The goal of this study is to explore the application of epigenetic markers in the identification of biofluids that are commonly found at the crime scene. A series of genetic loci were examined in order to define epigenetic markers that display differential methylation patterns between blood, saliva, semen, and epithelial tissue. Among the different loci tested, we have identified a panel of markers, C20orf117, ZC3H12D, BCAS4, and FGF7, that can be used in the determination of these four tissue types. Since methylation modifications occur at cytosine bases that are immediately followed by guanine bases (CpG sites), methylation levels were measured at CpG sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated CpG-associated cytosine bases to thymine bases. The bisulfite modified DNA was then amplified via nested PCR using a primer set of which one primer was biotin labeled. Biotinylated PCR products were in turn analyzed and the methylation level at each CpG site was quantitated by pyrosequencing. The percent methylation values at each CpG site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC3H12D and FGF7 loci differentiated sperm from blood, saliva, and epithelial cells. The C20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS4. The results of this study demonstrate the applicability of epigenetic markers as a novel tool for the determination of biofluids using bisulfite modification and pyrosequencing.

We report a large compilation of the internal validations of the probabilistic genotyping software STRmix™. Thirty one laboratories contributed data resulting in 2825 mixtures comprising three to six donors and a wide range of multiplex, equipment, mixture proportions and templates. Previously reported trends in the LR were confirmed including less discriminatory LRs occurring both for donors and non-donors at low template (for the donor in question) and at high contributor number. We were unable to isolate an effect of allelic sharing. Any apparent effect appears to be largely confounded with increased contributor number.

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Alu sequences represent the largest family of short interspersed repetitive elements (SINEs) in humans with 500 000 copies per genome. Recently, one Alu subfamily was found to be human specific (HS). We originally described the use of polymorphis HS Alu insertions as a tool in population studies and recently as tools in DNA fingerprinting and forensic analysis. In this report, we will use this simple polymerase chain reaction (PCR) base technique for the detection of HS Alu insertion polymorphisms. We will test the resolving power of this DNA profiling approach in both population genetics and paternity assessment. At the population level, we will describe the genotypic distribution of five polymorphic Alu insertions among 3 populations from the American continent, one of African origin, the other two Amerindians. Insight into their relationships will be provided. At the family level, we will examine one European American family of seven individuals and the same pedigree will also be characterized by way of the two systems currently and widely used to ascertain paternity: PCR-sequence specific oligonucleotide probe hybridization (PCR-SSO) and PCR-restriction fragment length polymorphism (PCR-RFLP) of human leucocyte antigen (HLA) class II molecules, and a standard RFLP protocol used in forensic casework and paternity studies. The importance and strengths of the methods as well as its perspectives for future use in filiation studies will be evaluated.