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BioSpotlight Citations

BioSpotlight Citations

Persistence of Biological Traces at Inside Parts of a Firearm from a Case of Multiple Familial Homicide

Backspatter from wounds caused by contact shots against a biological target had before been shown to be propelled into firearms' barrels where they can persist and be retrieved from as relevant forensic evidence. Herein, that insight was applied to the investigation of a case of multiple familial homicide with a firearm. Samples of backspatter were collected from the firearm using DNA-free swabs. DNA was extracted from the swabs, and 16 STR systems were PCR-amplified to generate DNA profiles of all victims shot by the firearm. The quality of the resulting DNA profiles was sufficient to exclude the perpetrator as donor and to differentiate the three closely related victims thereby proving that all three victims had been shot by the same firearm from very close or contact distance. A key insight gained from this case was that not only a firearms' barrel inside but other inner surfaces may be charged with profilable DNA.

Water yam (Dioscorea alata L.) diversity pattern in Brazil: an analysis with SSR and morphological markers

Substantial progress was made in the last decade in understanding the diversity in Dioscorea species. However, most of the studies so far concentrated in the ‘yam belt in Africa’. We present a genetic diversity and structure analysis among commercial and local varieties of water yam (Dioscorea alata L.) in Brazil using microsatellite and morphological markers. Twelve microsatellite primers were used to generate DNA profiles of 72 local varieties and 17 commercial accessions of water yam collected in four different regions in Brazil. Also, four morphological traits were evaluated on individual plants under field conditions. The morphological characterization showed considerable diversity. High polymorphism was found with 100 % polymorphism observed for 11 primers and a discriminating power value of 0.92, on average. We did not observe a strong population structure among sampling regions, while most of the genetic diversity was concentrated within regions (95.9 %). Analysis of the relationship between accessions did not clearly separate the local and commercial genotypes. However, the molecular analyses of D. alata showed high intraspecific diversity in local accessions from different regions in Brazil, with the highest Shannon index value (H′ = 0.41) for the Southeast region. These results confirmed an admixture of accessions in all sampling regions, consistent with the lack of a significant correlation between geographic and genetic distances, suggesting that farmers exchanged water yam materials extensively. The genetic diversity can be explained by the result of a continuous exchange of accessions by farmers throughout Brazil.

Técnicas de tipificación molecular para la vigilancia y control de la infección

Molecular typing methods are useful in the surveillance and control of nosocomial outbreaks because they can provide information on the clonal relatedness among isolates, identify reservoirs, and determine routes of transmission. The gold standard assay for molecular typing is pulsed-field gel electrophoresis (PFGE) due to its high discriminatory power. Some major disadvantages of PFGE include the high cost of the equipment, its labor intensiveness (the technique is not automated) and the time required to analyze the profiles of DNA bands (pulsotypes). Although there are many molecular typing methods based on polymerase-chain reaction (PCR), the most widely used is repetitive sequence-based PCR (REP-PCR). Most of the PCR techniques used for molecular typing have none of the limitations of PFGE as they are less expensive and labor intensive (some, such as bioMérieux's Diversilab system, are commercially available) and generate DNA profiles that are easier to interpret, depending on the microorganism. The discriminatory power of PCR is generally lower than or similar to that of PFGE. Both PFGE and PCR require optimal laboratory standardization to guarantee good reproducibility. PCR methods are preferable in the study of small, timelimited outbreaks. In more complex outbreaks of longer duration, in which clonal evolution and dynamics are studied, the use of PFGE is preferable. Molecular typing methods based on DNA sequencing, such as multilocus sequence typing, are applicable in global epidemiological studies or in analyses of the population structure of microorganisms.

Molecular characterization of selected wild species and miniature roses based on SSR markers

Interspecific hybridization in roses (Rosa spp.) has generated thousands of cultivars with commercial value, but only ten species are commonly represented in the pedigrees of modern cultivars. This would suggest that unexploited wild rose species have the potential to broaden the overall gene pool of rose breeding programs in general. We utilized previously described microsatellite (SSR) markers to generate DNA profiles in order to assess the genetic diversity of 44 selected rose genotypes among which are represented wild species, miniature roses and interspecific hybrids. A total of 83 alleles were scored using 15 SSR loci. The number of distinct alleles per locus ranged from three to eight, with an average of 5.5 per locus. Genetic distance matrices were used to generate dendrogram based on the procedure of minimum variance or Ward's method of clustering using the Multivariate Statistical Package. Cluster analysis groupings for the 44 genotypes segregated into three major categories which is consistent with taxonomy and their respective ploidy levels with only a few exceptions. Similarity coefficients determined for wild rose populations had a lower average value of 0.66 (ranged from 0.47 to 0.98) than those calculated for domesticated miniature roses and hybrid breeding lines which had an average value of 0.73 (ranged from 0.57 to 0.83). This indicates that modern rose hybrids have a narrower genetic base than wild relatives, and could benefit from the integration of valuable wild rose germplasm into the efforts of the hybrid rose breeding profession.

Automating a combined composite–consensus method to generate DNA profiles from low and high template mixture samples

We present an automated method to generate DNA profiles from replicate PCRs by combining advantages of the composite and consensus method by a system of brackets in which an allelic balance threshold is used as a variable to separate DNA-profiles of major from minor donors. Through the analysis of artificial low (125pg) and high (250pg) template three-person mixtures with low (1:1.5:3) and high (1:5:10) donor ratios we demonstrate the usefulness of a tool to determine the optimal allelic balance threshold within a locus. The automated extraction of dominant profiles saves considerable amounts of time when producing composite–consensus profiles. Drop-in/drop-out rates are produced and a comparison is made with an alternative open source script to evaluate the dominant profiles generated. By introducing this script into the forensic community we hope to increase awareness of much needed collaborative efforts with bioinformaticians and statisticians to develop forensic open source software scripts.

Persistence of biological traces in gun barrels—an approach to an experimental model

Traces of backspatter in gun barrels after homicidal or suicidal contact shots may be a valuable source of forensic evidence. Yet, a systematic investigation of the persistence and durability of DNA from biological traces in gun barrels is lacking. Our aim was to generate a realistic model to emulate blood and tissue spatters in gun barrels generated by contact gunshots at biological targets and to analyse the persistence and typability of DNA recovered from such stains. Herein, we devise and evaluate three different models for the emulation of backspatter from contact shots: a gelatine-based model with embedded blood bags, a model based on a spongious matrix soaked with blood and covered with a thin plastic membrane and a head model consisting of an acrylic half sphere filled with ballistic gelatine and with blood bags attached to the sphere under a 3-mm silicone layer. The sampling procedure for all three models: a first shot was fired with several types of guns at each model construction and subsequently a second shot was fired at a backstop. Blood samples were collected after each shot by probing the inner surface of the front and rear end of the respective gun barrel with a sterile swab. DNA was then extracted and quantified and up to 20 different short tandem repeat (STR) systems were amplified to generate DNA profiles. Although DNA quantity and STR typing results were heterogenous between the models, all models succeeded in delivering full STR profiles even after more than one shot. We conclude that biological traces in gun barrels are robust and accessible to forensic analysis and that systematic examination of the inside of gun barrels may be advisable for forensic casework.

A comparison between direct PCR and extraction to generate DNA profiles from samples retrieved from various substrates

Direct PCR generates DNA profiles from samples without using the extraction process. During sample extraction, DNA may be lost due to the methods used, which can affect the quality of the DNA profile obtained. This is not the case with direct PCR, where the sample is transferred directly into the PCR tube. Here, we report on the ability of direct PCR to generate DNA profiles from low amounts of control DNA retrieved from various surfaces using PowerPlex 16 HS. A comparison is made with samples undergoing a preliminary extraction stage using QiaAmp DNA Micro kits. Samples subjected to direct PCR generated DNA profiles with higher peak heights and lower allele dropout on all the different substrates tested when compared to the samples subjected to extraction. The amount of DNA retrieved from each substrate also varied even though the same amount of starting material was deposited, proving that the type of substrate can affect the retrieval of DNA.

DNA analysis and its application to forensic investigations at the victorian institute of forensic medicine

The use of DNA analysis has become an integral part of forensic investigations, with many forensic laboratories relying on DNA to assist with criminal investigations as well as the identification of deceased persons. Since the late 1990 s, the Victorian Institute of Forensic Medicine (VIFM) has provided DNA identification services for deceased persons to the Coroners Court of Victoria (CCOV), utilising nuclear DNA (nDNA) and/or mitochondrial DNA (mitDNA) in its analysis. Imperative to DNA analysis is not only the type of sample collected, but also the manner in which the sample is collected and ultimately processed. Through years of experience, the VIFM has optimised sample collection and analysis methods to achieve a high success rate for DNA profiling. In addition, the VIFM has developed standard operating procedures to minimise contamination during the collection of samples destined for DNA analysis during post-mortem examinations, as well as during the analysis process itself. nDNA analysis, in particular short tandem repeat (STR) analysis, is routinely used at the VIFM to generate DNA profiles for deceased persons and reference samples to assist in familial matching, and hence assist in the identification of the deceased person. The STR analysis is performed on 16 loci (15 autosomal and 1 sex determining loci) which provide sufficient discriminatory power to enable familial or kinship matching. In addition, the VIFM is accredited to perform mitDNA analysis for forensic applications. mitDNA is inherited through the maternal line, so unless a mutation has occurred, siblings and all maternal relatives share the same mitDNA sequence. Human mitDNA is comprised of a small circular genome (16,569 bp) that contains highly variable regions with sufficient variation to enable human identification. Its robustness has led to its use as a reliable forensic tool; in recent times enabling the analysis of human remains from casualties of war or mass disasters. Whilst it is not as informative as nDNA analysis, mitDNA is very useful when sufficient quantities of nDNA cannot be extracted for a case, be it from bone, teeth or decaying tissue. In recent years, mitDNA analysis has been used to assist in coronial case investigations ranging from missing persons, where putative mother/child relationship have been discounted or affirmed by mitDNA analysis, to homicides. Discussed will be the application of DNA analysis to forensic investigations at the VIFM to assist with coronial case and police investigations, from sample collection through to analysis. The use of DNA analysis has become an integral part of forensic investigations, with many forensic laboratories relying on DNA to assist with criminal investigations as well as the identification of deceased persons. Since the late 1990 s, the Victorian Institute of Forensic Medicine (VIFM) has provided DNA identification services for deceased persons to the Coroners Court of Victoria (CCOV), utilising nuclear DNA (nDNA) and/or mitochondrial DNA (mitDNA) in its analysis. Imperative to DNA analysis is not only the type of sample collected, but also the manner in which the sample is collected and ultimately processed. Through years of experience, the VIFM has optimised sample collection and analysis methods to achieve a high success rate for DNA profiling. In addition, the VIFM has developed standard operating procedures to minimise contamination during the collection of samples destined for DNA analysis during post-mortem examinations, as well as during the analysis process itself. nDNA analysis, in particular short tandem repeat (STR) analysis, is routinely used at the VIFM to generate DNA profiles for deceased persons and reference samples to assist in familial matching, and hence assist in the identification of the deceased person. The STR analysis is performed on 16 loci (15 autosomal and 1 sex determining loci) which provide sufficient discriminatory power to enable familial or kinship matching. In addition, the VIFM is accredited to perform mitDNA analysis for forensic applications. mitDNA is inherited through the maternal line, so unless a mutation has occurred, siblings and all maternal relatives share the same mitDNA sequence. Human mitDNA is comprised of a small circular genome (16,569 bp) that contains highly variable regions with sufficient variation to enable human identification. Its robustness has led to its use as a reliable forensic tool; in recent times enabling the analysis of human remains from casualties of war or mass disasters. Whilst it is not as informative as nDNA analysis, mitDNA is very useful when sufficient quantities of nDNA cannot be extracted for a case, be it from bone, teeth or decaying tissue. In recent years, mitDNA analysis has been used to assist in coronial case investigations ranging from missing persons, where putative mother/child relationship have been discounted or affirmed by mitDNA analysis, to homicides. Discussed will be the application of DNA analysis to forensic investigations at the VIFM to assist with coronial case and police investigations, from sample collection through to analysis.

Disaster victim investigation recommendations from two simulated mass disaster scenarios utilized for user acceptance testing CODIS 6.0

Members of the National DNA Data Bank (NDDB) of Canada designed and searched two simulated mass disaster (MD) scenarios for User Acceptance Testing (UAT) of the Combined DNA Index System (CODIS) 6.0, developed by the Federal Bureau of Investigation (FBI) and the US Department of Justice. A simulated airplane MD and inland Tsunami MD were designed representing a closed and open environment respectively. An in-house software program was written to randomly generate DNA profiles from a mock Caucasian population database. As part of the UAT, these two MDs were searched separately using CODIS 6.0. The new options available for identity and pedigree searching in addition to the inclusion of mitochondrial DNA (mtDNA) and Y-STR (short tandem repeat) information in CODIS 6.0, led to rapid identification of all victims. A Joint Pedigree Likelihood Ratio (JPLR) was calculated from the pedigree searches and ranks were stored in Rank Manager providing confidence to the user in assigning an Unidentified Human Remain (UHR) to a pedigree tree. Analyses of the results indicated that primary relatives were more useful in Disaster Victim Identification (DVI) compared to secondary or tertiary relatives and that inclusion of mtDNA and/or Y-STR technologies helped to link family units together as shown by the software searches. It is recommended that UHRs have as many informative loci possible to assist with their identification. CODIS 6.0 is a valuable technological tool for rapidly and confidently identifying victims of mass disasters.

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.

DNA profiling of complex bacterial populations: toxic cyanobacterial blooms

Cyanobacteria are prokaryotic photosynthetic living organisms that inhabit our planet for over three billion years. With a worldwide distribution, they can be found in all types of environments: fresh, brackish and saltwater as well as terrestrial. Though beneficial in the development of life on earth, they also constitute a serious risk to our ecosystems since they can biologically produce harmful secondary metabolites named cyanotoxins. When studying cyanobacteria and their cyanotoxins, several methodologies have been applied with an increasing relevance to molecular methods. Therefore, the aim of this review is to describe alternative molecular methods that can be used as alternative methods for the identification of cyanobacteria. More traditional chemotaxonomic methods are discussed briefly as are the standard and somewhat dated techniques for assessing genome content for taxonomic classification schemes. The use of DNA amplification technology has been applied to the systematics and phylogeny of many bacterial groups, and the optimisation of methods for rapid identification and classification of cyanobacteria are presented. Together with novel methods developed for these photosynthetic microorganisms, the generated DNA profiles have been utilised to study cyanobacterial bloom population diversity and prediction of strain toxigenicity. Finally, the genotypes found were applied to a variety of phylogenetic analyses; trees were reconstructed and compared to the current morphological system of classification. The ecology and diversity of the cyanobacteria is discussed with respect to the derived molecular phylogenies and systematics.

Forensic DNA Typing: Quo Vadis?

Since Alec Jeffreys coined the term individual-specific genetic fingerprints in 1985, DNA typing has become indispensable in forensic analysis, having as its central rationale the evidential power of the match between trace evi- dence and suspect. Established technological advances in this field include the adoption of the polymerase chain reaction (PCR) to generate DNA profiles from minute biological samples, the use of mitochondrial DNA to obtain information from old bones and the application of Y chromosomal polymorphisms in sexual assault cases. Present research questions focus on genetic markers for external visible traits and on improving the utility of poor quality samples with degraded DNA. The first valid marker for a physical trait was the sex marker amelogenin, which has been part of short tandem repeat (STR) multiplex PCRs for over a decade. Current studies are investigating markers for human pigmen- tation, predicting hair, iris and skin colour. The relationship between genome and pigmentation however is complex, involving numerous genes. Single nucleotide polymorphisms (SNPs) in these genes make promising candidates for predictive markers. Physical stature is also a complex genetic trait where study subjects can be phenotyped easily. Linkage analy- sis has revealed several short stature-quantitative trait loci, and polymorphisms in the fibrillin I gene are asso- ciated with tall stature.

Spatial Variation in Bacterial DNA Profiles for Forensic Soil Comparisons

ABSTRACTThe feasibility of using bacterial DNA profiling of soil in forensic investigations is evaluated. Terminal restriction fragment (TRF) analysis of 16S ribosomal DNA may be employed to generate DNA profiles for use to discriminate between soil samples. To assess the localisation of the profiles and strengthen any evidential value of TRF analysis, small-scale spatial variability has been examined. Soil samples were collected at intervals along 2 m horizontal transects of three different ecosystems—a field, a forest, and a dune system—and TRF profiles compared. 13% to 20% of TRFs were common to all sampling points, for all transects. The mean similarity for TRF profiles at all sampling points ranged from 49.3% for the dune system to 58.4% for the field transect. ‘Patch size’ (distance along the transect in which DNA profiles showed highest similarity) ranged from 20–80 cm for each ecosystem. This small- scale variability may cause problems if forensic ‘matching’ of soil DNA profiles is attempted, unless the precise location from which the soil sample was originally taken could be identified. The implications of these findings for forensic fingerprinting of soil samples are discussed.

The Successful DNA Typing of Samples Following a Thermal Cycler Power Loss*

An approach for generating DNA profiles when critical samples have been consumed and a power outage occurs during the polymerase chain reaction (PCR) amplification reaction is described. This study demonstrates that a complete and accurate DNA short tandem repeat profile can be obtained: (1) when single source DNA samples are amplified for 26, 27, or 28 cycles using the Profiler Plus and COfiler Amplification Kits after an interruption in amplification, (2) from mock samples when PCR amplification has been interrupted early (after five cycles) or late (after 18 cycles) and the sample is subjected to an additional round of amplification, even after incubation of the sample at room temperature overnight, and (3) from nonprobative casework samples interrupted after approximately 18 cycles of amplification, an overnight incubation at room temperature and subjected to one or two additional rounds of PCR amplification for approximately 26 total cycles. Samples interrupted before five completed cycles and subjected to additional PCR cycles yielded variable results.

PCR Marker-based Analysis of Wild and Cultivated Yams (Dioscorea spp.) in Nigeria: Genetic Relationships and Implications for ex situ Conservation

Reliable characterization of the variation among wild and cultivated yams in Nigeria is essential for improved management and efficient utilization of yam genetic resources. RAPD and double stringency PCR (DS-PCR) analyses were used to investigate genetic relationships and the extent of redundancy among 30 accessions of two cultivated, and 35 accessions of four wild yam species collected from Nigeria. Twenty-five selected random decamer and two microsatellite primers were used individually and in combination to generate DNA profiles for each accession of the six Dioscorea species. The number of amplified fragments varied from 7 to 18 fragments per primer/primer combination. Different levels of intraspecific genetic diversity were found, with Dioscorea rotundata Poir. being the most variable. Based on identical profiles for the RAPD and DS-PCR primers, 12 duplication groups consisting of a total number of 37 accessions were observed in the present study. An UPGMA analysis grouped the majority of plants according to the species. Cultivated yams belonging to the D. cayenensis–rotundata species complex, which were classified into seven morphotypes/varietal groups, could be clearly separated into two major groups corresponding to D. rotundata Poir. and D. cayenensis Lam. D. cayenensis cultivars exhibited a low level of intraspecific variation and were genetically close to the wild species Dioscorea burkilliana J. Miege. D. rotundata cultivars classified into six varietal groups showed a high degree of DNA polymorphism and were separated into two major groups that appeared most closely related to Dioscorea praehensilis Benth. and Dioscorea liebrechtsiana de Wild. We propose, based on these results, that cultivars classified into D. cayenensis should be considered as a taxon separate from D. rotundata. The implications of intraspecific variability for the ex situ conservation of wild and cultivated yam germplasm in Nigeria are discussed.

Forensics

Forensics

Authentication of scientific human cell lines: easy-to-use DNA fingerprinting.

Human cell lines are an important resource for research and most often used in reverse genetic approaches or as in vitro model systems of human diseases. In this regard, it is crucial that the cells faithfully correspond to the purported objects of study. A number of recent publications have shown an unacceptable level of cell lines to be false, in part as a result of the nonavailability of a simple and easy DNA profiling technique. We have validated different single- and multiple-locus variable numbers of tandem repeats (VNTRs) enabling the establishment of a noncommercial, but good laboratory practice, method for authentication of cell lines by DNA fingerprinting. Polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of six prominent and highly polymorphic minisatellite VNTR loci, requiring only a thermal cycler and an electrophoretic system, was proven as the most reliable tool. Furthermore, the generated banding pattern and the determination of gender allows for verifying the authenticity of a given human cell line by simple agarose gel electrophoresis. The combination of rapidly generated DNA profiles based on single-locus VNTR loci and information on banding patterns of cell lines of interest by official cell banks (detailed information at the website www.dsmz.de) constitute a low-cost but highly reliable and robust method, enabling every researcher using human cell lines to easily verify cell line identity.

Rapid identification of polymorphic microsatellite loci in a forest pathogen, Dothistroma pini, using anchored PCR

A microsatellite-based DNA profiling system was developed that can be used to distinguish genetically diverse isolates of a forest pathogen, Dothistroma pini. All isolates of this pathogen identified in New Zealand so far appear to be isogenic and the disease is kept under control by aerial applications of copper fungicide. Although New Zealand has strict importation controls in place, the possibility of genetically diverse isolates of D. pini being introduced from other countries poses a severe biosecurity threat to forest health. Therefore a DNA-based monitoring system was developed. Two informative microsatellite loci were found serendipitously in D. pini sequence data available in our laboratory. Further microsatellite loci were obtained using a rapid 5’-anchored PCR amplification technique. For each informative locus identified, specific primers were designed to flank the repeated sequence and subsequently used to generate DNA profiles for the D. pini strains. The profiles obtained from five microsatellite loci were sufficient to distinguish most isolates tested. The anchored primer technique provides an efficient tool for the identification of polymorphic loci that can be used to screen for genetic differences between fungi.

Loss of Heterozygosity Detected in a Short Tandem Repeat (STR) Locus Commonly Used for Human DNA Identification

Short tandem repeat (STR) markers are commonly used in basic genetic research and in human identification testing. Clinically, STRs can be used to study genetic alterations in tumors. A genetic deletion common to many types of cancer is referred to as the loss of heterozygosity (LOH). Numerous examples of LOH in cancer have been described and some have been mapped to areas located in close proximity to markers employed in human identity testing. Despite this fact, LOH has rarely been observed for STR loci commonly employed in forensic testing. Recently, for medico-legal purposes, we were asked to determine whether a tissue biopsy originated from a particular individual. For a reference source we assessed two specimens, one from normal tissue and one from cancerous tissue. When both reference specimens were used to generate DNA profiles, we observed LOH at one STR locus, D13S317. As demonstrated in other cancers only the cancerous biopsy demonstrated LOH. The forensic community should be cognizant of these unusual circumstances because, as identification of human DNA continues to be used more extensively, certain instances will arise in which reference material will not be readily available. In these situations, archived specimens may be employed as a reference source. Clinical specimens such as tissue biopsies should be used with caution if they have not been confirmed to contain normal tissue.