| Background The identification of body fluids/tissues at crime scenes is one of the important portions of criminal investigation. Recently, DNA methylation has become a hot topic in filed of forensic body fluids/tissues identification. A lot of researches show that numerous tDMRs exist in human genome and tDMRs display variety of methylation among different tissues/fluids. It is possible to identify different tissues and cells based on the theoretical foundation of tDMRs. Compared with the traditional methods and the technical methods that are based on mRNA-profiling or micro RNA-profiling for tissue identification,DNA methylation-based assay can provide direct link between DNA profile and its source by its good sensitivity and stability. Above all, this method does not need to consume additional material. With the development of methylation detection techniques, the methods of body fluids/tissues identification based on DNA methylation have been becoming more and more mature. Although many tDMRs markers were reported for forensic tissue identification, it is difficult to repeat the results of body fluids/tissues identification using some of these markers.Most of the markers only appear to have potential applicability for semen identification.Therefore, it is necessary to investigate additional differentially methylated loci and screen more novel tDMRs loci which showed high sensitivity and specificity for identification of different types of body fluids.If the richness of the methylation patterns in human body fluids/tissues can be increased greatly, it will contribute a new direction to promoting the forensic identification development.Obejective To screen the tDMRs from genome of human semen and saliva and provide new methylation markers for establishing a sensitive and efficient forensic body fluid/tissue identification method.Methods Blood, saliva, semen and muscle samples which are most common forensic biological samples were collected as research samples. Blood, saliva and semen were from healthy human. Muscle samples were from corpes without obvious illness. DNA was extracted from all samples by a standard phenol/chloroform and ethanol precipitation procedure. Genomic DNA of different types of body fluids/tissues was digested with Hpa?.RHpa24/11 adaptor was ligated to the digested genomic DNA by T4 DNA ligase. The ligation product was subsequently amplified into an “amplicon” by PCR with RHpa24 oligonucleotide as a universal primer. Comparing these amplicons which can be as the representative of the genomes: DNA pool of semen was first selected as a Tester and the Driver composed of mixing equal amounts of other three DNA pools. In positive subtractive hybridization, JHpa24/11, a new adaptor, was only ligated to Tester amplicon, and Tester amplicon was mixed with excess Driver amplicon without adaptor. Then the mixture underwent competitive hybridization by the best denatured and reannealing temperature.Products of hybridization were amplified by PCR using primer of JHpa24. The second cycle of subtractive hybridization was performed by switching the adaptor of the product to a new adaptor, NHpa24/11. The hybrid process was the same as the first cycle. By repeated positive subtractive hybridization, DNA fragments that are differentially methylated between the semen and other three sample pools(salive, blood and muscle) were isolated. Next the differentially methylation of other sample pools(salive, blood and muscle) was screend and compared with semen via exchange the type of Tester and Driver to complete the same experimental steps, which is product of reverse hybridization. On the basis of these steps,t DMRs from saliva were also screened. The final product was cloned into the pMD19-T Vector. After the plasmid DNA was transformed into Escherichia coli competent cells JM109(Takara Co., Japan), these clones were sequenced. Genomic origins of the isolated DNA fragments were examined by online BLAST tool and the chromosomal position and relativelocations to CGIs were discovered at the GeneBank web site. The methylation degree of these isolated fragments was roughly quantified by bisulfite sequencing PCR(BSP) in independent samples, respectiviely. The fragments that show obvious differential methylation between the semen and saliva were selected further to identify their specificity in other kinds of fluid samples by BSP.Results Differentially methylated fragments were preliminarily screened by MS-RDA:six differentially methylated regions which shows semen-specific(Se-1~Se-6) and six differentially methylated regions which shows saliva-specific(Sa-1~Sa-6). Methelation of CpGs in these fragments were obtained by BSP: ?In the six semen-specific candidate target bands, CpG sites of CpG11?CpG12?CpG13?CpG16? CpG17?CpG18 in the Se-3 fragment are unmethylated in semen and blood samples but show hypomethylation in saliva samples(Methylation Level:0.1~0.2);CpG sites of CpG 2?CpG3?CpG6?CpG9 in the Se-5 fragment are unmethylated in semen and saliva samples but exist hypomethylation in blood samples(Methylation Level:0.1~0.3); CpG sites of CpG2?CpG5? CpG6?CpG8?CpG11?CpG13?CpG17?CpG18 in the Se-6 fragment are unmethylated in semen samples but are hypermethylated in blood and saliva samples(Methylation Level:0.5~0.7). ? In the 6saliva-specific candidate target bands, CpG sites of CpG1?CpG4?CpG6?CpG7?CpG8 in the Sa-1 fragment are completely methylated in saliva samples and hypermethylated in blood and semen samples(Methylation Level:0.7~0.9); the methylation level of each CpG site in the Sa-6 fragment is very high in saliva and blood samples(Methylation Level:0.9)but is low in semen sample(Methylation Level:0.1).Conclusion The screened CpG sites of semen-specific unmethylation and CpG sites of saliva-specific hypermethylation in this research provide some effective methylation markers for identification of semen and saliva. In addition, this study provides a reference for establishing a high polymorphism and high specificity method of identification of tissues/fluids by DNA methylation in the field of forensic science. |
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