DNA identification of monozygotic twins.

This study details the differentiation of identical twins based on single mutational base differences. There were three pairs of male monozygotic (MZ) twins in this study. DNA samples from blood, a buccal swab or saliva from each individual were all initially genotyped using 22 autosomal STR and 27 Y-STR loci. Preliminary screening confirmed there were no differences in the STR data between each pair of MZ twins. Whole Genome Sequence (WGS) data were generated from DNA extracted from the three body fluids from each individual. Kinship coefficients with 0.4254, 0.4557 and 0.4543 from 3 twins were generated based on WGS data to further confirm that their relationship was that of MZ twins. The fastq data generated by the Illumina Hiseq 2000 between MZ twins were then treated as "normal" as opposed to "tumor" using commercially available software tools to identify mutational single base changes. Sanger DNA sequencing confirmed there were 1, 5 and 9 single base changes found in WGS data from each of the three MZ twin sets. There was individual variation in the mutational base changes when comparing data from the three body fluids. The methods used in this study to differentiate MZ twins based on WGS data can readily be performed in many operational forensic DNA laboratories using user friendly software.

Spermatozoa identification by the 3-plex MSRE-PCR assay: a collaborative exercise.

Identification of semen and spermatozoa is crucial in the forensic investigation of alleged sexual assault cases. In cases of alleged sexual assault where there is a long time gap between the incident and sample collection, or in cases of low sperm count, current methods have limitations of specificity, in the case of presumptive tests for semen, or the problem of recording spermatozoa by microscopy if they are few in number. A 3-plex MSRE-PCR (methylation-sensitive restriction enzyme-PCR) assay using a spermatozoa-specific DNA methylated marker to identify spermatozoa has been reported previously by our laboratory. A key advantage over current methods is the increased sensitivity and specificity. A transition from a research tool to operational use requires blind trial testing and inter-laboratory trials. We report on a collaborative exercise where reagents of the 3-plex MSRE-PCR were sent to six participating laboratories. Each laboratory used their own equipment, consumables, and the presumptive reagents conventionally for body fluid (such as acid phosphatase or PSA), DNA extraction, and quantification in practical casework. The reagents and protocol for the 3-plex MSRE-PCR assay and 9 samples were provided by the organizing laboratory. The participating laboratories were requested to fill in the questionnaire after testing. The reported results from all the six participating laboratories were concordant and the expected correct results for the presence of spermatozoa. These outcomes verified the reproducibility and feasibility of the 3-plex MSRE-PCR assay. The results also indicated that the 3-plex MSRE-PCR assay was readily accessible to forensic laboratories for integrating it into current forensic casework processes.

Evaluation of a fluorescent dye to visualize touch DNA on various substrates.

A wide variety of items are submitted as evidence in a forensic investigation. Identifying the location of DNA on such items is central to maximizing DNA profiling success and thus the ability to link a person of interest to a particular item or crime. Recent publications describe a fluorescent staining method using Diamond™ Dye (DD) to visualize cellular material on the surface of non-porous items (e.g., glass, plastic). However, substrates of varying porosity and background color have not yet been examined. Varying porous substrates (i.e., paper bank note, stamp, cigarette, wooden matchstick, and fabric) and non-porous substrates (i.e., enamel tooth and plastic bank note) were examined for their suitability with the use of DD. To improve the visualization of cellular material on the porous substrates, we also explored two DD diluents and adjusting image contrast. The results suggest the optimal diluent depends on the absorbent nature of the substrate. For example, ethanol was sufficient for visualization on the non-porous substrates, whereas water was better for the porous substrates. While cellular material was detected on the paper bank note, tooth, and stamp, background fluorescence or autofluorescence and surface type of matchstick prevented clear visualization on this substrate. It was also determined that by adjusting the contrast of images for tooth, paper bank note and matchstick aided in the detection of cellular material. Overall, this study extends the use of DD for latent DNA detection to absorbent substrates, highlights the limitations associated with these substrate types, and suggests modifications to improve visualization on these challenging substrates.

The detection and identification of saliva in forensic samples by RT-LAMP.

We report on a novel method for saliva identification by reverse transcription-loop-mediated isothermal amplification (RT-LAMP). In our previous report, real-time RT-LAMP was used for blood identification by using HBB detection as a model but in this advanced study, this method was refined for the identification of the more challenging body fluid of saliva. Expression of the18S rRNA gene was used as the internal control and the Statherin (STATH) gene as the saliva-specific marker. A turbidimeter was used for real-time detection of the RT-LAMP products, and confirmation was obtained that the real products were generated using: agarose gel electrophoresis, calcein fluorescence detection and/or enzymatic digestion. The specificity of the test was performed using 42 samples including 7 different body fluids, and the expression of STATH was only observed in all the saliva samples (6) with a threshold time of 39.4 ± 2.9 min. Sensitivity testing showed that RT-LAMP products for STATH were stably detected when the RNA template was not less than 6.25 ng. When the primer concentrations for STATH were two times that of 18S rRNA, saliva could be identified in the body fluid mixtures even at a ratio (saliva:semen) of 1:3 (without loop primer)/1:5 (with loop primer). A multiplex RT-LAMP was established to simultaneously amplify the 18S rRNA and STATH genes, and applied to the identification of saliva on ten non-probative cigarette butts. A positive result for saliva was obtained from all ten butts, even for those that returned a negative or ambiguous result using the amylase test. A direct RT-LAMP test is also reported where the RNA extraction step was omitted to speed the collection of data and all tests using either the simplex or multiplex RT-LAMP resulted in a positive response if saliva was present. Our data provide a simple and effective means to detect the presence of saliva.

A complementary forensic 'proteo-genomic' approach for the direct identification of biological fluid traces under fingernails.

Violent contact between individuals during a crime can result in body fluids becoming trapped under the fingernails of the individuals involved. The traces under fingernails represent valuable forensic evidence because DNA profiling can indicate from whom the trace originated and proteomic methods can be used to determine the type of fluid in the trace, thus providing evidence as to the circumstances surrounding the crime. Here, we present an initial study of an analytical strategy that involves two complementary techniques, direct PCR DNA profiling and direct mass spectrometry-based protein biomarker detection, for the comprehensive examination of traces of biological fluids gathered from underneath fingernails. With regard to protein biomarker detection, direct MALDI-ToF MS/MS is very sensitive, allowing results to be obtained from biological material present on only a few fibres plucked from a microswab used to collect the traces. Human cornulin, a protein biomarker for vaginal fluid, could be detected up to 5 h after it had been deposited under fingernails whereas haemoglobin, a biomarker for blood, is somewhat more persistent under fingernails and could be detected up to 18 h post-deposition. Bottom-up tandem mass spectrometry techniques were used to provide a high level of confidence in assigning the identity of protein biomarkers. nLC-ESI-qToF MS/MS offered higher levels of confidence and the ability to detect traces that had been present under fingernails for longer periods of time, but this performance came with the cost of longer analysis time and a more laborious sampling approach. Graphical abstract ᅟ.

A mass spectrometry-based forensic toolbox for imaging and detecting biological fluid evidence in finger marks and fingernail scrapings.

During a crime, biological material such as blood or vaginal fluid may become smeared on the fingers of the victim or suspect or trapped under their fingernails. The type of trapped fluid is extremely valuable forensic information. Furthermore, if either person touches an object at the crime scene with their 'contaminated' finger then a 'contaminated' finger mark may be deposited. Such marks have great value as they could identify not only who deposited the mark but also who they touched and which part of the body they touched. Here, we describe preliminary work towards a 'toolbox' of techniques based on mass spectrometry (MS) for the identification of biological fluid traces under fingernails or the imaging of them in finger marks. Liquid chromatography-multidimensional MS was effective for the detection of protein biomarkers characteristic of vaginal fluid and blood trapped under fingernails, even after hands had been washed. In regard to examination of finger marks for the presence of biological fluids, the most practical implementation of any technique is to integrate it with, but after, routine crime scene finger mark enhancement has been applied. Here, we demonstrate the usage of matrix-assisted laser desorption ionization-time of flight-MS for the detection and mapping of proteins and peptides from body fluids in finger marks, including marks enhanced using aluminium-containing magnetic powder and then 'lifted' with adhesive tape. Hitherto, only small molecules have been detected in enhanced, lifted marks. In a novel development, aluminium in the enhancement powder assisted ionization of small molecules in finger marks to the extent that conventional matrix was not required for MS.

The Influence of Selected Fingerprint Enhancement Techniques on Forensic DNA Typing of Epithelial Cells Deposited on Porous Surfaces.

Fingerprints deposited at crime scene can be a source of DNA. Previous reports on the effects of fingerprint enhancement methods have focused mainly on fingermarks deposited in blood or saliva. Here, we evaluate the effects of fingerprint enhancement methods on fingerprints deposited on porous surfaces. We performed real-time quantification and STR typing, the results of which indicated that two methods (iodine fuming and 1,2-indanedione in ethyl acetate enhancement) had no effect on the quantity of DNA isolated and resultant STR alleles when compared to control samples. DNA quantities and allele numbers were lower for samples enhanced with silver nitrate and 1,2-indanedione in acetic acid when compared to control samples. Based on DNA quantity, quality, and observable stochastic effects, our data indicated that iodine fuming and 1,2-indanedione in ethyl acetate were the preferred options for the enhancement of fingerprints on porous surfaces.

Sequence selective capture, release and analysis of DNA using a magnetic microbead-assisted toehold-mediated DNA strand displacement reaction.

This paper reports on the modification of magnetic beads with oligonucleotide capture probes with a specially designed pendant toehold (overhang) aimed specifically to capture double-stranded PCR products. After capture, the PCR products were selectively released from the magnetic beads by means of a toehold-mediated strand displacement reaction using short artificial oligonucleotide triggers and analysed using capillary electrophoresis. The approach was successfully shown on two genes widely used in human DNA genotyping, namely human c-fms (macrophage colony-stimulating factor) proto-oncogene for the CSF-1 receptor (CSF1PO) and amelogenin.

A highly polymorphic STR locus in Cannabis sativa.

We report on the first short tandem repeat (STR) locus to be isolated from the plant Cannabis sativa. The STR locus, isolated by a hybrid-capture enrichment procedure, was found to contain a simple sequence repeat motif of 6 bp. This 6 bp repeat motif showed no variation in repeat length but with minor variations in repeat unit sequences. The data show the locus to be highly polymorphic with the number of repeat units ranging from 3 to 40 in 108 screened samples. The observed heterozygosity was approximately 87.04%. The forward and reverse primers (CS1F and CS1R) produced no PCR products in cross-reaction study from 20 species of plants, including highly related species such as Humulus japonicus and Nicotiana tabacum. This hexanucleotide repeat DNA locus could be used to identify cannabis samples and predict their genetic relationship as the test is specific to C. sativa and is highly reproducible.