Independent Validation And Analysing Of Body-fluid Specific DNA Methylation Markers

Background The identifying of suspicious biological materials found at crime scenes can provide important clues for reconstructing crime scene and ascertaining the fact of case by linking DNA typing and actual criminal acts.In recent years,mRNA/microRNA-based molecular methods have been extensively studied.The specific and sensitivity are higher than that of conventional identification methods,but the stability is easily affected by ribonucleases present in the air,which prevents forensic experts from routinely integrating RNA analysis into a forensic casework scheme.The DNA methylation-based method has good specificity,high sensitivity and stability,no additional consumption of biological samples,and compatibility with the DNA typing platform of forensic standards laboratory.It has become a new studying goal for body fluids identification in forensic.There are a large number of tissue-specific differentially methylated regions(tDMRs)in the human genome,and different tissue types can be identified by comparing the methylation status of these regions.In recent years,forensic-related researches have reported some body fluid-specific DNA methylation markers,which can effectively identify the source of body fluids,but there are still many problems,such as differences in the specificity of different populations and the cross reaction among different body fluids.Therefore,it is necessary to verify and compare the existing research results on the same platform,and use accurate and feasible methylation quantitative analysis methods to screen out efficient body fluid identification markers for local population,and to provide a reliable method for the body fluid identification.Methods Body fluid-specific DNA methylation markers with good specificity from relevant literature were selected as candidate markers in this study.Five different kinds of common body fluids in human(peripheral blood,semen,saliva,vaginal fluid,and menstrual blood)were used as research samples(each of 20 cases).Methylation level of the candidate CpG sites were detected using methylation SNaPshot method by genomic DNA extraction,bisulfite conversion,PCR amplification,single base primer extension,and capillary electrophoresis analysis.Finally,the methylation specificity and individual stability of each candidate CpG site in different body fluids were analyzed,and the CpG sites with strong specificity and good stability were selected.Methylation level of the CpG sites were calculated as follows: Methylation % = C peak height /(C peak height + T peak height)× 100 = G peak height /(G peak height + A peak height)× 100.Results This paper screened nine CpG sites as more efficient body fluid-specific DNA methylation markers: cg22407458-288 d and cg05656364-283 d for semen-specific hypermethylation and hypomethylationmarkers;cg08792630 for blood-specific hypermethylation marker,cg26285698+39d and cg26285698-14 d for blood-specific unmethylated markers;cg09652652-2d for saliva-specific hypermethylation marker;cg26079753-7d for vaginal secretion-specific hypermethylation marker;cg09696411 and cg09696411 +25d for menstrual blood-specific methylation markers.Conclusion These nine screened markers were verified in 100 body fluid samples using methylation SNaPshot method,and all showed higher specificity and individual stability for identification of the target body fluid.We suggest that these nine DNA methylation markers may provide reliable reference for the construction of a more efficient body fulid identification assay for body fluids in local population.