Laser microdissection methodology in forensic casework

Laser microdissection (LMD) is a tool used in forensic laboratories for the analysis of DNA from specifically targeted cells. In our laboratory, LMD analysis is applied to case samples where it has been shown (or it is anticipated) that there are small numbers of target cells present, such as sperm. In this paper, we extend previous LMD technology developed in this laboratory¹. A method for improved recovery and visualisation of cells from substrates for downstream laser microdissection and DNA analysis is discussed and the optimal numbers of LMD-recovered sperm and epithelial cells for Low Copy Number (LCN) DNA analysis is determined.     

Sex-specific fluorescent labelling of cells for laser microdissection and DNA profiling

Sex-specific isolation of cells from mixtures would greatly facilitate forensic casework. Thus, male and female cell mixtures were marked with a fluorescent X/Y-probe CEP X SpectrumOrange/Y SpectrumGreen DNA probe kit for fluorescence in situ hybridization, and single cells were isolated via laser microdissection (LMD). DNA profiling of LMD isolated, hybridized cells showed usable short tandem repeat profiles for at least 20 cells, which are comparable with results from other studies. To simulate casework samples, the method was also optimized for air-dried samples.     

Validation of mitochondrial DNA sequencing for forensic casework analysis

Two sets of studies were performed to evaluate the forensic utility of sequencing human mitochondrial DNA (mtDNA) derived from various tissues and amplified by the polymerase chain reaction (PCR). Sequencing was performed on a Perkin-Elmer/Applied Biosystems Division (PE/ABD) automated DNA sequencer (model 373A). The first set of experiments included typical validation studies that had previously been conducted on forensic DNA markers, such as: chemical contaminant effects on DNA from blood and semen and the effect of typing DNA extracted from body fluid samples deposited on various substrates. A second set of experiments was performed strictly on human hair shafts. These studies included typing mtDNA from hairs that were: (1) from different body areas, (2) chemically treated, (3) from deceased individuals, and (4) deliberately contaminated with various body fluids. The data confirm that PCR-based mtDNA typing by direct automated sequencing is a valid and reliable means of forensic identification.     

Digoxigenin labelling and laser capture microdissection of male cells

Laser capture microdissection (LMD) is a relatively new technique for the isolation of single cells. The application in forensic investigations has become more and more widespread, especially to select spermatozoa out of mixtures with vaginal cells. In particular in cases with low numbers of sperm it could be profitable to isolate all male cells (e.g. sperm and male epithelial cells) instead of focussing on the sperm only. Therefore, the specific labelling and detection of the male cells in a male/female cell mixture is necessary. In order to label all cells carrying a Y-chromosome we used a digoxigenin labelled chromosome Y hybridisation probe (Q Biogen). The stained cells were isolated with the SL Cut LMD system from Molecular Machines & Industries AG (MMI). At least ten diploid male cells were required to obtain a partial STR profile, with 20 cells, a full profile could be obtained.     

Increasing amplification success of forensic DNA samples using multiple displacement amplification

Multiple displacement amplification (MDA) is capable of amplifying nanogram template amounts of DNA with high accuracy to generate micrograms of representative product. Although MDA is able to amplify small template amounts (<100 pg), increased levels of preferential amplification and allelic dropout are observed. The use of molecular crowders (polyethylene glycol 400) can decrease the amplification bias and increase amplification success. We describe the use of standard and crowded MDA on low-concentration casework samples originating from blood, semen, saliva, hair, and trace DNA. While standard MDA produced no significant increases in STR genotyping success, the use of crowded MDA yielded an average increase of 15% in the number of alleles, with a significant decrease in the amplification bias, resulting in clearer, more-complete profiles from forensically relevant samples. Presented at the 18th International Symposium on the Forensic Sciences organised by the Australian and New Zealand Forensic Science Society (ANZFS), Fremantle, Western Australia, Australia 2–7 April 2006.     

Degradation of forensic DNA profiles

Selected profiles typed at the Promega PowerPlex 21 (PP21) loci were examined to determine if a linear or exponential model best described the relationship between peak height and molecular weight. There were fewer large departures from observed and expected peak heights using the exponential model. The larger differences that were observed were exclusively at the high molecular weight loci. We conclude that the data supports the use of an exponential curve to model peak heights versus molecular weight in PP21 profiles. We believe this observation will improve our ability to model expected peak heights for use in DNA interpretation software.     

A new pentaplex PCR system for forensic casework analysis

In 1998 the Federal Criminal Police Office of Germany (BKA) established a central genetic database of offenders and suspects to facilitate comparisons with biological samples from future criminal offences. The five obligatory short tandem repeat (STR) loci in this database (TH01, SE33, vWA, FGA and D21S11) were co-amplified in a new PCR pentaplex analysing system together with the sex-specific locus amelogenin. Due to overlapping fragment sizes, amplification products were fluorescent dye-labelled with different colours, separated by electrophoresis and detected directly using the ABI PRISM 310 Genetic Analyzer. Reproducible and reliable results were obtained from as low as 125 pg template DNA, indicating high specificity and sensitivity of the assay. Environmental studies and enzymatic digest with DNase I revealed an excellent stability of the pentaplex system with typeable results even in cases of partially degraded DNA. Complete and reproducible DNA typing was possible in bloodstain mixtures with the minor component as low as 10%. Mean stutter peak intensities were analysed for all loci and ranged from 2.7 ± 0.8% (TH01) to 10.6 ± 1.6% (vWA) of the main signal intensity. Allele frequencies were determined in a North Bavarian population sample (n = 121). The combination of five systems resulted in a mean exclusion chance of 99.86% and a power of discrimination of 99.999996%. No deviation from Hardy-Weinberg equilibrium could be found. Received: 13 December 1999 / Accepted: 12 April 2000     

Assessment of DNA methylation markers for forensic applications

ABSTRACT

DNA methylation displays promise in a variety of forensic applications, including chronological age estimation and identical twin differentiation. Knowledge of the age of a body fluid donor would offer intelligence information, as well as provide context to physical characteristic markers. Differentiation of identical twins would also be of considerable benefit as the DNA profile of identical twins cannot be distinguished when a correspondence between DNA profiles recovered from crime scene samples and a person is identified. Our research investigates and develops a methodology for DNA methylation analysis that could be implemented into forensic casework. Our results show that the differentiation of identical twins and chronological age prediction are both possible, with similar accuracies to other international research. Targeted multiplexing of bisulphite converted DNA in combination with massively parallel sequencing is demonstrated as a viable alternative to traditional methylation analysis techniques.     

Developmental validation of DogFiler,a novel multiplex for canine DNA profiling in forensic casework

While the analysis of human DNA has been the focus of large-scale collaborative endeavors, non-human forensic DNA analysis has not benefited from the same funding streams and coordination of effort. Consequently, the development of standard marker panels, allelic ladders and allele-specific sequence data comparable to those established for human forensic genetics has lagged. To meet that need for domestic dogs, we investigated sequence data provided by the published 7.6X dog genome for novel short tandem repeat markers that met our criteria for sensitivity, stability, robustness, polymorphic information content, and ease of scoring. Fifteen unlinked tetranucleotide repeat markers were selected from a pool of 3113 candidate markers and assembled with a sex-linked marker into a multiplex capable of generating a full profile with as little as 60 pg of nuclear DNA. An accompanying allelic ladder was assembled and sequenced to obtain detailed repeat motif data. Validation was carried out according to SWGDAM guidelines, and the DogFiler panel has been integrated into forensic casework and accepted in courts across the U.S. Applying various formulae for calculating random match probabilities for inbred populations, estimates for this panel of markers have proven to be comparable to those obtained in human forensic genetics. The DogFiler panel and the associated allelic ladder represent the first published non-human profiling system to fully address all SWGDAM recommendations.     

Comparison of DNA polymerases for improved forensic analysis of challenging samples

Inhibitors of polymerase chain reaction (PCR) amplification often present a challenge in forensic investigations of e.g., terrorism, missing persons, sexual assaults and other criminal cases. Such inhibitors may be counteracted by dilution of the DNA extract, using different additives, and selecting an inhibitory resistant DNA polymerase. Additionally, DNA in forensic samples is often present in limited amounts and degraded, requiring special analyses of short nuclear targets or mitochondrial DNA. The present study evaluated the enzymes AmpliTaq Gold, HotStarTaq Plus, KAPA3G Plant, and KAPA2G Robust, with regard to their ability to overcome inhibitory effects. Our data showed that diluting the extracts and adding bovine serum albumin may increase the yield of the PCR product. However, the largest impact was observed when alternative enzymes were utilized, instead of the commonly used AmpliTaq Gold. KAPA2G Robust presented the highest amplification efficiency in the presence of the inhibitor ammonium nitrate. Moreover, the KAPA3G Plant enzyme had the highest efficiency in amplifying degraded DNA from old buried bone material. KAPA3G Plant and KAPA2G Robust may thus be useful for counteracting inhibitors and improving the analysis of challenging samples.     

The use of vacuum metal deposition for forensic casework in Canada

ABSTRACT

Canada has seen a positive shift in the use of vacuum metal deposition as a fingermark detection method. This article examines the current state of this technique in Canada and offers recommendations and strategies to overcome challenges still present in an attempt to further advance the application of this extremely versatile technique.     

An analysis of the success rate of 908 trace DNA samples submitted to the Crime Sample Database Unit in New Zealand

The Crime Sample Database Unit, part of the Forensic Biology group at the Institute of Environmental Science and Research Limited, receives samples predominantly from unsolved volume crime cases, such as burglaries and car theft, for inclusion on the New Zealand National DNA Databank. A high proportion of these samples are analysed for the presence of low or trace amounts of DNA. A study was undertaken to evaluate the success of such sample types in providing a DNA profile. Handled items expected to yield low or trace levels of DNA were found to be a commonly submitted type of item in this group, but one of the least successful in terms of DNA profiles obtained. These results are significant, as much time and money is invested in the analysis of such samples, resources that may be better employed elsewhere.     

Crowdsourced and crowdfunded: the future of forensic DNA?

ABSTRACT

Forensic DNA analysis is dependent on comparing the known and the unknown. Expand the number of known profiles, and the likelihood of a successful match increases. Forensic use of DNA is moving towards comparing samples of unknown origin with publicly available genetic data, such as the records held by genetic genealogy providers. Use of forensic genetic genealogy has yielded a number of recent high-profile successes but has raised ethical and privacy concerns. Navigating family trees is complex, even more so when combined with a comparison of genetic relationships. This intelligence-gathering process has led to occasional false leads, and its use also risks a public backlash, similar to concerns over Cambridge Analytica. A cautious approach to use of this technique is therefore warranted.     

Population data for 101 Austrian Caucasian mitochondrial DNA d-loop sequences: Application of mtDNA sequence analysis to a forensic case

The sequence of the two hypervariable segments of the mitochondrial DNA (mtDNA) control region was generated for 101 random Austrian Caucasians. A total of 86 different mtDNA sequences was observed, where 11 sequences were shared by more than 1 individual, 7 sequences were shared by 2 individuals and 4 sequences were shared by 3 individuals. One of the four most common mtDNA sequences in Austrians is also the most common sequence in both U.S. and British Caucasians, found in approximately 3.0% of Austrians, 4.0% of British, and 3.9% of U.S. Caucasians. Of the remaining three common Austrian sequences, one was not observed in either U.S. or British Caucasians. However, three British Caucasians exhibited a similar sequence type. Therefore, this particular cluster of sequence polymorphisms may represent a common “European” mtDNA sequence type. In general, Austrian Caucasians show little deviation from other Caucasian databases of European descent. Finally, mtDNA sequence analysis was applied to a forensic case, where hairs found at a crime scene matched the control hairs from the suspect. Received: 30 June 1997 / Received in revised form: 12 November 1997     

DNA transfer in forensic science: A review

Understanding the variables impacting DNA transfer, persistence, prevalence and recovery (DNA-TPPR) has become increasingly relevant in investigations of criminal activities to provide opinion on how the DNA of a person of interest became present within the sample collected. This review considers our current knowledge regarding DNA-TPPR to assist casework investigations of criminal activities. There is a growing amount of information available on DNA-TPPR to inform the relative probabilities of the evidence given alternative scenarios relating to the presence or absence of DNA from a specific person in a collected sample of interest. This information should be used where relevant. However, far more research is still required to better understand the variables impacting DNA-TPPR and to generate more accurate probability estimates of generating particular types of profiles in more casework relevant situations. This review explores means of achieving this. It also notes the need for all those interacting with an item of interest to have an awareness of DNA transfer possibilities post criminal activity, to limit the risk of contamination or loss of DNA.Appropriately trained forensic practitioners are best placed to provide opinion and guidance on the interpretation of profiles at the activity level. However, those requested to provide expert opinion on DNA-related activity level issues are often insufficiently trained to do so. We advocate recognition of DNA activity associated expertise to be distinct from expertise associated with the identification of individuals. This is to be supported by dedicated training, competency testing, authorisation, and regular fit for purpose proficiency testing.The possibilities for experts to report on activity-related issues will increase as our knowledge increases through further research, access to relevant data is enhanced, and tools to assist interpretations are better exploited. Improvement opportunities will be achieved sooner, if more laboratories and agencies accept the need to invest in these aspects as well as the training of practitioners.     

STR profiling of epithelial cells identified by X/Y-FISH labelling and laser microdissection using standard and elevated PCR conditions

During the investigation of allegations of sexual assault, samples are frequently encountered that contain DNA from a female and a male donor. These may represent contributions of DNA from the complainant and potentially, the offender. Many semen stained samples successfully undergo DNA analysis and interpretation using a differential extraction method that separates sperm from the epithelial cells present in the stain. However, for those mixed cell samples that contain only epithelial cells, separation of any male cells from female cells is problematic. This paper describes the application of fluorescent in situ hybridisation (FISH) for the gender identification of epithelial cells and subsequent recovery of target cells using laser microdissection (LMD). The profiling results obtained from samples of known cell numbers using the Identifiler™ multiplex at standard 28-cycle PCR conditions and, when cell numbers are low, the SGM Plus™ multiplex at elevated 34-cycle PCR conditions (also known as Low Copy Number DNA analysis (LCN)) are described.     

Methods enhancement for improved recovery of human DNA from forensic blood samples on different fabrics using the DNA IQ System

DNA typing of biological samples can be a challenging task due to the presence of small amounts of DNA and/or a high content of PCR inhibitors. Our aim is to develop a protocol to recover DNA suitable for DNA typing from blood-stained fabrics based on the DNA IQ System. Blood stains on different fabrics were buried in different types of soil and left for 1–7 days. The samples were then recovered and DNA was extracted with a modified DNA IQ System protocol, involving a modification in the preparation of the Wash Buffer. The samples extracted with the modified protocol showed a higher amount of recovered DNA compared with the recommended protocol. The removal of isopropanol from the Wash Buffer composition contributes to a higher amount of recovered DNA.     

Mitochondrial DNA polymorphisms: a study of 50 French Caucasian individuals and application to forensic casework

The analysis of mitochondrial DNA polymorphisms has proved to be efficient on highly degraded samples or samples having little or no genomic DNA such as hair shafts. In order to use this very sensitive method, the authors first established a database by analysing the mitochondrial DNA polymorphism in 50 French white Caucasian individuals, applied the analysis to different types of samples that can be found in forensic investigations and finally performed this method on two forensic cases involving the discovery of highly putrefied unidentified remains. Received: 10 March 1997 / Received in revised form: 4 February 1998     

A Bayesian approach to validating STR multiplex databases for use in forensic casework

A previous paper in this journal has described the conventional statistical analysis of three databases (Caucasian, Afro-Caribbean and Asians from the Indian subcontinent) where individuals are typed at six short tandem repeat (STR) loci. This paper presents a Bayesian analysis of the same data and the approach is centred on the concept of estimating coancestry coefficients from mixed databases. Posterior distributions for the three databases are presented and discussed and the consequences of implementing bootstrap estimation procedures are also shown. Received: 18 November 1996 / Received in revised form: 21 April 1997