Screening Of Age-related DNA Methylation Fragments

Background Age estimate is an important task in forensic science. Nowadays in practice, age is estimated mainly by measuring and calculating some osteal markers like bones and teeth in forensic anthropology. This method is admissible with its acceptable error. Because of the great dependence on these osteal markers, the application of this method is confined in certain cases. With the development of molecular biology, to estimate age by testing blood or soft tissues in a convenient way, researchers brought forward methods of age estimate based on telomere shortening or mitochondrial DNA's injury. To a certain extent, these two methods had received considerable attentions in the field of forensic science. But limited precision, heteroplasmy in mitochondrial and excessive statistical work made it difficult to apply them in practice work. In recent years, learners of epigenetics found that DNA methylation was widely distributed, and had strong correlation with age. So if the data model between DNA methylation profile and age can be set up by screening the DNA fragments correlative with aging, it would be possible to apply methylation in age estimate in the future.Objective To screen the methylation fragments changing with age from genome by comparing the DNA methylation profiles between different age sets, and make pre-preparation for the following exploring of the correlation between DNA methylation and age .Methods Eight peripheral blood samples from young(< 20years old) and old (>70 years old) healthy individuals were obtained from Hubei Tongji Center for Medicolegal Expertise. 4 samples from people born before 1949 were named as"old"and the rest samples from people born after 1990 were named as"young". Genomes DNA were extracted by standard hydroxybenzene-Chloroform procedure. Genome DNA were digested with enzyme NotⅠ(a kind of methylation sensitive restriction enzyme) based on the characteristic that methylation sensitive restriction enzyme could cleave the unmethylated sites, but didn't cleave methylated sites. Then, post-digestion DNA were linked with adaptors and amplified in order to enrich the amplicons. Firstly, amplicons from"old"was named as tester and amplicons from"young"was named as driver to take the following subtractive hybridization. After two or more rounds subtractive hybridizations, the"old"fragments specific to"young"was screened. Secondly, contrary subtractive hybridization was taken by exchanging the tester and driver. After all these screening stage, our second stage: cloning, sequencing, blasting and DNA methylation sequencing of the fragments got started.Results (1)10 candidate DNA fragments differently methylated between"old"and"young"were obtained by the methylation-sensitive-representational difference analysis (MS-RDA) technique. (2) Methylation sequencing revealed that three of 10 candidate target fragments had different degrees of methylation differences between the old and young groups.Conclusion Discrepancy of DNA methylation between the"old"and"young"is obvious. The results of this study lay a solid foundation for further study on correlation between DNA methylation and aging.