SallyAnn Harbison

The development of a mRNA multiplex RT-PCR assay for the definitive identification of body fluids.

With current methodology, DNA profiling can identify an individual from a sample of biological material but it does not reveal what body fluid or tissue source the DNA profile originated from. We have developed a multiplex PCR system using messenger RNA (mRNA) that can identify blood, saliva, semen and menstrual blood in individual stains or in mixtures of body fluids. Messenger RNA transcripts specific to each type of body fluid have been identified and a multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) system developed to identify these body fluids along with three housekeeping genes. This multiplex can detect semen and seminal fluid (semen without spermatozoa present). Furthermore, we have targeted the co-isolation of RNA and DNA from the same sample and, with the RT-PCR multiplex, we can determine the type of body fluid present as well as generate a DNA profile(s) from the same stain.

Characterising stutter in forensic STR multiplexes

Stutter is an artefact seen when amplifying short tandem repeats and typically occurs at one repeat unit shorter in length than the parent allele. In forensic analysis, stutter complicates the analysis of DNA profiles from multiple contributors, known as mixed profiles, a common profile type. Consequently it is important to both understand and predict stutter behaviour in order to improve our understanding of the resolution and interpretation of these profiles. Whilst stutter is well recognised and documented, little information is available that identifies and quantifies what influences the formation of stutter. In this work we use a novel approach to examine this. We have used synthetic oligonucleotides comprising multiple repeat units to test; the influence of repeat number, the influence of repeat sequence and the impact of interruptions to the repeat sequence length. Using multiple replicates allows detailed statistical analysis. We have confirmed a linear relationship between stutter ratio and repeat number. We have shown that increased A-T content increases stutter ratio and that interruptions in repeating sequences decreased stutter ratios to levels similar to the longest uninterrupted repeat stretch. We also found that there was no relationship between stutter ratio and repeat number for a repeat unit with an A-T content of 1/4 and that half of the interrupted repeat sequences stuttered significantly less than their longest uninterrupted repeat stretches. We have applied the knowledge gained to examine specific features of the loci present in the AmpFlSTR(®) SGM Plus(®) multiplex kit used in our laboratory.

The use of bacteria for the identification of vaginal secretions.

We have used the 16S-23S rRNA intergenic spacer region for identifying vaginal specific bacteria. Lactobacillus crispatus and Lactobacillus gasseri were detected in vaginal secretions but not in semen, blood or saliva. Our data indicated that both L. crispatus and L. gasseri were detected in vaginal secretions from women with different levels of expression of hormonal genes including pregnant, pre- and post-menopausal women, and a woman who has had a hysterectomy. Therefore, we have demonstrated that these Lactobacilli are promising new markers for the forensic identification of vaginal secretions. We have incorporated the Lactobacilli markers into a mRNA multiplex system to produce an 11-plex assay that can identify circulatory blood, menstrual blood, saliva, semen (in the presence and absence of spermatozoa) and vaginal secretions.

A method for DNA and RNA co-extraction for use on forensic samples using the Promega DNA IQ™ system

The use of messenger RNA profiling to identify the origin of biological samples (e.g. blood, semen and saliva) from crime scenes is now at the stage of being implemented into routine forensic casework. We report on the successful modification of the Promega DNA IQ™ system to enable co-extraction of DNA and RNA from the same sample without compromising the potential DNA profile. Using the protocol in our laboratory for extracting DNA using the DNA IQ™ system combined with the Zymo Research Mini RNA Isolation Kit™ II we demonstrate the simultaneous co-extraction of DNA and RNA from the same sample for routine DNA and mRNA profiling for the identification of both the individual and the biological stain.

A comparison of stochastic variation in mixed and unmixed casework and synthetic samples.

Understanding the behaviour of mixed DNA profiles is of paramount importance in forensic DNA analysis. Key parameters are those of heterozygote balance and mixture proportion and its variability. These parameters have been previously explored as a function of the average peak height of the active alleles in single source and mixed samples derived from pristine DNA. Here we report a comparison of this data with data obtained from casework samples. This allows an assessment of the difference in the distribution of heterozygote balance between mixed and single source stains and between casework mixtures and synthetic mixtures constructed from pristine DNA.

A time-course analysis of mRNA expression during injury healing in human dermal injuries

The determination of dermal injury age is important in forensic practice. It helps answer questions that are important to an investigation such as the timing of the injury and incident, the order of infliction (where there is more than one injury), the survival time after injury (post-infliction interval) and the relation of the injury to the incident. Despite the importance of injury age determination, there currently exists no reliable method to estimate dermal injury age. In this study, the expression of the following 14 mRNAs was studied in human dermal injuries and their usefulness in the estimation of human dermal injury age was evaluated: dual specificity phosphate 1 (DUSP1), interleukin 7 (IL7), vascular cell adhesion molecule 1, tenascin C, cluster of differentiation 14, interleukin 6, tumour necrosis factor alpha (TNFα), interleukin 1beta (IL1β), chymase 1 (CMA1), collagen type III alpha I, interleukin 2, collagen type I alpha I, collagen type I alpha II and vascular endothelial growth factor A (VEGFA). DUSP1, IL7, TNFα and VEGFA showed an initial decrease in expression during the early stages followed by an increase in expression towards the middle and late phases. IL1β and CMA1 expression was limited to specific time points. The remaining markers either showed inconsistent expression or were undetected in our samples. The expression patterns of the detected markers suggest they have potential to predict injury age, especially during the initial stages of injury healing, if used in combination with one another.

The Effect of Cleaning Agents on the Ability to Obtain DNA Profiles Using the Identifiler™ and PowerPlex® Y Multiplex Kits

Abstract:  A year after the introduction of Identifiler™ into the forensic DNA laboratories of the Institute of Environmental Science and Research Limited (ESR), increasing occurrences of dropout of the three loci, D7S820, D18S51, and FGA, were observed in samples where the DNA was not degraded and sufficient DNA was present that full DNA profiles were to be expected. The dropout was either partial or complete at these loci. Full profiles could sometimes be obtained by reamplification of samples using the same input amount of DNA. After a thorough investigation of the methods and procedures used in the laboratory, the cause of this inhibition was identified as the cleaning agent TriGene™ ADVANCE. This was determined after the deliberate addition of varying amounts of different cleaning reagents into the DNA amplification reactions. At concentrations of 0.004% TriGene™ ADVANCE caused inhibition resulting in tri‐loci dropout. At concentrations of 0.04% and higher, complete inhibition was observed. An effect was also seen on the amplification of samples using the Y STR profiling system PowerPlex®Y. This work highlights the importance of checking all reagents and chemicals prior to use, even those with no apparent direct influence on the DNA profiling process.

The development of a method of suspension RNA-FISH for forensically relevant epithelial cells using LNA probes.

Messenger RNA profiling is becoming a common method for body fluid identification in forensic science but there are disadvantages when cell mixtures are present from more than one individual. A method that could identify and separate such cell mixtures would simplify downstream analysis. To do this, we have developed a novel method of RNA suspension-fluorescent in situ hybridization (RNA S-FISH) using a locked nucleic acid (LNA) probe for the keratin 10 (KRT10) mRNA that is suitable as a potential marker for epithelial cells. As sample size may be restricted in forensic samples, this method has focused on minimizing cell loss whilst maintaining signal strength. Furthermore, we have shown that it is possible to obtain full DNA profiles from 150 RNA S-FISH labeled cells isolated using laser microdissection.

A review of bioinformatic methods for forensic DNA analyses

Short tandem repeats, single nucleotide polymorphisms, and whole mitochondrial analyses are three classes of markers which will play an important role in the future of forensic DNA typing. The arrival of massively parallel sequencing platforms in forensic science reveals new information such as insights into the complexity and variability of the markers that were previously unseen, along with amounts of data too immense for analyses by manual means. Along with the sequencing chemistries employed, bioinformatic methods are required to process and interpret this new and extensive data. As more is learnt about the use of these new technologies for forensic applications, development and standardization of efficient, favourable tools for each stage of data processing is being carried out, and faster, more accurate methods that improve on the original approaches have been developed. As forensic laboratories search for the optimal pipeline of tools, sequencer manufacturers have incorporated pipelines into sequencer software to make analyses convenient. This review explores the current state of bioinformatic methods and tools used for the analyses of forensic markers sequenced on the massively parallel sequencing (MPS) platforms currently most widely used.

Massively parallel sequencing for the forensic scientist – sequencing archived amplified products of AmpFlSTR Identifiler and PowerPlex Y multiplex kits to capture additional information

In the last few years the cost and ease of massively parallel sequencing (MPS) has reduced dramatically to the point that it can now be considered as a tool for use in forensic case work. An important consideration for the implementation of any new forensic technology is the ability to remain compatible with previous technology. During this study we sequenced the amplicons of two commercial forensic short tandem repeat (STR) multiplexes AmpFlSTR Identifiler and PowerPlex Y using the Illumina MiSeq and Ion PGM Sequencer (Life Technologies) and characterised the sequence data from a forensic perspective. Using the MPS data from both platforms we determined the STR genotypes of forensic samples and found previously undocumented sequence variation in seven STR alleles. By characterising features of the DNA sequence profiles, such as stutter and locus imbalance we identified areas for future development that will be needed prior to casework implementation. The rapid development of this technology has meant many in the forensic community have been ‘left behind’. We also provide an explanation, for forensic scientists, of what is happening at the different stages of the MPS workflow, from library preparation through to bioinformatics, and how this may affect the results.