title: The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing.
abstract: 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.
authors: Department of Chemistry and Biochemistry, Florida International University, Miami, FL, USA.
authors: Tania
authors: T
authors: Madi
authors: Kuppareddi
authors: K
authors: Balamurugan
authors: Robin
authors: R
authors: Bombardi
authors: George
authors: G
authors: Duncan
authors: Bruce
authors: B
authors: McCord
mesh_terms: D000328:Adult; D000704:Analysis of Variance; D018899:CpG Islands; D004247:DNA / Q000032:analysis* / Q000097:blood / Q000737:chemistry / Q000235:genetics; D019175:DNA Methylation; D057890:Epigenomics; D004847:Epithelial Cells / Q000737:chemistry; D005260:Female; D053803:Forensic Genetics / Q000379:methods*; D005819:Genetic Markers / Q000235:genetics; D006801:Humans; D008297:Male; D008875:Middle Aged; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012463:Saliva / Q000737:chemistry; D017422:Sequence Analysis, DNA / Q000379:methods*; D013094:Spermatozoa / Q000737:chemistry; D013447:Sulfites
chemical_list: D005819:Genetic Markers; D013447:Sulfites; D004247:DNA; C042345:hydrogen sulfite