Common cloning is often associated with instability of certain classes of DNA. Here we report on IS1 transposition as possible source of such instability. During the cloning of Arabidopsis thaliana gene into commercially available vector maintained in widely used Escherichia coli host the insertion of complete IS1 element into the intron of cloned gene was found. The transposition of the IS1 element was remarkably rapid and is likely to be sequence-specific. The use of E. coli strains that lower the copy number of vector or avoiding the presence of the problematic sequence is a solution to the inadvertent transposition of IS1. The transposition of IS1 is rare but it can occur and might confound functional studies of a plant gene.
Understanding the factors that may impact the transfer, persistence, prevalence and recovery of DNA (DNA-TPPR), and the availability of data to assign probabilities to DNA quantities and profile types being obtained given particular scenarios and circumstances, is paramount when performing, and giving guidance on, evaluations of DNA findings given activity level propositions (activity level evaluations). In late 2018 and early 2019, three major reviews were published on aspects of DNA-TPPR, with each advocating the need for further research and other actions to support the conduct of DNA-related activity level evaluations. Here, we look at how challenges are being met, primarily by providing a synopsis of DNA-TPPR-related articles published since the conduct of these reviews and briefly exploring some of the actions taken by industry stakeholders towards addressing identified gaps. Much has been carried out in recent years, and efforts continue, to meet the challenges to continually improve the capacity of forensic experts to provide the guidance sought by the judiciary with respect to the transfer of DNA.
Currently, promising new tools are under development that will enable crime scene investigators to analyze fingerprints or DNA-traces at the crime scene. While these technologies could help to find a perpetrator early in the investigation, they may also strengthen confirmation bias when an incorrect scenario directs the investigation this early. In this study, 40 experienced Crime scene investigators (CSIs) investigated a mock crime scene to study the influence of rapid identification technologies on the investigation. This initial study shows that receiving identification information during the investigation results in more accurate scenarios. CSIs in general are not as much reconstructing the event that took place, but rather have a “who done it routine.” Their focus is on finding perpetrator traces with the risk of missing important information at the start of the investigation. Furthermore, identification information was mostly integrated in their final scenarios when the results of the analysis matched their expectations. CSIs have the tendency to look for confirmation, but the technology has no influence on this tendency. CSIs should be made aware of the risks of this strategy as important offender information could be missed or innocent people could be wrongfully accused.
Routine neuropathology diagnostic methods are limited to histological staining techniques or directed PCR for pathogen detection and microbial cultures of brain abscesses are negative in one-third of the cases. Fortunately, due to improvements in technology, metagenomic sequencing of a conserved bacterial gene could provide an alternative diagnostic method. For histopathological work up, formalin-fixed paraffin-embedded (FFPE) tissue with highly degraded nucleic acids is the only material being available. Innovative amplicon-specific next-generation sequencing (NGS) technology has the capability to identify pathogens based on the degraded DNA within a few hours. This approach significantly accelerates diagnostics and is particularly valuable to identify challenging pathogens. This ensures optimal treatment for the patient, minimizing unnecessary health damage. Within this project, highly conserved primers in a universal PCR will be used, followed by determining the nucleotide sequence. Based on the obtained data, it is then precisely determined which microorganism(s) is/are responsible for the infection, even in cases of co-infection with multiple pathogens. This project will focus to answer the following research question; how can a new form of rapid molecular diagnostics contribute to the identification of microbial pathogens in CNS infections? The SME partner Molecular Biology Systems B.V. (MBS) develops and sells equipment for extremely rapid execution of the commonly used PCR. In this project, the lectorate Analysis Techniques in the Life Sciences (Avans) will, in collaboration with MBS, Westerdijk Institute (WI-KNAW) and the Institute of Neuropathology (Münster, DE) establish a new molecular approach for fast diagnosis within CNS infections using this MBS technology. This enables the monitoring of infectious diseases in a fast and user-friendly manner, resulting in an improved treatment plan.
