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Jawad Salehi, Shalini Behl, Rania Gomaa.
Department of Biotechnology
University of Modern Sciences, Dubai, U.A.E.
Abstract
Epigenetic changes arising due to DNA methylation and histone acetylation can be used
for body fluid identification to overcome the limitations that arise from presently
available standard identification tests. Markers DACT1, USP49, ZC3H12D, FGF7,
Cg23521140, Cg17610929, chromosome 4 (25287119–25287254), chromosome 11
(72085678–72085798, 57171095–57171236, 1493401–1493538), and chromosome 19
(47395505–47395651) are currently being used for semen identification. Markers
Cg26107890, cg20691722, Cg01774894 and cg14991487 are used to differentiate saliva
and vaginal secretions from other body fluids. However, such markers show overlapping
methylation pattern. Using DNA methylation to identify the body-fluids can give
Figure 1: Schematic of epigenetic changes in DNA.
accurate results without much evidence consumption.
Results and Concluding Remarks
Background
The use of DNA methylation for body tissue analysis in forensic field is relatively new; it was
first used by Frumkin et al. (2011). Wasserstrom et al. (2012) using Frumkin et al. approach
DNA methylation is involved in complex biological processes like cell differentiation
developed a kit for the identification of semen using DNA methylation pattern. This kit is
and aging [1]. Cell differentiation is regulated by gene expression which leads to the
commercially available as Nucleix DNA source identifier (DSI)-SemenTM kit and can replace the
development of different types of cells. This in turn is regulated by DNA methylation as
microscopic examination of samples used to differentiate semen from non-semen samples [10].
illustrated in figure 1, which is influenced by environment and age [2, 3]. Since each
Park et al. (2014) conducted genome wide profiling for various body fluids and were able to
biological fluid is produced at different locations of body and for different functions,
identify novel DNA methylation markers for blood, saliva, semen and vaginal secretions. [11].
they should have differential methylation patterns. This difference can be used to identify
Lee et al. (2012) studied 5 tissue-specific differentially methylated regions (tDMRs) to
the nature of the biological fluid found at crime scene. Most currently used body-fluid
differentiate body fluids. The tDMRs from DACT1 showed 93% unmethylation for semen
identification tests are enzyme and protein based that gives presumptive result and
samples but hypermethylation in other body fluids. Whereas tDMRs from USP49 showed
consumes the available evidence. Protein based assays are qualitative and not
hypomethylation in 97% of clones from semen and hypermethylation in other body fluid samples
quantitative due to which statistical confidence level cannot be calculated, but DNA
[12].
methylation provides quantitative results allowing for the calculation of statistical
confidence level of the results [4]. In addition, DNA methylation levels can be used to
estimate the age, gender and ethnicity of the individual [5-7]. This information is of great
value to either convict or exonerate the individual. The relation of age, gender and
ethnicity with DNA methylation is still under initial investigations which might serve
great potentials in forensic investigations.
Madi et al. (2012) studied 4 markers (C20orf117, BCAS4, FGF7, ZC3H12D) to differentiate
body luids (blood, semen, saliva/buccal cells). Gene C20orf117 showed hypermethylation for
blood, and successfully differentiated it from other samples. Marker ZC3H12D showed
hypomethylation for semen samples and successfully differentiated it from other body fluids. [8].
Previous studies were unable to find unique markers that can differentiate saliva from vaginal
secretions [8, 12]. Therefore further studies are needed that incorporate next-generation
sequencing to find more reliable markers that can differentiate between saliva and vaginal
secretion sample.
Methodology
There is also a need to develop multiplex assay for pyrosequencing which will greatly reduce the
time and starting amount of DNA needed [11].
Most common method of analyzing DNA methylation is by chemically modifying
References
cytosine residues using sodium bisulfate [8]. The non-methylated cytosines are
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[1]
Madi et al. (2012) used bisulfite treatment to modify the DNA samples and then
[2]
[3]
amplified the region of interest using PCR. The reverse primers were biotin labeled in
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[4]
[5]
Park et al. (2014) also used sodium bisulfide treatment to modify the DNA samples and
[6]
then amplify using the InfiniumTM Methylation Assay kit (Illumina). Amplified DNA
[7]
was then hybridized to Human Methylation 450 kTM bead array (Illumina) and scanned
[8]
with Illumina iSCAN system to identify body-fluid specific DNA methylation markers.
Selected marker candidates were then validated using pyrosequencer.
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Wasserstrom et al. (2013) used forward and reverse primers in order to amplify CpG
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methylation between semen and non-semen samples. [9].
[9]
[10]
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PP 7.2
DNA Methylation as a Biomarker for Body Fluid Identification
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The International Arab Forensic Science and Forensic Medicine Conference / 8-10 November 2015