An examination of differences in epigenetic methylation of saliva type samples based on collection method.

In the context of forensic casework, it is imperative to both establish a DNA profile from biological specimens and accurately identify the specific bodily fluid source. To achieve this, DNA methylation markers have been developed for the differentiation of blood, semen, vaginal epithelial secretions, and saliva samples. Saliva, alternatively referred to as oral fluid, is recognized for its heterogeneous cellular composition, characterized by a mixture of epithelial, leukocytic, and bacterial cells. Consequently, our research has revealed variations in methylation percentages that correlate with the method employed for collecting saliva samples. To investigate these concepts, we scrutinized four CpG markers situated within or in proximity to the BCAS4, SLC12A8, SOX2OT, and FAM43A genes. Subsequently, we designed primers based on bioinformatically transformed reference sequences for these markers and rigorously assessed their quality by examining dimer and hairpin formation, melting temperature, and specificity. These loci were identified as saliva markers based on either buccal swabs or spit collection. Yet, there has been minimal or no research conducted to explore the variations in methylation between different collection methods. For this study, buccal, lip, tongue, spit, and nasal swabs were collected from 20 individuals (N = 100). Mock forensic samples, which include chewing gum (N = 10) and cigarettes (N = 10), were also tested. DNA was extracted, bisulfite converted, then amplified using in-house designed assays, and pyrosequenced. The methylation levels were compared to other body fluids (semen, blood, vaginal epithelia, and menstrual blood [N = 32]). A total of 608 pyrosequencing results demonstrated that sampling location and collection method can greatly influence the level of methylation, highlighting the importance of examining multiple collection/deposition methods for body fluids when developing epigenetic markers.

High-resolution melt analysis for the detection of skin/sweat via DNA methylation.

The determination of tissue type is important when reconstructing a crime scene as skin cells may indicate innocent contact, whereas other types of cells, such as blood and semen, may indicate foul play. Up to now, there has been no specific DNA methylation-based marker to distinguish skin cell DNA from other body fluids. The goal of this study is to develop a DNA methylation-based assay to detect and identify skin cells collected at forensic crime scenes for use in DNA typing. For this reason, we have utilized a DNA methylation chip array-based genome-wide association study to identify skin-specific DNA methylation markers. DNA obtained from skin along with other body fluids, such as semen, saliva, blood, and vaginal epithelia, were tested using five genes that were identified as sites for potential new epigenetic skin markers. Samples were collected, bisulfite converted, and subjected to real-time polymerase chain reaction (PCR) with high-resolution melt analysis. In our studies, when using WDR11, PON2, and NHSL1 assays with bisulfite-modified PCR, skin/sweat amplicons melted at lower temperatures compared to blood, saliva, semen, and vaginal epithelia. One-way analysis of variance demonstrates that these three skin/sweat markers are significantly different when compared with other body fluids (p < 0.05). These results demonstrate that high-resolution melt analysis is a promising technology to detect and identify skin/sweat DNA from other body fluids.

A study of the potential application of digital PCR in the detection of fecal contamination of strawberries using Bacteroides markers.

Food-borne illnesses can result from contamination of agricultural products. In this study, we examined nanoplate digital PCR (dPCR) to test for fecal contamination of agricultural products. In nanoplate technique, the PCR mastermix is divided into 8.526,000 partitions, providing direct detection of individual DNA molecules, with correction by Poisson distribution. In this project, strawberries were inoculated with fecal material from animals, and the result detected by nanoplate digital PCR. A detection limit of 250 fg/uL was determined. Overall, dPCR offers a quick and sensitive method to detect contaminated produce.

Development of a microwave-based extraction for forensic biological samples.

In this study, a quick microwave-based treatment was developed as a front end for DNA analysis of forensic samples. The effect of microwave treatment is to cause cell disruption which can improve the release of DNA during direct PCR as well as with extraction methods. Exposure to microwave preprocessing improved the quality of rapid genotyping, particularly when used with low level samples. Optimal results were obtained when samples were microwaved at 300W for 40 s, resulting in improved allele detection. Overall, the addition of this simple preprocessing step improves sensitivity and allele recovery for low level DNA samples when combined with expedited DNA analysis workflows. Its main advantages include speed, low cost, compatibility with downstream DNA methods and application to a wide variety of samples.