Screening Of Differentially Methylated DNA Fragments From Human Body Fluids/Tissues

Background Tissue identification of biological materials at crime scenes is an important part of forensic examination, which still show variety of shortages in traditional methods. Though mRNA and miroRNA have been well performed recently in tissue identification, RNA is still not stable enough to corrupted samples and also consume additional samples. So it is of great importance to search for new methods and ideal genetic markers in tissue identification. Recent advances in whole genome epigenetic analysis indicate that numerous tDMRs exist in mammalian and human. Different tDMRs show different DNA methylation profiles according to the type of cells or tissues and may discriminate their certain types in theory, which lay the theoretical foundation for DNA methylation markers in tissue identification. If the varying patterns in DNA methylation and their expression patterns in human body fluids/tissues can be ascertained, a brand-new tissue identification method and idea in genetic level can be performed to significantly improve the forensic identification level.Objective To screen the differentially methylated DNA fragments from human body fluids/tissues and to lay the foundation for further fragment screening to establish an efficient and sensitive forensic fluids/tissues identification method.Methods Six different kinds of fluids/tissues (blood, brain, muscle skin, liver and vaginal secretion) were obtained to extract genome DNA. The genome DNA were digested by MSRE HpaII and the digested DNA fragments were linked with adaptors and then amplified to enrich amplicons. Firstly, amplicons from blood samples were named as tester and the others were together named as driver to finish the forward subtractive hybridization. After two or three rounds, the "blood" fragments specific to other five tissues were screened. Secondly, the reverse subtractive hybridization was taken by exchanging the tester and driver and the other five tissues’fragments specific to the "blood" were screened. Then amplicons from muscle samples were named as tester to finish the forward and reverse subtractive hybridization and two opposite specific fragments were screened. After all, four groups of screened specific fragments were purified, cloned and sequenced to make comparison and analyzation with the sequences in GenBank by BLAST software.Results By the MS-RDA technique with MSRE HpaⅡ, differently methylated DNA fragments were preliminarily obtained:5between "blood" and other five tissues and11between "muscle" and other five tissues.Conclusion There are obvious different DNA methylation sites between Human blood and muscle. The screened DNA methylated fragments in this research lay a foundation for their further application in forensic fluids/tissues identification.