Cpg methylase m sssi

Manufactured by New England Biolabs

CpG methylase M.SssI is an enzyme that catalyzes the transfer of a methyl group from S-adenosylmethionine to the C-5 position of cytosine residues within the dinucleotide sequence 5'-CG-3' (CpG sites) in DNA.

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Lab products found in correlation

Dual luciferase reporter assay system, by Promega (2 mentions) Pgl3 basic luciferase vector, by Promega (1 mentions) Pcdna3, by Thermo Fisher Scientific (1 mentions)

Spelling variants of this product

M.SssI (63 citations) SssI methylase (34 citations) M.SssI methylase (16 citations)

3 protocols using cpg methylase m sssi

Investigating SP1 Regulation of MIR155HG

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To construct SP1 overexpression vector, the amplified coding region of the human SP1 gene was subcloned into a pcDNA3.1 vector. The MIR155HG proximal promoter region sequences containing the CpG islands (+400 bp to −400 bp) were amplified and inserted into a pGL3-basic luciferase vector (Promega), named pGL3-MIR155HG-WT. Generation of the mutated version (pGL3-MIR155HG-Mut) was achieved by mutating the putative binding sites of SP1. For in vitro DNA methylation, pGL3-MIR155HG-WT was treated with the CpG methylase M.SssI (NEB, Ipswich, MA). Then, 48 h post-transfection, the Dual Luciferase Reporter Assay System (Promega, Madison, WI) was used to assess luciferase activities.

Wu X., Wan Q., Wang J., Hou P., Zhang Q., Wang Q, & Lu X. (2022). Epigenetic Activation of lncRNA MIR155HG Mediated by Promoter Hypomethylation and SP1 is Correlated with Immune Infiltration in Glioma. OncoTargets and therapy, 15, 219-235.

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Dual-Luciferase Assay for Epigenetic Regulation

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The reporter plasmid pCpGL-CMV-firefly luciferase was generated by subcloning the Cytomegalovirus (CMV) promoter from pcDNA3.1 (Invitrogen) into the CpG-free pCpGL-basic vector (33 (link)). By replacing the firefly luciferase gene with the renilla luciferase gene, an analogous control reporter plasmid pCpGL-CMV-renilla luciferase was constructed. The firefly plasmid was in vitro methylated with CpG methylase M.SssI (NEB) and the complete methylation was verified by digestion with methylation sensitive enzymes TaiI (Fermentas). HEK293T cells were transiently transfected in 12-well plate with 500 ng expression constructs (Tet2/TDG/Gadd45a) each, 20 ng methylated firefly luciferase reporter and 0.2 ng unmethylated renilla luciferase reporter as an internal control for normalization. Forty-six hours after transfection, luciferase activities were measured using the dual-luciferase reporter assay system (Promega) according to the manufacturer's instructions. Each experiment was repeated at least three times.

Li Z., Gu T.P., Weber A.R., Shen J.Z., Li B.Z., Xie Z.G., Yin R., Guo F., Liu X., Tang F., Wang H., Schär P, & Xu G.L. (2015). Gadd45a promotes DNA demethylation through TDG. Nucleic Acids Research, 43(8), 3986-3997.

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Methylation-Specific PCR for Tumor DNA

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Methylation-specific PCR was performed using genomic DNA from the tumor cells, which was modified with bisulfate using an EZ DNA Methylation Kit according to the manufacturer's instructions. For detection of unmethylated DNA, the forward primer was 5'-agttattttaaaggaggtgggatgg-3', and the reverse primer was 5'-aaacaaaacccctaaacaacaa-3'. For detection of methylated DNA, the forward primer was 5'-gagttattttaaaggaggcgggac-3', and the reverse primer was 5'-gaaacccctaaacgacaacgac-3'. The primers were designed using the MethPrimer software program as described previously (31 (link)). The negative control was water. The positive control was genomic DNA that was methylated using the CpG methylase M.Sss I (New England Biolabs). The PCR conditions consisted of an initial 5 minutes at 95°C; 40 cycles of 94°C for 25 seconds, 58°C for 25 seconds, and 72°C for 30 seconds; and a final extension step at 72°C for 10 minutes. The PCR products were confirmed using 2% agarose gel electrophoresis and ethidium bromide staining.

Quan M., Cui J., Xia T., Jia Z., Xie D., Wei D., Huang S., Huang Q., Zheng S, & Xie K. (2015). Merlin/NF2 Suppresses Pancreatic Tumor Growth and Metastasis by Attenuating the FOXM1-Mediated Wnt/β-catenin Signaling. Cancer research, 75(22), 4778-4789.

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