Lindsey Welch

European Network of Forensic Science Institutes (ENFSI): Evaluation of new commercial STR multiplexes that include the European Standard Set (ESS) of markers

To support and to underpin the European initiative to increase the European set of standard markers (ESS), by the addition of five new loci, a collaborative project was organised by the European Network of Forensic Science Institutes (ENFSI) DNA working group in order to assess the new multiplex kits available. We have prepared allele frequency databases from 26 EU populations. Concordance studies were carried out to verify that genotyping results were consistent between kits. Population genetics studies were conducted and it was estimated that F(ST)<0.001. The results showed that the kits were comparable to each other in terms of performance and major discrepancy issues were highlighted. We provide details of allele frequencies for each of the populations analysed per laboratory.

A comparison of methods for forensic DNA extraction: Chelex-100® and the QIAGEN DNA Investigator Kit (manual and automated)

Efficient isolation of DNA from a sample is the basis for successful forensic DNA profiling. There are many DNA extraction methods available and they vary in their ability to efficiently extract the DNA; as well as in processing time, operator intervention, contamination risk and ease of use. In recent years, automated robots have been made available which speed up processing time and decrease the amount of operator input. This project was set up to investigate the efficiency of three DNA extraction methods, two manual (Chelex(®)-100 and the QIAGEN DNA Investigator Kit) and one automated (QIAcube), using both buccal cells and blood stains as the DNA source. Extracted DNA was quantified using real-time PCR in order to assess the amount of DNA present in each sample. Selected samples were then amplified using AmpFlSTR SGM Plus amplification kit. The results suggested that there was no statistical difference between results gained for the different methods investigated, but the automated QIAcube robot made sample processing much simpler and quicker without introducing DNA contamination.

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.

Evaluation of nucleosome forming potentials (NFPs) of forensically important STRs

Degraded forensic samples have proved difficult to analyze and interpret. New analysis techniques are constantly being discovered and improved but researchers have overlooked the structural properties that could prevent or slow the process of degradation. In theory, DNA that are bound to histones as nucleosomes are less prone to degradation, because nucleosomes prevent DNA from being exposed to degradative enzymes. In this study we determined the probability of 60 forensic DNA markers to be bound to histones based on their base sequence composition. Two web-based tools - NXSensor and nuScore - were used to analyze four hundred base pairs surrounding each DNA marker for properties that inhibit or promote the binding of DNA to histones. Our results showed that the majority of markers analyzed were likely to be bound as nucleosomes. Selection of the markers that are more protected to form a multiplex could increase the chance of obtaining a better balanced, easier to interpret DNA profile from degraded samples.

Using the Taguchi method for rapid quantitative PCR optimization with SYBR Green I

Here, we applied the Taguchi method, an engineering optimization process, to successfully determine the optimal conditions for three SYBR Green I-based quantitative PCR assays. This method balanced the effects of all factors and their associated levels by using an orthogonal array rather than a factorial array. Instead of running 27 experiments with the conventional factorial method, the Taguchi method achieved the same optimal conditions using only nine experiments, saving valuable resources.

A comparison of mini-STRs versus standard STRs—Results of a collaborative European (EDNAP) exercise

here is a general agreement by the European DNA Profiling Group (EDNAP) and the European Network of Forensic Science Institutes (ENFSI) that the reason to implement new DNA markers is to increase the chance of obtaining DNA profiles from highly degraded DNA rather than to increase the discriminating power gained from full DNA profiles. Previous collaborative exercises have demonstrated that smaller amplicons tend to provide improved amplification when the DNA template has been degraded [1] and [2]. These exercises have led to the commercial development of ‘mini-STR’ multiplex kits.

A proof of principal study on the use of direct PCR of semen and spermatozoa and development of a differential isolation protocol for use in cases of alleged sexual assault

Sexual assault samples are some of the most common samples encountered in forensic analysis. These samples can require a significant time investment due to differential extraction processes. We report on the first record of successful direct amplification of semen for STR analysis. Neat seminal fluid, dilutions ranging from 1:5 to 1:160 and GEDNAP samples were successfully amplified using a direct method. A mild differential isolation technique to enrich spermatozoa was developed and successfully implemented to separate and directly amplify a mixture of semen and female epithelial cells. Aliquots of samples subjected to the differential isolation protocol were stained with Haemotoxylin and Eosin for sperm scoring. Samples stained after PCR showed a complete lack of intact spermatozoa demonstrating that the cells are lysed during the PCR process. This paper demonstrates the potential to incorporate direct PCR in cases of sexual assault to more rapidly obtain results and achieve a higher sensitivity.

Recovery of human DNA profiles from poached deer remains part 2: Improved recovery protocol without the need for LCN analysis

Although poaching is a common wildlife crime, the high and prohibitive cost of specialised animal testing means that many cases are left un-investigated. We previously described a novel approach to wildlife crime investigation that looked at the identification of human DNA on poached animal remains (Tobe, Govan and Welch, 2011). Human DNA was successfully isolated and amplified from simulated poaching incidents, however a low template protocol was required which made this method unsuitable for use in many laboratories. We now report on an optimised recovery and amplification protocol which removes the need for low template analysis. Samples from 10 deer (40 samples total - one from each leg) analysed in the original study were re-analysed in the current study with an additional 11 deer samples. Four samples analysed using Chelex did not show any results and a new method was devised whereby the available DNA was concentrated. By combining the DNA extracts from all tapings of the same deer remains followed by concentration, the recovered quantity of human DNA was found to be 29.5pg±43.2pg, 31× greater than the previous study. The use of the Investigator Decaplex SE (QIAGEN) STR kit provided better results in the form of more complete profiles than did the AmpFℓSTR® SGM Plus® kit at 30cycles (Applied Biosystems). Re-analysis of the samples from the initial study using the new, optimised protocol resulted in an average increase of 18% of recovered alleles. Over 17 samples, 71% of the samples analysed using the optimised protocol showed sufficient amplification for comparison to a reference profile and gave match probabilities ranging from 7.7690×10(-05) to 2.2706×10(-14). The removal of low template analysis means this optimised method provides evidence of high probative value and is suitable for immediate use in forensic laboratories. All methods and techniques used are standard and are compatible with current SOPs. As no high cost non-human DNA analysis is required the overall process is no more expensive than the investigation of other volume crime samples. The technique is suitable for immediate use in poaching incidents.

Highly potent human haemopoietic stem cells first emerge in the intraembryonic aorta-gonad-mesonephros region

Abstract Background Haemopoietic stem cells (HSCs) are used in the clinic to treat various haematological disorders. These cells emerge during early embryogenesis and maintain haemopoiesis in the adult organism. In the vertebrate embryo, HSCs develop in multiple locations. Little is known about the embryonic development of human HSCs. Methods Human embryonic and fetal tissues were obtained after elective termination of pregnancy. Preconditioned immunodeficient mice were used as recipients for human HSCs. Transplanted mice were bled every 1–2 months to assess human HSC contribution. Findings We have found that human HSCs emerge first in the aorta-gonad-mesonephros (AGM) region and only later appear in the yolk sac, liver, and placenta. Transplantation of human AGM region cells into immunodeficient mice provides long-term high-level multilineage haemopoietic repopulation. We have shown that, despite the low number of HSCs in the human AGM region, their self-renewal potential is enormous. A single HSC derived from the AGM region generates around 600 daughter HSCs in primary recipients, which disseminate throughout the entire recipient bone marrow and are retransplantable. Interpretation We provide a systematic spatiotemporal analysis of HSC emergence in the early human embryo and identify the AGM region as the primary source of powerful HSCs with enormous self-renewal capacity. This high potency of the first HSCs sets a new standard for in-vitro generation of HSCs from pluripotent stem cells for the purpose of regenerative medicine. Funding UK Medical Research Council.