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The determination of tissue‐specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing
Author(s) -
Madi Tania,
Balamurugan Kuppareddi,
Bombardi Robin,
Duncan George,
McCord Bruce
Publication year - 2012
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.201100711
Subject(s) - dna methylation , cpg site , methylation , bisulfite , biology , bisulfite sequencing , microbiology and biotechnology , pyrosequencing , epigenetics , saliva , primer (cosmetics) , illumina methylation assay , dna , genetics , gene , biochemistry , chemistry , gene expression , organic chemistry
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, C 20orf117, ZC 3 H 12 D , BCAS 4, and FGF 7, 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 ( C p G sites), methylation levels were measured at C p G sites spanning each marker. Up to 11 samples of each tissue type were collected and subjected to bisulfite modification to convert unmethylated C p G ‐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 C p G site was quantitated by pyrosequencing. The percent methylation values at each C p G site were determined and averaged for each tissue type. The results indicated significant methylation differences between the tissue types. The methylation patterns at the ZC 3 H 12 D and FGF 7 loci differentiated sperm from blood, saliva, and epithelial cells. The C 20orf117 locus differentiated blood from sperm, saliva, and epithelial cells and saliva was differentiated from blood, sperm, and epithelial cells at a fourth locus, BCAS 4. 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.