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. (150

Independent validation of body fluid-specific CpG markers and construction of a robust multiplex assay

Potential forensic use of tissue-specific DNA methylation markers has recently been discussed for the identification of the biological source of a stain. In this study 13 promising markers were evaluated to identify suitable candidate markers for the development of a robust and reliable multiplex assay. The results of this study suggest that a combination of only four highly informative markers will be enough for clear body fluid identification. A multiplex assay was developed for the identification of menstrual blood, saliva, semen, and venous blood. This assay was successfully applied to the identification of these body fluids in mixtures and crime scene stains. The multiplex assay aids in the identification of not only single source body fluids but also of body fluid mixtures. The main advantage of using DNA methylation assays over alternative tests is that it can be applied at a later time point in the investigative process since testing is possible even after DNA analysis