Body fluid Identification using epigenetic methylation markers and pyrosequencing

In certain criminal cases- especially sexual assaults, the evidentiary value of DNA is greatly enhanced if the type of stain (e.g. blood, semen, saliva), can be identified. Examples of such scenarios include sexual assaults in which the presence of sperm or saliva indicates a crime was committed but skin cells might be consistent with innocent contact. Similarly blood under a fingernail would be consistent with a violent act but skin cells might simply indicate a handshake. Current procedures for detection of body fluids are insensitive and may fade with time. We propose the use of DNA methylation patterns to detect body fluids and dried stains. Thus it is the goal of this project to develop epigenetic methylation markers for detection of different tissue types present in forensic evidence. Epigenetics is a rapidly expanding field, especially in cancer diagnostics, in which aberrant methylation sites in the DNA can be used to probe cell differentiation.. Generally speaking, methylation of DNA at promoter regions blocks transcription and silences gene expression, permitting cell differentiation. As a result of this process, certain locations in the genome known as CpG islands are methylated in a tissue specific fashion. These loci can be exploited using bisulfite modification which converts unmethylated cytosines to lower melting uracils followed by PCR and analysis using pyrosequencing or real time PCR melt curves. We can use this information to identify body fluid stains. The procedure utilizes DNA extraction, bisulfite modification and PCR amplification. Detection of methylation sites will be performed by pyrosequncing and high resolution melting real time PCR. In the project we will work with the University of Southern Mississippi, the Broward Sheriffs Office DNA laboratory, and Qiagen Inc. to develop forensic loci which are differentially methylated in blood, skin, saliva, sperm and vaginal epithelia cells. Preliminary findings using a panel of markers, that include C20orf117, ZC3H12D, BCAS4 and FGF7 demonstrates our capability to accurately and specifically detect these fluids in clinical samples. To find additional loci we will utilize next generation sequencing to scan a population of 4-5 individuals to detect methylation specific differences in their chromosomes. Once loci have been identified we will develop methylation specific PCR primers capable of identifying semen, saliva, blood, vaginal epithelial cells and skin cells. Then using bisulfite modification and multiplex PCR amplification followed by pyrosequencing and real time PCR we will determine cell specific methylation patterns permitting us to identify the source of single and mixed biofluids at crime scenes. As shown in our preliminary data, the results produce clear and distinguishable differences between cell types with a sensitivity approaching 1 ng/ìL of genomic DNA. Thus the goal of this project is to develop a database of forensic methylation markers for application to identification of trace DNA collections. To do this we will screen a small population database to increase the number of markers and cell types we can identify. In addition, we will work to increase the sensitivity of the procedure, perform a larger population study that includes a variety of ages and racial groups and finally complete a full developmental validation of the process that includes adjudicated forensic samples, mixtures, aged and environmentally challenged samples. ca/ncf