, and TRIM59) resulted in a RMSE of 4.19 years and MAD of 3.24 years for the test set (cross validation training set: RMSE 4.63 years, MAD 3.64 years). The amplified region was additionally investigated for occurrence of SNPs in case of an aberrant DNAm result, which in some cases can be an indication for a deviation in DNAm. Our approach uncovered well-known DNAm age-dependent markers, as well as additional new age-dependent sites for improvement of the model, and allowed the creation of a reliable and accurate epigenetic tool for age-prediction without restriction to a linear change in DNAm with age., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

DNA analysis has become an essential intelligence tool in the criminal justice system for the identification of possible offenders. However, it appears that about half of the processed DNA samples contains too little DNA for analysis. This study looks at DNA success rates within 28 different categories of trace exhibits and relates the DNA concentration to the characteristics of the DNA profile. Data from 2260 analyzed crime samples show that cigarettes, bloodstains, and headwear have relatively high success rates. Cartridge cases, crowbars, and tie-wraps are on the other end of the spectrum. These objective data can assist forensics in their selection process.The DNA success probability shows a positive relation with the DNA concentration. This finding enables the laboratory to set an evidence-based threshold value in the DNA analysis process. For instance, 958 DNA extracts had a concentration value of 6 pg/μL or less. Only 46 of the 958 low-level extracts provided meaningful DNA profiling data., (© 2016 American Academy of Forensic Sciences.)

Mobile Rapid-DNA devices have recently become available on the market. These devices can perform DNA analyses within 90min with an easy 'sample in-answer out' system, with the option of performing comparisons with a DNA database or reference profile. However, these fast mobile systems cannot yet compete with the sensitivity of the standard laboratory analysis. For the future this implies that Scene of Crime Officers (SoCOs) need to decide on whether to analyse a crime sample with a Rapid-DNA device and to get results within 2h or to secure and analyse the sample at the laboratory with a much longer throughput time but with higher sensitivity. This study provides SoCOs with evidence-based information on DNA success rates, which can improve their decisions at the crime scene on whether or not to use a Rapid-DNA device. Crime samples with a high success rate in the laboratory will also have the highest potential for Rapid-DNA analysis. These include samples from e.g. headwear, cigarette ends, articles of clothing, bloodstains, and drinking items., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Different portions of the feline mitochondrial DNA control region (CR) were evaluated for their informative value in forensic investigations. The 402bp region located between RS2 and RS3 described most extensively in the past is not efficient for distinguishing between the majority of Dutch cats, illustrated by a random match probability (RMP) of 41%. Typing of the whole region between RS2 and RS3, and additional typing of the 5'portion of the feline CR decreases the RMP to 29%, increasing the applicability of such analyses for forensic investigations. The haplotype distribution in Dutch random bred cats (N=113) differs greatly from the distributions reported for other countries, with a single haplotype NL-A1 present in 54% of the population. The three investigated breeds showed haplotype distributions differing from each other and the random bred cats with haplotype NL-A1 accounting for 4%, 29% and 32% of Maine Coon, Norwegian forest cats and Siamese & Oriental cats. These results indicate the necessity of validating haplotype frequencies within continents and regions prior to reporting the value a mtDNA match. In cases where known purebred cats are involved, further investigation of the breed may be valuable., (Copyright © 2015. Published by Elsevier Ireland Ltd.)

Current figures on the efficiency of DNA as an investigative tool in criminal investigations only tell part of the story. To get the DNA success story in the right perspective, we examined all forensic reports from serious (N = 116) and high-volume crime cases (N = 2791) over the year 2011 from one police region in the Netherlands. These data show that 38% of analyzed serious crime traces (N = 384) and 17% of analyzed high-volume crime traces (N = 386) did not result in a DNA profile. Turnaround times (from crime scene to DNA report) were 66 days for traces from serious crimes and 44 days for traces from high-volume crimes. Suspects were truly identified through a match with the Offender DNA database of the Netherlands in 3% of the serious crime cases and in 1% of the high-volume crime cases. These data are important for both the forensic laboratory and the professionals in the criminal justice system to further optimize forensic DNA testing as an investigative tool., (© 2015 American Academy of Forensic Sciences.)

Identifying the victims of a disaster is important for the next of kin, to issue a death certificate and, if necessary, for forensic investigations. In the Netherlands victims are identified by the Dutch disaster victim identification team, which is part of the national forensic investigation team ('Landelijk Team Forensische Opsporing'). Ante-mortem data are collected during the identification process; these include the victim's specific medical characteristics and the DNA profile of the victim and their family members. The victim's own doctor can play an important role in the ante-mortem investigation because of his or her knowledge of their personal medical details, and of the possible availability of samples for establishing a DNA profile. The ante-mortem data are then compared with post-mortem data. For a definitive identification at least 1 primary identification characteristic has to be established from the physical remains - dermatoglyphics, the DNA profile or the dental status.

In the examination of sexual assault cases, DNA typing of vaginal samples mostly occurs after differential DNA extraction. Notwithstanding the differential extraction method, the DNA profiles from the seminal fraction often show the male alleles at low-level in combination with female alleles. This unfavorable ratio male to female DNA is due to a limited amount of sperm cells and an overwhelming quantity of female cells. In this study, we compared standard cotton and nylon flocked swabs for post-coital vaginal sampling. Twelve couples donated 88 vaginal swabs - 44 cotton, 44 nylon flocked - which were taken with a time since intercourse (TSI) up to 84 h. These vaginal swabs were sorted into categories on the basis of the TSI and submitted to (1) microscopic examination for the presence of male cells, (2) presumptive tests for the detection of seminal fluid and (3) DNA typing. Cellular elution was found to be 6-fold more efficient from the nylon flocked swabs. This makes microscopic analysis less time consuming as the higher cell yield and better cell morphology simplify detection of male cells. Both swab types reveal similar results regarding presumptive tests and male DNA typing. Positive presumptive tests (RSID-semen and PSA) were obtained up to 60 h TSI and male autosomal profiles up to 72 h TSI. Interestingly, over 50% of the samples negative for both presumptive tests resulted in informative male STR profiles. After differential extraction, less DNA was left on the nylon flocked swabs and more male DNA was isolated. Our results imply that the use of nylon flocked swabs for vaginal sampling will improve microscopic analysis and DNA typing in the medical forensic investigation of sexual assault cases.

Background: The identification and use of Ancestry-Sensitive Markers (ASMs), i.e. genetic polymorphisms facilitating the genetic reconstruction of geographical origins of individuals, is far from straightforward., Results: Here we describe the ascertainment and application of five different sets of 47 single nucleotide polymorphisms (SNPs) allowing the inference of major human groups of different continental origin. For this, we first used 74 cell lines, representing human males from six different geographical areas and screened them with the Affymetrix Mapping 10K assay. In addition to using summary statistics estimating the genetic diversity among multiple groups of individuals defined by geography or language, we also used the program STRUCTURE to detect genetically distinct subgroups. Subsequently, we used a pairwise F(ST) ranking procedure among all pairs of genetic subgroups in order to identify a single best performing set of ASMs. Our initial results were independently confirmed by genotyping this set of ASMs in 22 individuals from Somalia, Afghanistan and Sudan and in 919 samples from the CEPH Human Genome Diversity Panel (HGDP-CEPH) CONCLUSION: By means of our pairwise population F(ST) ranking approach we identified a set of 47 SNPs that could serve as a panel of ASMs at a continental level.

In this study, we have evaluated the efficacy and the validity of the AmpFISTR SGM plus multiplex PCR typing system when Low Copy Number (LCN) amounts of DNA are processed. The characteristics of SGM plus profiles produced under LCN conditions were studied on the basis of heterozygote balance, between loci balance and stutter proportion based on profiles that were obtained from a variety of mock casework samples. These experiments clearly showed that LCN DNA profiles carry their own characteristic features, which must be taken into account during interpretation. Herewith, we confirmed the data of recent other studies that a comprehensive interpretation strategy is dependent upon multiple replication of the PCR using the same extract together with the proper use of extraction and amplification controls. The limitations of LCN DNA analysis were further studied in a series of single cell PCR experiments using an amplification regime of 34 PCR cycles. The allele dropout phenomenon was demonstrated to its full extent when single cells were analysed. However, the "consensus profile" which was obtained from separate single cell PCR experiments matched the actual profile of the cell donor. Single cell PCR experiments also showed that a further increase of the number of PCR cycles did not result in enhanced sensitivity and had a highly negative effect on the balance of this multiplex PCR system which hampered correct interpretation of the profile. Also, the potential of LCN typing in analysing mixtures of DNA was investigated. It was clearly shown that LCN typing had no advantages over 28 cycles amplification in the detection of the minor component of DNA-mixtures. In addition to the 34 cycles PCR amplification regime, the utility of a new approach that involved reamplification of the 28 cycle SGM plus PCR products with an extra 6 PCR cycles after the addition of fresh AmpliTaq Gold DNA Polymerase was investigated. This approach provides the scientist with an extra typing result that enhances the reliability of the consensus profile, which is commonly retrieved from two separate 34 cycle PCR results. Furthermore, the 28 + 6 cycles approach may be used to screen LCN samples for their potential to produce a 34 PCR cycle profile. Finally and as a last resort the 28 + 6 cycles approach can be used in those cases where no further extract from the crime sample is available. Finally, the potential of LCN typing was demonstrated in typing samples from non-probative and actual casework examples. From a high proportion of samples that failed to demonstrate SGM plus typing results using the standard protocol of 28 cycles, at least partial profiles could be obtained after LCN methods were used. For example, LCN typing was applied in a case where 10-year old samples from bones and teeth that were retrieved from a mass grave had to be identified. This study resulted in the positive identification of a number of victims by comparing the LCN DNA profiles with the profiles from putative relatives. The value of LCN DNA typing was further demonstrated in a strangulation case. The throat of the victim was sampled and only after 34 PCR cycles were we able to reveal that the evidential sample contained a distinct mixture of the victim's own DNA and the DNA of the defendant.

Y-chromosome linked short tandem repeat (STR) loci are inherited as a closely linked haplotype, which appears to remain stable in a given paternal lineage over many generations. In forensic cases, Y-linked STRs are particularly useful for the identification of human remains as well as in rape cases with mixed male/female stain samples. DYS385 is derived from tandemly duplicated segments of the Y chromosome thus giving rise to two fragments of variable length which do not behave like alleles but genotypes. The European DNA Profiling (EDNAP) group has carried out a collaborative exercise among 14 participating laboratories using DYS385 for typing of five unknown bloodstains and a control sample. Furthermore, population data from eight different European countries with samples sizes between 91 and 150 male individuals were collected. The results confirm previous observations that DYS385 is one of the most informative Y-linked STR loci. It could also be demonstrated that reproducible results can be obtained independently from the electrophoretic separation and detection methods used. Thus DYS385 may serve as a useful complementation to the routinely used autosomal STR systems in special cases.

If the DNA profiles of a crime stain and the reference sample from the suspect do not match, the suspect is excluded as the donor of the crime stain. However, in some situations the DNA evidence can suggest that a close relative of the suspect might match the stain, in particular when the reference sample from the suspect and the crime stain share rare alleles. This finding can be important for the authorities. The forensic scientist has to decide whether or not to notify the authorities in these circumstances. To the best of our knowledge there is not yet an objective rule for making this decision. We propose such a decision rule for brothers of the suspect, investigate its performance and address some ethical, legal, and practical aspects. Our calculations can be simply adjusted for other relatives of the suspect.

This paper describes the results of three collaborative exercises which continues the EDNAP theme to explore whether uniformity of DNA profiling results could be achieved between European laboratories using STRs. In an earlier exercise, complex hypervariable AAAG-repeat STR loci were investigated, but reproducibility was found to be poor because of the variation of techniques used by participating laboratories. In the exercise reported here, an internal allelic ladder composed of ACTBP2 and D11S554 fragments was distributed. This ladder was used to size ACTBP2 analysed by a "singleplex" PCR amplification and D11S554 combined with APOAI1 in a separate "duplex" reaction. Laboratories were asked to test 7 blood stains, one of which was a known control, and to report the results to the co-ordinating laboratory. The exercise demonstrated that ACTBP2 showed good reproducibility between laboratories, whereas further testing would be needed to validate APOAI1 and D11S554 for interlaboratory comparisons. In separate exercises, the simple loci D12S391 and D1S1656 were tested; both of these showed excellent reproducibility between laboratories.

This paper describes a collaborative exercise which was intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using two complex short tandem repeat (STR) loci. The loci D21S11 and HUMFIBRA (FGA) were chosen because they are commonly used by different European laboratories. D21S11 has approximately 14 common alleles (f > 0.001), whereas HUMFIBRA has 19 common alleles. Laboratories were asked to test seven blood stains, one of which was a known control, and to report the results to the coordinating laboratory. The exercise demonstrated that complex STRs were amenable to standardisation.

The European DNA Profiling Group (EDNAP) has previously carried out collaborative exercises to determine which STR systems will produce results that can be reproduced by different laboratories. The first EDNAP exercise involving STR systems focused on different types of loci: a simple locus with six common alleles (HUMTH01) and a complex locus with > 35 alleles (ACTBP2). Generally the simpler STR system was found to be readily amenable for use across a wide range of different technologies, whereas a more complex locus presented difficulties. The second EDNAP STR exercise was intended to take the process of investigation a stage further. Some laboratories are developing automation, coupled with fluorescent methods of detection and multiplex applications, whereas others use manual methods involving visual detection techniques such as silver staining. The purpose of this exercise was to determine whether loci amenable to multiplexing with automation (as a quadruplex reaction) could also be successfully used with manual methods, either by multiplexing in duplex reactions or alternatively by using just a single pair of PCR primers.

To introduce the loci LDLR, GYPA, HBGG, D7S8, and GC (PM loci) in Dutch forensic identity testing, allele and genotype frequencies were determined in a Dutch Caucasian population sample, which had previously been typed for the HLADQA1 locus [12]. All 6 loci met Hardy-Weinberg equilibrium expectations, and there is little evidence for association between pairs of loci. The combined power of discrimination for all 6 loci is 0.9997. The allele frequencies of the PM loci were similar to 2 other Caucasian populations [3, 10], and differed from 3 non-Caucasian populations [3].

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profiling results could be achieved between European laboratories using short tandem repeat (STR) loci. Two different STRs were chosen--HUMTH01 and the AT-rich HUMACTBP2 (SE33). The former locus has only five common alleles, whereas the latter is complex and has at least 30 alleles. Laboratories were asked to test seven blood stains and to report the results to the coordinating laboratory. The exercise demonstrated that the simple STR systems such as HUMTH01 are more amenable to adoption as standard loci than complex AT-rich systems.

The HLA DQ alpha amplification and typing kit has been designed to be used by the forensic community for purposes of identity testing. The introduction of any new DNA marker in forensic identity testing requires the establishment of a population database for the relevant population(s). To this end allele and genotype frequencies for the HLA DQ alpha locus were determined in a Dutch Caucasian population sample and compared with 7 other population genetic studies. In our population sample the HLA DQ alpha genotype frequencies did not deviate from Hardy-Weinberg expectations and for this locus the power of discrimination is 0.94. A test for homogeneity of the HLA DQ alpha population data based on the allele frequency counts for 8 Caucasian population samples was performed and significant differences were found (P = 0.007). The differences in the frequency of the HLA DQ alpha 2 and 3 alleles are the major cause of this deviation. No deviation from population homogeneity was observed when we compared the genotype frequency distributions among the 8 Caucasian population samples. Combined with the extensive validation studies from Comey and Budowle and Helmuth et al. this population genetic study will allow HLA DQ alpha typing to be used in forensic identity testing in the Netherlands.

A series of experiments has been performed to evaluate amplification and typing of the D1S80 VNTR locus. The validation study that has been carried out showed that correct D1S80 typing results can be obtained when a defined amplification protocol and a high-resolution polyacrylamide gel electrophoresis method are used. The use of the Chelex extraction protocol has substantially reduced the processing time. DNA-extraction, amplification and subsequent typing can be performed in one day. The discrimination power of this locus is 0.94 in a Dutch Caucasian population sample. The system is extremely sensitive: 0.1 ng of genomic DNA gave a correct typing result. The test could also detect the correct genotypes in mixed samples containing DNA from different individuals. Even if the major type was in a 20-fold excess, the minority type could still be amplified and typed correctly. We have found no deviation from Hardy-Weinberg equilibrium in a Dutch Caucasian population sample. Evidence for the somatic stability of this locus was obtained from a set of experiments where we compared DNA-profiles from corresponding blood, semen and saliva samples. The results of this study suggest that in the near future analysis of the D1S80 locus by DNA-amplification can be applied in actual forensic case work.

This paper describes a collaborative exercise intended to demonstrate whether uniformity of DNA profile results could be achieved between different European laboratories. It was shown that this goal can be obtained provided that a common protocol is followed (specifically the use of a common electrophoretic buffer as being the most important parameter). Generally, lower molecular weight loci (with lower molecular weight fragments) such as YNH24 perform better than higher molecular weight loci such as MS43a. The results of the exercise are discussed in relation to the objectives of the European DNA profiling group (EDNAP).

A collaborative exercise was carried out in 1989 among 12 European forensic laboratories using the single locus VNTR probe pYNH24, the restriction enzyme HinfI, the same set of human genomic DNA samples, and a standardized DNA size marker. The objectives of the exercise were: (1) to study the degree of variation within and between laboratories, (2) to obtain information on requirements for technical standardization allowing the exchange of typing results and (3) to compare different approaches for the identification of allelic DNA fragments of unknown size. Each laboratory carried out up to 10 independent typing experiments using the same DNA samples. The results were analysed independently by two laboratories using three different methods. The results of the exercise demonstrate the correlation of typing that can be achieved within and between laboratories under conditions of minimal standardization.

We investigated whether a radioimmunoassay for prostatic acid phosphatase might be used as a more specific test for the identification of semen in samples from cases of sexual assault than the measurement of total acid phosphatase enzyme activity. The results of the measurement of acid phosphatase by enzyme assay in semen and vaginal swab extracts were compared with the results of the radioimmunoassay. It was found that the radioimmunoassay is a sensitive and more specific method than the enzymic determination of acid phosphatase. Incidentally we have found that a low concentration of an immunological cross reacting acid phosphatase is present in semen free vaginal swab extracts.

The phospholipid exchange proteins of rat liver that catalyze the transfer of phosphatidylcholine and phosphatidylinositol from rat liver microsomes to liposomes, have been purified and characterized. Two proteins were detected with dual specificities catalyzing the transfer of both phosphatidylinositol and phosphatidylcholine. Both proteins showed a strong preference for phosphatidylinositol transferring 8 to 9 times as much of the microsomal phosphatidylinositol pool as the microsomal phosphatidylcholine pool. The two proteins had iso-electric points of 5.1 and 5.3 and were purified 300-fold and 500-fold, respectively. A protein that catalyzed specifically the transfer of phosphatidylcholine, was purified 7000-fold. This protein had an iso-electric point of 8.4 and a molecular weight of approximately 16000 calculated from Sephadex G-50 chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis; the amino acid composition was determined. An antiserum against this protein was raised in rabbits. Treatment of a rat liver supernatant fraction with the antiserum immunoglobulin fraction demonstrated that 60% of the phosphatidylcholine transfer activity is due to this protein.