{"id":12,"date":"2019-06-13T14:24:48","date_gmt":"2019-06-13T11:24:48","guid":{"rendered":"https:\/\/www.epigendx.online\/?page_id=12"},"modified":"2021-06-08T11:37:50","modified_gmt":"2021-06-08T08:37:50","slug":"%d0%b3%d0%bb%d0%b0%d0%b2%d0%bd%d0%b0%d1%8f","status":"publish","type":"page","link":"https:\/\/www.epigendx.online\/en\/","title":{"rendered":"Home"},"content":{"rendered":"

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Research article<\/a><\/span><\/div>\t\t\t\t<\/a>\n\t\t\t\t\t\t\t<\/div>\n\t\t

\t\t\n\t\t\tDetermination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data\t\t<\/a>\n\t\t<\/h2>\t\t
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\n\t\t\t\tM.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev\r\n\r\nSibEnzyme Ltd, Novosibirsk\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\nCorresponding author: M.A. Abdurashitov, SibEnzyme Ltd., 2\/12 Ak. Timakova Street,Novosibirsk 630117, Russia; Tel.: +7 (383) 333-4991; Fax: +7 (383) 333-6853; E-mail: abd@sibenzyme.ru\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n \r\nAbstract\r\nThe 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves DNA sites with the sequence 5\u2032-R(5mC)GY-3\u2032 \/ 3\u2032-YG(5mC)R-5\u2032, which are formed de novo in the human genome by the DNA methyltransferase DNMT3. Owing to this specificity, GlaI is efficiently used to determine the methylation status of RCGY sites in the human genome. Genome-wide mapping of R(5mC)GY sites can be performed by next-generation sequencing (NGS) of DNA hydrolysates generated by GlaI digestion, whereas analysis of individual DNA fragments can be carried out using GlaI-PCR analysis.\r\nIn this study, we compared the results of NGS analysis of GlaI DNA hydrolysates with data obtained by…\t\t\t<\/div>\n\t\t\t\t\tRead More...<\/a>\n\t\t\t\t<\/div>\n\t<\/article >\n\t<\/div>\n\t<\/div>[\/vc_column][vc_column width=”1\/3″][vc_column_text]<\/p>\n

New methods of DNA methylation analysis<\/a><\/h3>\n

[\/vc_column_text][rev_slider_vc alias=”gladpcr_eng” title=”GLAD PCR Assay”][rev_slider_vc alias=”glaipcr_eng” title=”GlaI PCR Assay”][\/vc_column][\/vc_row][vc_row][vc_column width=”2\/3″]

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\t\t\n\t\t\tDetermination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data\t\t<\/a>\n\t\t<\/h2>\t\t
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Research article<\/a><\/span><\/div>\t\t\t<\/a>\n\t\t\t\t\t\t<\/div>\t\t\t
\n\t\t\t\tM.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev\r\n\r\nSibEnzyme Ltd, Novosibirsk\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\nCorresponding author: M.A. Abdurashitov, SibEnzyme Ltd., 2\/12 Ak. Timakova Street,Novosibirsk 630117, Russia; Tel.: +7 (383) 333-4991; Fax: +7 (383) 333-6853; E-mail: abd@sibenzyme.ru\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n\r\n \r\nAbstract\r\nThe 5-methylcytosine-dependent DNA endonuclease GlaI recognizes and cleaves DNA sites with the sequence 5\u2032-R(5mC)GY-3\u2032 \/ 3\u2032-YG(5mC)R-5\u2032, which are formed de novo in the human genome by the DNA methyltransferase DNMT3. Owing to this specificity, GlaI is efficiently used to determine the methylation status of RCGY sites in the human genome. Genome-wide mapping of R(5mC)GY sites can be performed by next-generation sequencing (NGS) of DNA hydrolysates generated by GlaI digestion, whereas analysis of individual DNA fragments can be carried out using GlaI-PCR analysis.\r\nIn this study, we compared the results of NGS analysis of GlaI DNA hydrolysates with data obtained by GlaI-PCR analysis of regulatory regions of 11 tumor suppressor genes in DNA from human cell lines L68 (normal lung fibroblasts), Raji (Burkitt lymphoma), and U937 (histiocytic lymphoma). It was shown that in L68 cells most of the analyzed regulatory regions are unmethylated, whereas methylation of these regions is observed in the malignant cell lines Raji and U937. With the exception of a single region, for all other analyzed fragments the methylation status determined by GlaI-PCR analysis fully correlates with the number of R(5mC)GY sites detected by NGS.\r\nThe only discrepancy between the two methods was identified for the regulatory region of the RASSF1A gene in L68 cells and was attributed to the presence of allele-specific methylation, which was confirmed by additional real-time GlaI-PCR analysis. Taking allele-specific methylation of the RASSF1A regulatory region into account, complete concordance was observed between the data obtained by NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis of regulatory regions of the studied tumor suppressor genes in the human cell lines L68, Raji, and U937.\r\nThus, NGS analysis of GlaI DNA hydrolysates and GlaI-PCR analysis are complementary approaches that enable reliable identification of aberrantly methylated genomic regions in normal and malignant cells, making them promising tools for epigenetic research and DNA-based diagnostics.\r\nKeywords: 5-methylcytosine; DNA methylation; methyl-dependent restriction endonucleases;GlaI; NGS analysis; GlaI-PCR analysis; epigenetic DNA diagnostics; allele-specific methylation.\r\n\r\nDOI: 10.26213\/3034-4298.2025.6.1.001\r\n\r\nCitation: M.A. Abdurashitov, A.G. Akishev, S.Kh. Degtyarev (2025) Determination of the Methylation Status of RCGY Sites in the Human Genome: A Comparison of NGS Analysis of GlaI DNA Hydrolysates and GlaI-PCR Data, Epigenetic DNA diagnostics, vol 2025(1), DOI: 10.26213\/3034-4298.2025.6.1.001\r\n\r\n\r\nThis work is available at\u00a0Creative Commons…\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t<\/article >\n\t
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\t\t\n\t\t\tDetermination of the risk of breast and stomach cancer by identifying variants of the 5mC\/T polymorphism in the intron of the KIF26B gene at position xp1: 245618129\t\t<\/a>\n\t\t<\/h2>\t\t
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Diagnostics<\/a><\/span><\/div>\t\t\t<\/a>\n\t\t\t\t\t\t<\/div>\t\t\t
\n\t\t\t\tWe used GlaI\/FatI-PCR assay for determination of 5mC\/T polymorphism frequencies at position xp1: 245618129 (according to the GRCh38.p13 genomic assembly) in DNA preparations isolated from blood cells of 51 breast cancer (BC) patients and 63 gastric cancer patients (GC). The data obtained were compared with those previously obtained for 92 healthy blood donors.\r\nIt was shown that the cytosine in this position is predominantly is methylated in all DNA samples. The incidence of a diploid C\/C set in this position in patients with breast cancer and gastric cancer is 9.8% and 7.94%, respectively. This is significantly different from the incidence of C homozygotes in healthy women (26.09%) and healthy donors of both sexes (22.83%). Thus, C\/C homozygotes at position xp1: 245618129 is an favorable sign indicating a lower risk (2.7-2.9 times) of two types of oncological diseases. At the same time, the incidence of C\/T heterozygotes and T\/T homozygotes in patients with gastric cancer and breast cancer exceeds similar indicators for control groups of healthy people by 5.5-10.7%.\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t<\/article >\n\t
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\t\t\n\t\t\tStudy of the methylation of RCGY sites in the regulatory region of the FAM19A4 gene in human cell line dna preparations by GLAD-PCR assay\t\t<\/a>\n\t\t<\/h2>\t\t
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Research article<\/a><\/span><\/div>\t\t\t<\/a>\n\t\t\t\t\t\t<\/div>\t\t\t
\n\t\t\t\tThe method of GLAD-PCR analysis was used to determine the methylation of RCGY sites in the regulatory region of the FAM19A4 gene (Xp3: 68932451 - 68932800) in the DNA preparations of the malignant human cell lines Raji, Jurkat, HeLa, U-937 and the control DNA of the lung fibroblast cell line L68.\r\nIt is shown that the octanucleotide GCGCGCGC (Xp3: 68932500), located in the first exon of the FAM19A4 gene, is cleaved approximately equally in the DNA of all 4 malignant human cell lines, whereas in the DNA of L68 this site is hydrolyzed 10-15 times weaker. These results are consistent with data of epigenomic sequencing of L68, Raji, and U-937 DNA.\r\nMethylation of CG-dinucleotides at positions 68932727 and\/or 68932740 in the promoter of the FAM19A4 gene occurs only in tumor DNA and is not observed in L68 DNA, whereas methylation of CG-dinucleotide at position 68932704 is observed only in HeLa DNA.\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t<\/article >\n\t
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\t\t\n\t\t\tDetermination of SNP 5mC\/T in position Chr1: 245618129 in DNA samples from the human blood by GlaI- and FatI-PCR assay\t\t<\/a>\n\t\t<\/h2>\t\t
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Diagnostics<\/a><\/span><\/div>\t\t\t<\/a>\n\t\t\t\t\t\t<\/div>\t\t\t
\n\t\t\t\tSNP 5mC\/T (Chr1: 245618129) in DNA samples from the human blood of 92donors was studied by GlaI and FatI-PCR assay. The work includes a) isolation of DNA from the blood cells, b) GlaI and FatI-PCR assay of DNA fragment Chr1: 245617889 - 245618464, c) determination of 5mC (Chr1: 245618129) and thymine in DNA preparations and d) comparative analysis of the obtained results.\r\n\r\nIt was shown that in position (Chr1: 245618129) 43 donors (46,74%) have a heterozygote (5mC\/T), 28 donors (30,43%) have homozygote (T\/T) and 21 donors (22,83%) have homozygote (5mC\/5mC). Thus, taking into account a diploid set of chromosomes in the blood cells, SNP 5mC\/T is observed in 99 positions from 184 (53,8%).\r\n\r\nThe results obtained have shown that cytosine in position Chr1: 245618129 in the most of the blood DNA is methylated.\t\t\t<\/div>\n\t\t\t\t<\/div>\n\t<\/article >\n\t<\/div>\n\t<\/div>[\/vc_column][vc_column width=”1\/3″][vc_widget_sidebar sidebar_id=”primary-sidebar”][\/vc_column][\/vc_row]<\/p>","protected":false},"excerpt":{"rendered":"

[vc_row][vc_column width=”2\/3″][\/vc_column][vc_column width=”1\/3″][vc_column_text] New methods of DNA methylation analysis [\/vc_column_text][rev_slider_vc alias=”gladpcr_eng” title=”GLAD PCR Assay”][rev_slider_vc alias=”glaipcr_eng” title=”GlaI PCR Assay”][\/vc_column][\/vc_row][vc_row][vc_column width=”2\/3″][\/vc_column][vc_column width=”1\/3″][vc_widget_sidebar sidebar_id=”primary-sidebar”][\/vc_column][\/vc_row]<\/p>\n","protected":false},"author":1,"featured_media":0,"parent":0,"menu_order":0,"comment_status":"closed","ping_status":"closed","template":"","meta":[],"coauthors":[26,15,16,17],"_links":{"self":[{"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/pages\/12"}],"collection":[{"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/pages"}],"about":[{"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/types\/page"}],"author":[{"embeddable":true,"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/comments?post=12"}],"version-history":[{"count":21,"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/pages\/12\/revisions"}],"predecessor-version":[{"id":471,"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/pages\/12\/revisions\/471"}],"wp:attachment":[{"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/media?parent=12"}],"wp:term":[{"taxonomy":"author","embeddable":true,"href":"https:\/\/www.epigendx.online\/en\/wp-json\/wp\/v2\/coauthors?post=12"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}