Andrew Hopwood

Integrated Microfluidic System for Rapid Forensic DNA Analysis: Sample Collection to DNA Profile

We demonstrate a conduit for the delivery of a step change in the DNA analysis process: A fully integrated instrument for the analysis of multiplex short tandem repeat DNA profiles from reference buccal samples is described and is suitable for the processing of such samples within a forensic environment such as a police custody suite or booking office. The instrument is loaded with a DNA processing cartridge which incorporates on-board pumps and valves which direct the delivery of sample and reagents to the various reaction chambers to allow DNA purification, amplification of the DNA by PCR, and collection of the amplified product for delivery to an integral CE chip. The fluorescently labeled product is separated using micro capillary electrophoresis with a resolution of 1.2 base pairs (bp) allowing laser induced fluorescence-based detection of the amplified short tandem repeat fragments and subsequent analysis of data to produce a DNA profile which is compatible with the data format of the UK DNA database. The entire process from taking the sample from a suspect, to database compatible DNA profile production can currently be achieved in less than 4 h. By integrating such an instrument and microfluidic cartridge with the forensic process, we believe it will be possible in the near future to process a DNA sample taken from an individual in police custody and compare the profile with the DNA profiles held on a DNA Database in as little as 3 h.

DNA typing from human faeces

A method has been developed for the forensic analysis of faeces by DNA amplification and direct sequencing of a polymorphic segment of mitochondrial DNA. Starting from as little as 10 mg wet weight of faeces, DNA was extracted by a variety of protocols and amplified using primers specific to hypervariable region I of the mitochondrial control region. The resulting amplification products were sequenced in solid phase using an automated DNA sequencer. In total, mtDNA sequences were generated from the faeces of nine Caucasians and compared with sequences generated from their respective blood samples. Sequences of faeces and blood samples from the same individual were identical in every case, but a range of 1–10 nucleotide differences was observed between individuals, with an average sequence variation of approximately 4.88 per 400 bp. Of the various extraction protocols assessed in this study, greatest success rates were achieved using magnetisable beads to bind and purify the DNA. STR analysis of DNA extracted from faeces was not routinely possible.

An automated instrument for human STR identification: Design, characterization, and experimental validation

The microfluidic integration of an entire DNA analysis workflow on a fully integrated miniaturized instrument is reported using lab‐on‐a‐chip automation to perform DNA fingerprinting compatible with CODIS standard relevant to the forensic community. The instrument aims to improve the cost, duration, and ease of use to perform a “sample‐to‐profile” analysis with no need for human intervention. The present publication describes the operation of the three major components of the system: the electronic control components, the microfluidic cartridge and CE microchip, and the optical excitation/detection module. Experimental details are given to characterize the level of performance, stability, reliability, accuracy, and sensitivity of the prototype system. A typical temperature profile from a PCR amplification process and an electropherogram of a commercial size standard (GeneScan 500™, Applied Biosystems) separation are shown to assess the relevance of the instrument to forensic applications. Finally, we present a profile from an automated integrated run where lysed cells from a buccal swab were introduced in the system and no further human intervention was required to complete the analysis.

D-dimer assays for the identification of menstrual blood

A method to reliably distinguish menstrual blood from blood in the normal circulation (peripheral blood) would be of considerable use in the forensic analysis and interpretation of evidence in sexual offence investigations. Previous attempts to address this issue have explored microscopy, lactate dehydrogenase isozyme identification, mRNA and miRNA profiling, and identification of the products of fibrinolysis. Here, four assays for D-dimer, a terminal degradation product of fibrinolysis, are evaluated for their specificity and sensitivity in detection of menstrual blood. In addition the effect of exercise, and sample storage upon D-dimer detection was investigated. Comparison of different assays revealed significant differences in results given. Nevertheless, no positive results for D-dimer were obtained using peripheral blood, mixtures of peripheral blood with semen, or peripheral blood taken from donors after moderate exercise. D-dimer was found to be detectable in 100% of menstrual blood samples after 1 week at room temperature and also in samples stored long-term (>3 years) at -20 °C. D-dimer may be an effective, simple to use tool for the presumptive identification of menstrual blood identification.

Consideration of the probative value of single donor 15-plex STR profiles in UK populations and its presentation in UK courts

The adoption of new 15 locus STR multiplex systems into UK forensic science would be facilitated by agreed guidelines for reporting the strength of DNA evidence using likelihood ratios. To facilitate such an agreement, we present an analysis of previously published UK allele frequencies for white Caucasian, Afro-Caribbean and Indo-Pakistani populations and investigate their effect on likelihood ratios for single donor profiles. We consider the implication of the five additional loci and suggest a procedure for reporting likelihood ratios for 15-plex STR profiles.

Forensic DNA research: keeping it real

With any research project, a successful outcome is dependant firstly on a proper understanding of the problem you are trying to solve. It pays to think long and hard about the way an experiment should be designed, and in a forensic context, the design should emulate as far as possible the real world scenarios likely to be encountered. In our experience, the choice of sample used in a research project is critical to the successful implementation of a DNA-based technique into forensic science. Whilst the crude development of an end-to-end process can be performed using a general sample type, it is essential that the optimisation of the process is performed on samples that mimic the real sample type as closely as possible. A number of sample types might be discussed, including unadulterated blood vs EDTA anticoagulated blood, DNase-degraded DNA samples vs degraded body fluid samples, and post-coital samples vs cellular samples seeded with semen. As an example, we consider the consequences of the sampling decisions we make when developing protocols for the examination of post-coital (PC) samples submitted for DNA analysis.