Genotyping from samples containing different types of body fluids is a major difficulty in forensic investigations. Recently, CpG sites that are specifically methylated or unmethylated in different types of body fluids have been reported as novel markers for body fluid identification. In this study, we hypothesized that the simultaneous analysis of CpGs and neighboring polymorphic sites on the same molecule could be useful for individual DNA typing from mixed samples. We performed a proof-of-concept study of this approach by searching the genome-wide methylation dataset deposited at the National Center for Biotechnology Information Gene Expression Omnibus repository for semen-specific CpG markers adjacent to common single nucleotide polymorphisms. From the identified candidates, we selected 5 regions on different chromosomes and validated the presence of semen-specific methylation or unmethylation in each region by pyrosequencing analyses. By combining methylation-specific polymerase chain reaction and pyrosequencing technology, we developed a semen-specific DNA typing method for two semen-specific methylated regions and one semen-specific unmethylated region. Finally, the method successfully identified semen-derived alleles from mixed stains, indicating that this methylation-based approach can be applicable to actual forensic samples. Since existing separation techniques physically isolate cells derived from each type of body fluid, this approach may be useful when existing methods cannot be performed due to the degradation of samples.
Keywords: Body fluid identification; DNA methylation; DNA mixture; DNA typing; Semen-specific DNA typing.
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