cpg methyltransferase m sssi (New England Biolabs)
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CpG Methyltransferase M SssI
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CpG Methyltransferase M SssI 500 units
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M0226L
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500 units
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DNA Methylases
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New England Biolabs
cpg methyltransferase m sssi
CpG Methyltransferase M SssI 500 units
https://www.bioz.com/result/cpg methyltransferase m sssi/product/New England Biolabs
Average 99 stars, based on 68 article reviews
Price from $9.99 to $1999.99
CpG Methyltransferase M SssI 500 units
https://www.bioz.com/result/cpg methyltransferase m sssi/product/New England Biolabs
Average 99 stars, based on 68 article reviews
Price from $9.99 to $1999.99
cpg methyltransferase m sssi - by Bioz Stars,
2019-12
99/100 stars
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Images
1) Product Images from "Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor"
Article Title: Epigenetic Control of Viral Life-Cycle by a DNA-Methylation Dependent Transcription Factor
Journal: PLoS ONE
doi: 10.1371/journal.pone.0025922
Figure Legend Snippet: Evaluation of predicted ZREs in three EBV promoters. A. Summary of the known (filled box) and predicted (open box) core ZRE sequences within the proximal 500 nucleotides of the indicated BZLF1 , BRLF1 and BMRF1 promoters. The arrows represent the transcription start sites. Stars represent CpG ZREs. B. Double strand oligonucleotides were generated with the core ZRE sequence and at least 10 nucleotides of cognate sequence on either side. Following radio labeling, these were incubated with in vitro translated Zta and subject to EMSA. The reactions contained no protein, 0, control lysate, C or Zta, Z. The DNA probes are indicated above with their originating promoters. C. Double strand oligonucleotides were generated with the core ZRE sequence and 10 nucleotides of cognate sequence on either side. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
Techniques Used: Generated, Sequencing, Labeling, Incubation, In Vitro, Methylation
Figure Legend Snippet: Zta recognition and methylation dependence of PFM CpG5 predicted CpG containing ZREs. A. Flow diagram illustrating the information flow from the PFM to the predictions of novel ZREs in the EBV genome and their subsequent evaluation. B. Core heptamer sequences, in both forward and reverse complement, of PFM CpG5 predicted CpG containing ZREs within the EBV genome. C. PFM CpG5 was used to predict the potential for further ZREs in the EBV genome. Double strand oligonucleotides were generated. Following radio labeling, these were subject to in vitro methylation with SssI methyl transferase (+), or a mock reaction (−). Subsequently, they were incubated with in vitro translated Zta and subject to EMSA. The reactions contained control lysate, C; or Zta, Z.
Techniques Used: Methylation, Flow Cytometry, Generated, Labeling, In Vitro, Incubation
2) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"
Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
Journal: Nature biotechnology
doi: 10.1038/nbt.3073
Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification
3) Product Images from "Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing"
Article Title: Single-base resolution analysis of active DNA demethylation using methylase-assisted bisulfite sequencing
Journal: Nature biotechnology
doi: 10.1038/nbt.3073
Figure Legend Snippet: MAB-seq strategy and quantitative mapping of active DNA demethylation ( a ) Schematic diagram of MAB-seq. DNMT methylates unmodified C to generate 5mC, which can be successively oxidized by TET to generate 5hmC/5fC/5caC. Highly oxidized cytosine derivatives, 5fC and 5caC, are repaired by TDG/BER to regenerate unmodified C. ( b ) M.SssI exhibits robust methylase activity towards unmodified cytosines within CpGs, but has significantly lower activity for cytosines in non-CpG contexts (CHG or CHH, H=A, T, C). M.SssI methylase activity was measured by MAB-seq analysis (Illumina deep sequencing) of unmethylated lambda DNA. Standard bisulfite sequencing (BS-seq) confirmed nearly complete conversion of unmethylated C to T at CpG and non-CpG sites. ( c ) Locus-specific MAB-seq analysis of 5fC/5caC at Tbx5 by Illumina sequencing in control ( shCtrl ) and Tdg knockdown ( shTdg ) mouse ESCs. For comparison, also shown are 5fC/5caC antibody DNA immunoprecipitation (DIP) based maps of 5fC and 5caC in control and Tdg -depleted mouse ESCs. DIP-seq tracks are represented in normalized read density (reads per 10 million reads, rp10m) and the vertical axis range of all DIP-seq tracks is from 1 to 25. Black horizontal bars denote 5fC/5caC-enriched regions identified by 5fC/5caC DIP-seq. The level of 5fC/5caC (only Watson strand shown) is displayed as the percentage of total C modified as 5fC/5caC, and background signals detected in Tet1/2 −/− mouse ESCs was subtracted. ( d ) Statistical calling of 5fC/5caC-modified CpGs in locus-specific MAB-seq analysis. Shown are 5fC/5caC levels (background corrected using raw MAB-seq signals in Tet1/2 −/− ) of 11 CpG sites from the Tbx5 locus in shCtrl, shCtrl +VC, shTdg and shTdg +VC mouse ESCs. shTdg was compared with shCtrl while shTdg +VC was compared with shCtrl +VC. An asterisk indicates that a CpG is statistically enriched for 5fC/5caC (multiple comparison corrected P
Techniques Used: Activity Assay, Sequencing, Lambda DNA Preparation, Methylation Sequencing, Immunoprecipitation, DNA Immunoprecipitation Sequencing, Modification
4) Product Images from "Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells"
Article Title: Retinoic Acid and GM-CSF Coordinately Induce Retinal Dehydrogenase 2 (RALDH2) Expression through Cooperation between the RAR/RXR Complex and Sp1 in Dendritic Cells
Journal: PLoS ONE
doi: 10.1371/journal.pone.0096512
Figure Legend Snippet: Methylation of the CpG island in the Aldh1a2 promoter prohibits Sp1 to activate the promoter, whereas the Aldh1a2 promoter is largely unmethylated in BM-pDCs as well as in BM-cDCs. ( A ) pCpGL-basic and pCpGL-RALDH2 (−873) reporter vectors were methylated with 1.25 µg of M.SssI. COS-7 cells were transfected with methylated or unmethylated pCpGL-basic or pCpGL-RALDH2 (−873) reporter vector in combination with or without the 0.5 µg of pCMV-Myc-Sp1 expression vector. One day after transfection, luciferase activity was measured. Relative promoter activities were calculated by arbitrarily defining the activity of pCpGL-basic alone as 1. ( B ) COS-7 cells were transfected with pCMV-Myc-Sp1. One day after transfection, cell lysates were analyzed for DNA binding activity by DNAP assay using DNA Probe B and Probe C methylated with M.SssI or left unmethylated. The bound proteins were analyzed by SDS-PAGE followed by Western blotting with anti-Myc Ab. ( C ) Flt3L-generated BM-DCs were stained with allophycocyanin-labeled anti-CD11c Ab and phycoerythrin-labeled anti-B220 Ab, and were sorted to cDC and pDC fractions with a FACSAria. ( D ) Sorted BM-pDCs and BM-cDCs were cultured for 16 h with or without 10 ng/ml GM-CSF. Expression of Aldh1a2 mRNA was analyzed by real-time PCR. Relative expression levels were calculated by defining the Aldh1a2 mRNA expression in the cells incubated with medium alone for 16 h was set to 1. Data in (A and D) are presented as mean + SD of triplicate cultures. Statistical significance between two groups was determined by the Student's t test (*** p
Techniques Used: Methylation, Transfection, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Binding Assay, SDS Page, Western Blot, Generated, Staining, Labeling, Cell Culture, Real-time Polymerase Chain Reaction, Incubation
5) Product Images from "Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource"
Article Title: Independence between pre-mRNA splicing and DNA methylation in an isogenic minigene resource
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkx900
Figure Legend Snippet: Determination of de novo and maintenance CpG methylation in Tnp1 and Pfn3 minigenes with distinct splicing capacities. ( A ) Schematic representation of regions interrogated by bisulfite-pyrosequencing (BS-pyroseq) in the Tnp1 and Pfn3 minigenes. ( B ) Per-base heatmap for percent CpG methylation at the 7 interrogated sites within Tnp1 exon 2. Values represent averages from duplicate BS-pyroseq performed on biological replicates of Dox-induced Tnp1 host cells transfected with vector encoding DNMT3A, DNMT3B or control (left). Average methylation across the seven sites in DNMT3-transfected and control cells (right), mean ± SEM. ( C ) BS-pyroseq of the 18 CpGs overlapping the Pfn3 ORF, as in (A). P -values = two-tailed Student's t -test comparing average methylation DNMT3-transfected cells versus control, as indicated. ( D ) CpG-free Tnp1 expression vector was methylated in vitro using M.SssI and stably transfected into 293T cells for random integration. ( E ) MSRE-qPCR for relative Tnp1 DNA methylation from all stable gDNA and ΔSS minigene clones at 21, 49 and 77 days post-transfection summarized in boxplot; white diamonds indicate average Tnp1 methylation from seven and six independent Tnp1 gDNA and ΔSS clones, respectively. Student's t -test comparing mean methylation between indicated pairs yielded no significant differences (NS).
Techniques Used: CpG Methylation Assay, Transfection, Plasmid Preparation, Methylation, Two Tailed Test, Expressing, In Vitro, Stable Transfection, Real-time Polymerase Chain Reaction, DNA Methylation Assay, Clone Assay
6) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"
Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
Journal: Journal of Cellular and Molecular Medicine
doi: 10.1111/j.1582-4934.2010.01124.x
Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase
7) Product Images from "The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes"
Article Title: The Epigenetic Landscape of Latent Kaposi Sarcoma-Associated Herpesvirus Genomes
Journal: PLoS Pathogens
doi: 10.1371/journal.ppat.1000935
![Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.](https://storage.googleapis.com/bioz_article_images/PMC2880564/ppat.1000935.g001.jpg "... of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells ...")
Figure Legend Snippet: Experimental design of MeDIP analysis. A : Schematic representation of the experimental setup for the analysis of CpG methylation patterns. The KSHV episome in infected cells is expected to be partially methylated, as indicated by black and white circles which symbolize methylated or unmethylated CpG dinucleotides, respectively. Genomic DNA was isolated from such cells and the samples were subjected to immunoprecipitation using a methylcytidine specific antibody (MeDIP procedure), followed by hybridization of the precipitated samples versus the input on tiling microarrays. For each probe, an enrichment score E S was calculated, which represents the ratio of MeDIP over input fluorescence signals. The efficiency of the immunoprecipitation depends on the total number of methylated CpG motifs in a given fragment and E S is thus a function of the extend of methylation as well as local CpG frequencies. Therefore, to obtain reference values which signify maximum methylation for each probe, we generated a positive control by subjecting KSHV bacmids to CpG methylation in vitro . The bacmid was mixed with cellular DNA to simulate the host background and subjected to the same MeDIP procedure as samples from infected cells. Similarly, a negative control of unmethylated bacmid was prepared to control for cross-hybridization of unspecific background. After normalization of the array data using a spike-in control (see Material Methods for details), background-corrected methylation values M S and M P were calculated for each probe by subtraction of the corresponding negative control value. B : Confirmation of successful in vitro methylation of KSHV bacmids used as a positive control. A bacmid carrying the complete KSHV genome (BAC36 [36] ) was methylated using M.SssI, a methyltransferase specific for CpG dinucleotides. Methylated or unmethylated bacmids were subjected to restriction digestion using the methylation sensitive enzyme HpaII and its isoschizomer MspI, which cuts regardless of methylation. Methylated bacmids were resistant to HpaII digestion, signifying complete methylation.
Techniques Used: Methylated DNA Immunoprecipitation, CpG Methylation Assay, Infection, Methylation, Isolation, Immunoprecipitation, Hybridization, Fluorescence, Generated, Positive Control, In Vitro, Negative Control
8) Product Images from "Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing"
Article Title: Hydrophobicity of Methylated DNA as a Possible Mechanism for Gene Silencing
Journal:
doi: 10.1088/1478-3975/9/6/065001
Figure Legend Snippet: The 2905 bp DNA template (A) showing Ava1 restriction sites. The sequence contains 951 cytosines (32.2% C content) and 259 occurrences of the 5’CG3’ motif (the target for CpG Methyltransferase) on one strand. (B) Ava1 digests of DNA incubated
Techniques Used: Sequencing, Incubation
9) Product Images from "Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation"
Article Title: Human Vav1 Expression in Hematopoietic and Cancer Cell Lines Is Regulated by c-Myb and by CpG Methylation
Journal: PLoS ONE
doi: 10.1371/journal.pone.0029939
Figure Legend Snippet: Methylation of CpG sites in the vav 1 promoter impairs expression of the reporter gene in various cell lines. (A) Le2 plasmid, either un-treated or methylated by CpG methyltransferase (M.SssI), was incubated with HpaII and analyzed on a gel. The plasmid treated with M.SssI was not digested by HpaII, indicating that methylation was successful. (B) Unmethylated or methylated Le2 was transfected into Jurkat T cells, U937 myeloid cells and H441 lung cancer cells. The luciferase activity of these plasmids was measured 24 hr after transfection. Fold induction of luciferase activity was calculated relative to the activity in cells transfected with unmethylated Le2. Each point is the mean of three experiments. (***) indicates p
Techniques Used: Methylation, Expressing, Plasmid Preparation, Incubation, Transfection, Luciferase, Activity Assay
10) Product Images from "Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma"
Article Title: Promoter hypomethylation up-regulates CD147 expression through increasing Sp1 binding and associates with poor prognosis in human hepatocellular carcinoma
Journal: Journal of Cellular and Molecular Medicine
doi: 10.1111/j.1582-4934.2010.01124.x
Figure Legend Snippet: Methylation status interfered the Sp1 binding to the CD147 gene promoter and inhibited CD147 transcriptional activity in vitro . (A) Methylation status interfered the Sp1 binding to the CD147 gene promoter in vitro . The binding of Sp1 to the CD147 gene promoter was determined by electrophoretic mobility shift analysis (EMSA). By using biotin-labelled 30-bp double-stranded oligonucleotides containing wild, mutated or methylated Sp1-binding sites as probes, EMSAs were performed with the same amount of nuclear extracts from HepG2 cells, and the products were separated on a 5% polyacrylamide gel (lanes 2–7). Lane 1, free probe; lane 2, biotin-labelled wild-type Sp1 consensus oligonucleotides were mixed with nuclear proteins; lane 3, the same reaction was performed as that in lane 2, except for the presence of a 100-fold excess of unlabelled wild-type Sp1 consensus oligonucleotides as a competitor; lanes 4, binding assays of biotin-labelled mutant-type Sp1 consensus oligonucleotides mixed with nuclear proteins; lanes 5–6, 1 μg each of IgG and anti-Sp1 antibody were added to the binding reaction mixtures with biotin-labelled wild-type probe; lane 7, binding assays of biotin-labelled methylated Sp1 consensus oligonucleotides mixed with nuclear proteins. (B) Analysis for the effect of in vitro DNA methylation on the CD147 promoter activity through the transfection of HEK-293 cells with methylated CpG reporter constructs. CD147P/pGL3 treated or untreated with SssI methylase was co-transfected with the pcDNA3.1 or Sp1/pcDNA3.1 into HEK-293 cells with pGL3-Basic as control. The relative luciferase activity was denoted as above-mentioned method and also expressed as the mean ± S.D. for three independent experiments. * P
Techniques Used: Methylation, Binding Assay, Activity Assay, In Vitro, Electrophoretic Mobility Shift Assay, Mutagenesis, DNA Methylation Assay, Transfection, Construct, Luciferase
11) Product Images from "Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer"
Article Title: Hypomethylation at non-CpG/CpG sites in the promoter of HIF-1α gene combined with enhanced H3K9Ac modification contribute to maintain higher HIF-1α expression in breast cancer
Journal: Oncogenesis
doi: 10.1038/s41389-019-0135-1
Figure Legend Snippet: Non-CpHs methylation can silence HIF-1α gene transcription. a Analyzing in vitro DNA-methyltransferase, MSP I and MSss I recognition sites within HIF-1α gene promoter. b The methylation at CpC and CpG sites could cause transcriptional repression of HIF-1α gene by using luciferase activity assay. Data are presented as the means ± SD of three independent experiments. * p
Techniques Used: Methylation, In Vitro, Luciferase, Activity Assay
12) Product Images from "Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat"
Article Title: Epigenetic Control of the Vasopressin Promoter Explains Physiological Ability to Regulate Vasopressin Transcription in Dehydration and Salt Loading States in the Rat
Journal: Journal of Neuroendocrinology
doi: 10.1111/jne.12371
Figure Legend Snippet: Methylation of CpG (cytosine‐phosphate‐guanine) sites on the Avp promoter in vitro . The substitution (C‐A) at CpG sites in the Avp promoter by overlap extension polymerase chain reaction was used to prevent methylation at specific CpG sites. The mutation sites are shown in ( a ). The mutated plasmids were subsequently methylated by methyltransferase enzyme. ( b ) Successful methylation was determined using methylation sensitive restriction enzyme Pml I. (c, d ) Luciferase assays were performed by co‐transfection of plasmid expressing Creb3l1 and 350 bp Avp promoter contructs with ( c ) unmethylated and ( d ) methylated plasmid. Error bars indicate the mean ± SEM (n = 4 per group). *P
Techniques Used: Methylation, In Vitro, Overlap Extension Polymerase Chain Reaction, Mutagenesis, Luciferase, Cotransfection, Plasmid Preparation, Expressing
Figure Legend Snippet: Demethylation of the Avp promoter dramatically increases Avp transcription in hypothalamic 4B cells. ( a ) Tile diagram showing the methylation status of CpG (cytosine‐phosphate‐guanine) sites for individual clones of the Avp promoter from the hypothalamic 4B cells. ( b ) Treatment of hypothalamic 4B cells with DNA methyltransferase inhibitor 5‐Aza‐dc (1–10 μ m ) increases Avp synthesis. ( c ) Forskolin (10 μ m ) induced Avp synthesis was further enhanced by 5‐Aza treatment. Error bars indicate the mean ± SEM (n = 4 per group). ***P
Techniques Used: Methylation, Clone Assay
Related Articles
Methylation Sequencing:Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using Clone Assay:Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Amplification:Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum Article Snippet: Paragraph title: Amplicon-specific TAB pyrosequencing ... 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Human genomic DNA treated in vitro with Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by Synthesized:Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Electrophoresis:Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer Article Snippet: DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. DNA from lymphocytes of healthy volunteers treated with Incubation:Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: The PCR condition consisted of one incubation of 10 minutes at 95°C, followed by 35 cycles of 94°C for 30 s, 62°C for 30 s, and 72°C for 30 s, and a final extension at 72°C for 10 minutes. .. Genomic DNA, methylated in vitro by Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: The end-labeled probe was incubated with HeLa nuclear extract (5 µg; Millipore) for 40 minutes at room temperature in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and poly dI-dC), and resolved on a 5% native polyacrylamide gel. .. Methylated probe for EMSA was generated via in vitro methylation using Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA Article Snippet: Sat2R: 5′–ATCAG ATTCC ATTCG AATCC ATTCG AAAAT GATTA CATTC GAATC CATTC GAAGA TTCCA TTTGA GCCTG TTCGA AAATT CCATT TGAGT CCAAC CAATG ATTCC ATTCA TTTCC ATTCA ATGAT TCCAT TCGAA TCCAT TTGGA T–3′. .. CpG methylation was introduced by an incubation with the Luciferase:Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Paragraph title: In vitro methylation, transient transfection and Luciferase assay ... Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Paragraph title: In vitro methylation, transient transfection and luciferase assay ... Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Activity Assay:Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Mass Spectrometry:Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using Modification:Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum Article Snippet: The distribution of 5-hmC and 5-mC in the GAD1 promoter amplicon was determined by a modification of TAB-seq. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer Article Snippet: DNA samples were bisulphite treated using the MethylEasy Xceed Rapid DNA Bisulphite Modification kit (Human Genetic Signatures, Sydney, Australia) following the manufacturer's instructions. .. Normal DNA samples were treated using Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer Article Snippet: Water blanks were included in each assay. .. DNA from lymphocytes of healthy volunteers treated with Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity Article Snippet: 0.5 µg of each complete plasmid were methylated via Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by Whole Genome Amplification:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: For each assay, a standard curve of 0, 25, 50, 75 and 100% methylated DNA was measured. .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Bisulfite Sequencing:Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Methylation analysis of CTCFL/BORIS NCBI RefSeq NR_072975, spanning nucleotides chr20:56099842-56100217 on GRCh37/hg19 assembly was performed in bisulphite sequencing assay. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Transfection:Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Paragraph title: In vitro methylation, transient transfection and Luciferase assay ... Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Paragraph title: In vitro methylation, transient transfection and luciferase assay ... Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of SYBR Green Assay:Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Generated:Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using CpG Methylation Assay:Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA Article Snippet: Sat2R: 5′–ATCAG ATTCC ATTCG AATCC ATTCG AAAAT GATTA CATTC GAATC CATTC GAAGA TTCCA TTTGA GCCTG TTCGA AAATT CCATT TGAGT CCAAC CAATG ATTCC ATTCA TTTCC ATTCA ATGAT TCCAT TCGAA TCCAT TTGGA T–3′. .. CpG methylation was introduced by an incubation with the Sequencing:Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Paragraph title: DNA extraction, sequencing and methylation analysis ... Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Binding Assay:Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: The end-labeled probe was incubated with HeLa nuclear extract (5 µg; Millipore) for 40 minutes at room temperature in binding buffer (10 mM Tris, pH 7.5, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, and poly dI-dC), and resolved on a 5% native polyacrylamide gel. .. Methylated probe for EMSA was generated via in vitro methylation using Cellular Antioxidant Activity Assay:Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: The primer sequences for the methylated form were 5′-TGC GAG CGT TTT TTT AAA TGC-3′ (sense) and 5′-TAA ACT CGC AAA CCA ACT CG-3′ (antisense) (74 bp), and the primer sequences for the unmethylated form were 5′- GTT TGG TTG TTG TTT ATT GGT TG-3′ (sense) and 5′-CCT AAA CTC ACA AAC CAA CTC A-3′ (antisense) (115 bp). .. Genomic DNA, methylated in vitro by Magnetic Cell Separation:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the DNA Extraction:Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Paragraph title: DNA extraction, sequencing and methylation analysis ... Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Nucleic Acid Electrophoresis:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Fluorescence:Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The bisulfite-modified DNA was used as a template for fluorescence-based real-time PCR, as previously described [ ]. .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Methylation:Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by Article Title: Analysis of the Mitogen-activated protein kinase kinase 4 (MAP2K4) tumor suppressor gene in ovarian cancer Article Snippet: Paragraph title: MAP2K4 promoter methylation analysis by Methylation-Specific Single Strand Conformation Polymorphism (MS-SSCP) and bisulfite sequencing ... Normal DNA samples were treated using Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: For each assay, a standard curve of 0, 25, 50, 75 and 100% methylated DNA was measured. .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines Article Snippet: In cases with diverging results from the two rounds of MSP, we did a third independent MSP round. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: Paragraph title: Methylation-specific polymerase chain reaction (MSP) ... Human genomic DNA treated in vitro with Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity Article Snippet: Plasmid DNA from transformed liquid cultures was extracted using the QIAprep Spin Miniprep Kit (Qiagen, USA), and the direction of each insert within the plasmid was determined by Sanger sequencing using the forward primer previously listed for each region. .. 0.5 µg of each complete plasmid were methylated via Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker Article Snippet: Samples were composed of normal and tumor tissues along with blood of healthy donors as negative control. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Mutagenesis:Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using Isolation:Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Subcloning:Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: 200 ng of gDNA was treated in DNA methylation Direct kit by Zymo Research, following PCR by specific primers (available upon request) employing HotSTAR polymerase (Qiagen), and subcloning in pGEM-T-easy vector (Promega). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Size-exclusion Chromatography:Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Electrophoretic Mobility Shift Assay:Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: Paragraph title: Electrophoretic Mobility Shift Assay ... Methylated probe for EMSA was generated via in vitro methylation using Purification:Article Title: Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3 Article Snippet: DNA was extracted and purified from an aliquot of 200 µl of cervical swab sample in Specimen Transport Medium with QIAamp DNA Mini kit (Qiagen, Valentia, CA) and suspended in 100 µl AE buffer (10 mM Tris·Cl, 0.5 mM EDTA, pH 9.0). .. SiHa cellular DNAs with and without a treatment by Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA Article Snippet: The DNA fragments were purified by the same methods as described above. .. CpG methylation was introduced by an incubation with the Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Polymerase Chain Reaction:Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by Article Title: Inverse Association between Methylation of Human Papillomavirus Type 16 DNA and Risk of Cervical Intraepithelial Neoplasia Grades 2 or 3 Article Snippet: SiHa cellular DNAs with and without a treatment by Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: A 231-bp PCR fragment (position −222 to +9 relative to the initiation codon) containing the putative CTCF binding site in the 5′UTR of the FXN gene was used as the probe. .. Methylated probe for EMSA was generated via in vitro methylation using Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer Article Snippet: Paragraph title: Methylation-Specific Polymerase Chain Reaction (MSP) ... DNA from lymphocytes of healthy volunteers treated with Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines Article Snippet: Paragraph title: Methylation-specific PCR (MSP) ... Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: Paragraph title: Methylation-specific polymerase chain reaction (MSP) ... Human genomic DNA treated in vitro with Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker Article Snippet: Paragraph title: Methylation Specific PCR ... In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the FACS:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Activated Clotting Time Assay:Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: The primer sequences for the methylated form were 5′-TGC GAG CGT TTT TTT AAA TGC-3′ (sense) and 5′-TAA ACT CGC AAA CCA ACT CG-3′ (antisense) (74 bp), and the primer sequences for the unmethylated form were 5′- GTT TGG TTG TTG TTT ATT GGT TG-3′ (sense) and 5′-CCT AAA CTC ACA AAC CAA CTC A-3′ (antisense) (115 bp). .. Genomic DNA, methylated in vitro by Hot Start PCR:Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: Human genomic DNA treated in vitro with Plasmid Preparation:Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos Article Snippet: In the present study, the plasmid pIRES2-EGFP with CMV promoter was treated by bisulfite modification and analyzed as a control to assess the specificity of HIV LTR methylation. .. The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Article Title: Influence of DNA methylation on positioning and DNA flexibility of nucleosomes with pericentric satellite DNA Article Snippet: For the Sat2L and Sat2R DNA fragments, eight Sat2L (145 base pairs) or Sat2R (146 base pairs) DNA fragments were tandemly ligated into the pGEM-T Easy vector. .. CpG methylation was introduced by an incubation with the Article Title: Ethnicity-specific epigenetic variation in naïve CD4+ T cells and the susceptibility to autoimmunity Article Snippet: Plasmid DNA from transformed liquid cultures was extracted using the QIAprep Spin Miniprep Kit (Qiagen, USA), and the direction of each insert within the plasmid was determined by Sanger sequencing using the forward primer previously listed for each region. .. 0.5 µg of each complete plasmid were methylated via Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: 200 ng of gDNA was treated in DNA methylation Direct kit by Zymo Research, following PCR by specific primers (available upon request) employing HotSTAR polymerase (Qiagen), and subcloning in pGEM-T-easy vector (Promega). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Software:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: PCR products were checked by gel electrophoresis, pyrosequenced using PyroMark Gold Q24 Reagents (Qiagen) on a PyroMark Q24 instrument and quantified with the PyroMark Q24 software (Qiagen). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Sequences were inspected with Sequence Scanner software 1.0 (Applied Biosystems), excluding variants present in the SNP database (dbSNP) http://www.ncbi.nlm.nih.gov/snp ). .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Real-time Polymerase Chain Reaction:Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The bisulfite-modified DNA was used as a template for fluorescence-based real-time PCR, as previously described [ ]. .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Negative Control:Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: Human genomic DNA from peripheral blood lymphocytes, treated with bisulfite, was used as the negative control. .. Human genomic DNA treated in vitro with Article Title: Qualitative analysis of Adenomatous Polyposis Coli promoter: Hypermethylation, engagement and effects on survival of patients with esophageal cancer in a high risk region of the world, a potential molecular marker Article Snippet: In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by CpG methylase (New England Biolabs) were used as positive control and control of bisulfite treatment efficiency. .. In addition, methylated DNA (CpGenome Universal methylated DNA, Chemicon) and blood DNA modified by Recombinant:Article Title: Relationship between LTR Methylation and gag Expression of HIV-1 in Human Spermatozoa and Sperm-Derived Embryos Article Snippet: Paragraph title: Methylation of the Recombinant Plasmids ... The methylation of the pIRES2-EGFP and pIRES2-EGFP-LTR-gag plasmids was performed in a 50 µl reaction containing the plasmid DNA (0.5 µg/µl, 5 µl), 10× NEBuffer (5 µl) (New England BioLabs, Beijing, China), S-adenosylmethionine (SAM, 1.6 mM, 5 µl), In Vitro:Article Title: Increased binding of MeCP2 to the GAD1 and RELN promoters may be mediated by an enrichment of 5-hmC in autism spectrum disorder (ASD) cerebellum Article Snippet: To determine the efficiency of each enzymatic step, spike-in controls were added and independently scored by conventional Sanger sequencing. .. Unmethylated λDNA (cI857 Sam7, Promega, Madison, WI, USA) was methylated in vitro with Article Title: Aberrant Methylation of Thrombospondin-1 and Its Association with Reduced Expression in Gastric Cardia Adenocarcinoma Article Snippet: PCR products were analyzed on 2% agarose gels with ethidium bromide and visualized under UV illumination. .. Genomic DNA, methylated in vitro by Article Title: Epigenetic Silencing in Friedreich Ataxia Is Associated with Depletion of CTCF (CCCTC-Binding Factor) and Antisense Transcription Article Snippet: Cold competition was performed using excess unlabeled probe, a CTCF consensus oligonucleotide (sc-2613; Santa Cruz), or a mutant CTCF oligonucleotide (sc-2614; Santa Cruz). .. Methylated probe for EMSA was generated via in vitro methylation using Article Title: Epigenetic Regulatory Effect of Exercise on Glutathione Peroxidase 1 Expression in the Skeletal Muscle of Severely Dyslipidemic Mice Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: Lower Methylation of the ANGPTL2 Gene in Leukocytes from Post-Acute Coronary Syndrome Patients Article Snippet: Cloned vectors were isolated by Qiagen QIAprep Spin Miniprep kit (Qiagen). .. Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines Article Snippet: In cases with diverging results from the two rounds of MSP, we did a third independent MSP round. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: Human genomic DNA from peripheral blood lymphocytes, treated with bisulfite, was used as the negative control. .. Human genomic DNA treated in vitro with Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Article Title: Microplate-based platform for combined chromatin and DNA methylation immunoprecipitation assays Article Snippet: For MethyLight, 1 µl of bisulfite-converted DNA solution was combined with 600 nmol/l of each primer and 200 nmol/l of probe in ABI Universal PCR Master Mix and amplified under the default cycling conditions for Prism HT7900 instrument. .. Human sperm DNA and human sperm DNA methylated in vitro using the Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the DNA Methylation Assay:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: Genomic DNA (500 ng) was extracted from MACS- or FACS-purified nuclei (AllPrep DNA/RNA Mini Kit, Qiagen) and was bisulfite converted with the EZ DNA Methylation Kit (D5001, Zymo Research). .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Article Title: Gene methylation profile of gastric cancerous tissue according to tumor site in the stomach Article Snippet: The methylation status of the MLH1 , MGMT , and DAPK-1 promoters was determined by treating the DNA with bisulfite, using the EZ DNA Methylation Gold Kit™ (Zymo Research), according to the manufacturer’s instructions. .. Human genomic DNA treated in vitro with Article Title: Hypomethylation of the CTCFL/BORIS promoter and aberrant expression during endometrial cancer progression suggests a role as an Epi-driver gene Article Snippet: Up to 12 colonies were sequenced from each tumor/case for the analysis of CpG-site specific DNA methylation in the CTCFL/BORIS promoter regions. .. Methylation positive and methylation depleted control PCR products (primers available upon request) spanning the CTCFL/BORIS promoter region were gel purified and in vitro methylated in the presence or absence of the Concentration Assay:Article Title: Dynamic DNA methylation orchestrates cardiomyocyte development, maturation and disease Article Snippet: The concentration of converted DNA was adjusted to 5 ng μl−1 and DNA was amplified with the PyroMark PCR Kit (Qiagen) using a three-primer approach with a universal biotinylated primer . .. The GenomePlex Complete WGA Kit (Sigma) was used to generate unmethylated DNA and the Article Title: The Diagnosis Value of Promoter Methylation of UCHL1 in the Serum for Progression of Gastric Cancer Article Snippet: The final reaction mixture contained 600 nmol/L of each primer (Invitrogen, Carlsbad, CA), 1 unit of platinum Taq polymerase (Invitrogen), a 200 μ mol/L concentration each of dATP, dCTP, dGTP, and dTTP, 16.6 mmol/L ammonium sulfate, 67 mmol/L Trizma, 6.7 mmol/L magnesium chloride, 10 mmol/L mercaptoethanol, and 1 × SYBR Green I dye (Sigma-Aldrich, St. Louis, MO, USA). .. PCR was conducted under the following conditions: 1 cycle at 95°C for 5 min followed by 45 cycles at 95°C for 10 sec, 58°C for 10 sec, 72°C for 20 sec, and 81°C for 1 sec. Leukocyte DNA from a healthy individual was methylated in vitro with excess Staining:Article Title: CDH1 promoter hypermethylation and E-cadherin protein expression in infiltrating breast cancer Article Snippet: DNA from lymphocytes of healthy volunteers treated with SssI methyltransferase (New England Biolabs, Beverly, MA, USA) and then subjected to bisulfite modification was used as positive controls for methylated alleles. .. DNA from lymphocytes of healthy volunteers treated with Article Title: An ANOCEF genomic and transcriptomic microarray study of the response to radiotherapy or to alkylating first-line chemotherapy in glioblastoma patients Article Snippet: PCR products were separated on 2% agarose gels stained with ethidium bromide and visualized under UV illumination. .. As a positive control for methylated alleles, we used DNA from lymphocytes treated with Article Title: A CpG island hypermethylation profile of primary colorectal carcinomas and colon cancer cell lines Article Snippet: PCR products were loaded onto 7.5% polyacrylamide gels, stained with ethidium bromide, and visualized by UV illumination. .. Human placental DNA (Sigma Chemical Co., St. Louis, MO) treated in vitro with Variant Assay:Article Title: DNA Methylation-Mediated Downregulation of DEFB1 in Prostate Cancer Cells Article Snippet: A 790-bp fragment harboring 14 CpG sites located at the 5′-end region of DEFB1 , a 5′ BamHI site, and a 3′ HindIII site was synthesized and cloned into the pCpGfree-basic-Lucia reporter plasmid (InvivoGen, San Diego, CA, USA) that codes for a secreted coelenterazine-utilizing variant of luciferase. .. Briefly, 5 μg of the Lucia reporter plasmid was methylated using 12 U of |
