Mapping of R(5mC)GY Sites in the Genome of Human Malignant Cell Line Raji

Abdurashitov M.A., Tomilov V.N., Gonchar D.A., Kuznetsov V.V., Degtyarev S.Kh. 
Biology and Medicine, Volume 7, Issue 4, BM-135-15 (2015)

The purpose of this study was to develop a method for the wide-scale determination of methylated RCGY sites in a genome using the next-generation sequencing (NGS) approach. We hydrolyzed genomic DNA from human, malignant cell line Raji with methyl-directed site-specific endonuclease GlaI and isolated DNA fragments 140-400 bp in length. DNA structure of 75 bp was determined for both ends of each DNA fragment based on NGS technology with Illumina MiSeq. The mapping of the obtained sequences on the human reference genome allowed to define the positions of more than one million R(5mC)GY sites represented in Raji genome with relatively high frequency. These data were compared with the results of the methylation study of regulatory regions of tumor suppressor genes studied earlier with a new version of PCR analysis. We have concluded that a suggested method of R(5mC)GY sites determination in a whole genome may be used for epigenetic studies, particularly for determination of methylation of regulatory regions of genes, which may affect the gene expression.

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