Methylation-sensitive/-dependent restriction enzyme (MSRE/MDRE) PCR can be performed to detect hypomethylated or hypermethylated CpG sites. With the combined use of different tissue-specific CpG markers, MSRE/MDRE-PCR leads to tissue-specific methylation patterns (TSMPs), enabling the correlation of DNA samples to their source tissue. MSRE/MDRE assays can use the same platform as forensic STR typing and offer many advantages in the field of forensic body fluid detection. In the present study, we aimed to establish MSRE assays for the detection of blood, saliva, vaginal secretion, and semen, using markers from literature and from our own database search. We designed two different MSRE test-sets, which include two novel Y-chromosomal non-semen markers, and enable differentiation between female and male non-semen samples. Furthermore, we established an MSRE/MDRE semen approach, which includes only Y-chromosomal non-semen and semen markers. This Y-semen multiplex PCR utilizes the novel combination of the methylation-sensitive enzyme SmaI and the methylation-dependent enzyme GlaI, which enables more sensitive detection of male body fluids within male/female DNA mixtures. Our validation tests confirmed that MSRE/MDRE assays exhibit high sensitivity, similar to that of STR typing.
