human dna c 5 mtase dnmt1  (New England Biolabs)


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    Name:
    Human DNA c 5 MTase Dnmt1
    Description:
    Human DNA c 5 MTase Dnmt1 250 units
    Catalog Number:
    m0230l
    Price:
    500
    Size:
    250 units
    Category:
    DNA Methylases
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    New England Biolabs human dna c 5 mtase dnmt1
    Human DNA c 5 MTase Dnmt1
    Human DNA c 5 MTase Dnmt1 250 units
    https://www.bioz.com/result/human dna c 5 mtase dnmt1/product/New England Biolabs
    Average 95 stars, based on 30 article reviews
    Price from $9.99 to $1999.99
    human dna c 5 mtase dnmt1 - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine"

    Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine

    Journal: Oncotarget

    doi:

    Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.
    Figure Legend Snippet: Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.

    Techniques Used: Genome Wide, DNA Methylation Assay, Expressing, Mutagenesis, Western Blot

    2) Product Images from "DNMT1-interacting RNAs block gene specific DNA methylation"

    Article Title: DNMT1-interacting RNAs block gene specific DNA methylation

    Journal: Nature

    doi: 10.1038/nature12598

    Genome-wide alignment of DNMT1 bound and unbound transcripts, DNA methylation, and gene expression a , Two-way Venn diagram showing DNMT1 specific peaks overlapping with transcribed elements identified in HL-60 total and poly(A+)-depleted RNA-Seq libraries. b , Cloud plots representing genes within DNMT1 unbound, bound and all RRBS-covered groups stratified by DNA methylation and expression levels. All genes are presented in Supplementary Excel File 2 . c , Examples of genes from the C ( CEBPA ) and B ( USP29 ) clusters. Peaks are visualized using the SSIRs 48 . d , Model of DNMT1 sequestration. Upper panel: DNMT1 can access transcriptionally inactive hemimethylated genomic regions. Lower panel: DNMT1 cannot access transcriptionally active hemimethylated genomic regions.
    Figure Legend Snippet: Genome-wide alignment of DNMT1 bound and unbound transcripts, DNA methylation, and gene expression a , Two-way Venn diagram showing DNMT1 specific peaks overlapping with transcribed elements identified in HL-60 total and poly(A+)-depleted RNA-Seq libraries. b , Cloud plots representing genes within DNMT1 unbound, bound and all RRBS-covered groups stratified by DNA methylation and expression levels. All genes are presented in Supplementary Excel File 2 . c , Examples of genes from the C ( CEBPA ) and B ( USP29 ) clusters. Peaks are visualized using the SSIRs 48 . d , Model of DNMT1 sequestration. Upper panel: DNMT1 can access transcriptionally inactive hemimethylated genomic regions. Lower panel: DNMT1 cannot access transcriptionally active hemimethylated genomic regions.

    Techniques Used: Genome Wide, DNA Methylation Assay, Expressing, RNA Sequencing Assay

    ecCEBPA –DNMT1 interactions; DNMT1 binds to RNA with greater affinity than to DNA a , Diagram showing position of qRT-PCR primers used in RIP, double-headed arrow; RNA and DNA oligonucleotides used in EMSA and REMSA. Asterisks indicate position of methylated cytosines; umDNA, hmDNA, and mDNA refer to unmethylated, hemimethylated, and methylated DNA probes, respectively; b , ecCEBPA is immunoprecipitated with anti-DNMT1 antibody. qRT-PCR, bars indicate mean ± s.d.; c , RNA- DNMT1 binding is not affected by the absence of CpG dinucleotides (right panel). Left and middle panels: RNA oligonucleotide R2 and its mutated form mut R2 (asterisks indicate cytosines substituted into uridines), both able to form stem-loop-structures; d , RNA oligonucleotide able to form stem-loop structure bind DNMT1 (R6); e , R5 RNA oligonucleotide forming stem-loop structure (R5) has a greater DNMT1 affinity compared to mut R5, unable to fold into stem-loop, (taken in equimolar amounts), at the same DNMT1 concentration; f , Left four panels: REMSA and EMSA performed with the fixed concentration of ssRNA and dsDNA oligonucleotides (1 nM) and increasing concentrations of DNMT1 protein; Right panel: Nonlinear regression analysis of bound RNA/DNA versus DNMT1 concentrations. Error bars indicate s.d. from two independent experiments; g , REMSA showing that RNA oligonucleotide R4, which is unable to form stem-loop structure, displays lower DNMT1 affinity as compared to R5 (Fig. 3f left panel) at the same DNMT1 concentrations; h , Left panel: Schematic diagram showing the DNMT1 domains and the GST-DNMT1 isolated fragments (F1–F5); Right panel: GST-DNMT1 pull down assay demonstrating binding of the folded RNA oligonucleotide R5 to the catalytic domain of DNMT1.
    Figure Legend Snippet: ecCEBPA –DNMT1 interactions; DNMT1 binds to RNA with greater affinity than to DNA a , Diagram showing position of qRT-PCR primers used in RIP, double-headed arrow; RNA and DNA oligonucleotides used in EMSA and REMSA. Asterisks indicate position of methylated cytosines; umDNA, hmDNA, and mDNA refer to unmethylated, hemimethylated, and methylated DNA probes, respectively; b , ecCEBPA is immunoprecipitated with anti-DNMT1 antibody. qRT-PCR, bars indicate mean ± s.d.; c , RNA- DNMT1 binding is not affected by the absence of CpG dinucleotides (right panel). Left and middle panels: RNA oligonucleotide R2 and its mutated form mut R2 (asterisks indicate cytosines substituted into uridines), both able to form stem-loop-structures; d , RNA oligonucleotide able to form stem-loop structure bind DNMT1 (R6); e , R5 RNA oligonucleotide forming stem-loop structure (R5) has a greater DNMT1 affinity compared to mut R5, unable to fold into stem-loop, (taken in equimolar amounts), at the same DNMT1 concentration; f , Left four panels: REMSA and EMSA performed with the fixed concentration of ssRNA and dsDNA oligonucleotides (1 nM) and increasing concentrations of DNMT1 protein; Right panel: Nonlinear regression analysis of bound RNA/DNA versus DNMT1 concentrations. Error bars indicate s.d. from two independent experiments; g , REMSA showing that RNA oligonucleotide R4, which is unable to form stem-loop structure, displays lower DNMT1 affinity as compared to R5 (Fig. 3f left panel) at the same DNMT1 concentrations; h , Left panel: Schematic diagram showing the DNMT1 domains and the GST-DNMT1 isolated fragments (F1–F5); Right panel: GST-DNMT1 pull down assay demonstrating binding of the folded RNA oligonucleotide R5 to the catalytic domain of DNMT1.

    Techniques Used: Quantitative RT-PCR, Methylation, Immunoprecipitation, Binding Assay, Concentration Assay, Isolation, Pull Down Assay

    Transcription impedes DNA methylation a–d , Diagram showing the parallel in vitro transcription-methylation assays performed on a hemimethylated template containing the T7 promoter ( Supplementary Fig. 4 ) with and without combinations of RNA polymerase, DNMT1, or both; e , DNMT1 exerts enzymatic activity only in the absence of transcription. COBRA analysis of methylation patterns acquired in reactions shown in b–d; f , DNA methylation changes as are shown as the ratios of methylated to unmethylated CpGs in all clones analyzed per each construct (n=5). The same effect was observed with two different RNA polymerases: T7 and Sigma-Saturated (σ70)-Holoenzyme ( E. coli RNA Polymerase). DNA methylation changes were analyzed by Fisher’s exact test (* P
    Figure Legend Snippet: Transcription impedes DNA methylation a–d , Diagram showing the parallel in vitro transcription-methylation assays performed on a hemimethylated template containing the T7 promoter ( Supplementary Fig. 4 ) with and without combinations of RNA polymerase, DNMT1, or both; e , DNMT1 exerts enzymatic activity only in the absence of transcription. COBRA analysis of methylation patterns acquired in reactions shown in b–d; f , DNA methylation changes as are shown as the ratios of methylated to unmethylated CpGs in all clones analyzed per each construct (n=5). The same effect was observed with two different RNA polymerases: T7 and Sigma-Saturated (σ70)-Holoenzyme ( E. coli RNA Polymerase). DNA methylation changes were analyzed by Fisher’s exact test (* P

    Techniques Used: DNA Methylation Assay, In Vitro, Methylation, Activity Assay, Combined Bisulfite Restriction Analysis Assay, Clone Assay, Construct

    3) Product Images from "Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *"

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M113.525279

    Cellular location and colocalization of PHF20L1a. A , the top panel shows pericentric localization of PHF20L1a. DNA staining is DAPI. In the middle panel , 5mC is shown in green and DNMT1K142me1 in red . In the bottom panel are shown CFP-SET7 in blue along with PHF20L1a in green and DNMT1K142me1 in red . The merge panel of DNMT1K142me1, PHF20L1a, and SET7 shows circular pericentric heterochromatin matching to the bright field image. Percentage perinucleolar localization was calculated from 200 transfected cells. B , colocalization of endogenous DNMT1, PHF20L1, and 5-methylcytosine in pericentric heterochromatin in HeLa cells. DAPI staining shows the heterochromatic regions.
    Figure Legend Snippet: Cellular location and colocalization of PHF20L1a. A , the top panel shows pericentric localization of PHF20L1a. DNA staining is DAPI. In the middle panel , 5mC is shown in green and DNMT1K142me1 in red . In the bottom panel are shown CFP-SET7 in blue along with PHF20L1a in green and DNMT1K142me1 in red . The merge panel of DNMT1K142me1, PHF20L1a, and SET7 shows circular pericentric heterochromatin matching to the bright field image. Percentage perinucleolar localization was calculated from 200 transfected cells. B , colocalization of endogenous DNMT1, PHF20L1, and 5-methylcytosine in pericentric heterochromatin in HeLa cells. DAPI staining shows the heterochromatic regions.

    Techniques Used: Staining, Transfection

    PHF20L1a inhibits DNMT1K142me1 ubiquitination and degradation. A , FLAG-PHF20L1a overexpression prevents ubiquitination of DNMT1K142me1. Overexpression ( O/E ) of GFP, GFP-SET7, FLAG-PHF20L1a, or a combination of GFP-SET7 and FLAG-PHF20L1a are indicated at the top of the Western blot. Overexpression of DsRed-DNMT1 and HA-ubiquitin and subsequent treatment with the MG132 proteasome inhibitor were carried out for all samples. Immunoprecipitations ( IP ) with anti-DNMT1 antibody were subsequently performed, and the precipitate was analyzed by Western blot. Input was revealed with anti-PHF20L1a, anti-SET7, anti-DsRed-DNMT1, anti-DNMT1K142me1, and anti-histone H3 antibodies, whereas DNMT1 immunoprecipitations were revealed with anti-HA-ubiquitin antibody (indicated on the right ). Full-length HA-ubiquitinated DNMT1 was present (*), but smaller degradation products were prevalent. Densitometry measurements of anti-HA-ubiquitin, normalized to histone H3, were done within the indicated brackets to show levels of ubiquitinated full-length DNMT1 (arbitrary units). B , loss of chromatin-bound DNMT1 in response to PHF20L1 knockdown is mediated by proteasomal degradation. siRNA-mediated knockdown of PHF20L1 and subsequent treatment with the MG132 proteasomal inhibitor are indicated at the top of the Western blot. Anti-PHF20L1a, anti-DNMT1, and anti-histone H3 antibodies used in the Western blot are indicated to the right of the blot. C , LC-MS analysis reveals that siRNA-mediated knockdown of PHF20L1 significantly reduces global levels of 5-methylcytosine. Genomic DNA was digested to single nucleosides for LC-MS analysis. Methylation changes were normalized to total levels of corresponding nucleotides. Total levels of 5-methylcytosine were significantly lower ( p = 0.029) after transfection of PH20L1 siRNA compared with control siRNA. Data represent two experimental replicates and four technical replicates. Error bars , S.D.
    Figure Legend Snippet: PHF20L1a inhibits DNMT1K142me1 ubiquitination and degradation. A , FLAG-PHF20L1a overexpression prevents ubiquitination of DNMT1K142me1. Overexpression ( O/E ) of GFP, GFP-SET7, FLAG-PHF20L1a, or a combination of GFP-SET7 and FLAG-PHF20L1a are indicated at the top of the Western blot. Overexpression of DsRed-DNMT1 and HA-ubiquitin and subsequent treatment with the MG132 proteasome inhibitor were carried out for all samples. Immunoprecipitations ( IP ) with anti-DNMT1 antibody were subsequently performed, and the precipitate was analyzed by Western blot. Input was revealed with anti-PHF20L1a, anti-SET7, anti-DsRed-DNMT1, anti-DNMT1K142me1, and anti-histone H3 antibodies, whereas DNMT1 immunoprecipitations were revealed with anti-HA-ubiquitin antibody (indicated on the right ). Full-length HA-ubiquitinated DNMT1 was present (*), but smaller degradation products were prevalent. Densitometry measurements of anti-HA-ubiquitin, normalized to histone H3, were done within the indicated brackets to show levels of ubiquitinated full-length DNMT1 (arbitrary units). B , loss of chromatin-bound DNMT1 in response to PHF20L1 knockdown is mediated by proteasomal degradation. siRNA-mediated knockdown of PHF20L1 and subsequent treatment with the MG132 proteasomal inhibitor are indicated at the top of the Western blot. Anti-PHF20L1a, anti-DNMT1, and anti-histone H3 antibodies used in the Western blot are indicated to the right of the blot. C , LC-MS analysis reveals that siRNA-mediated knockdown of PHF20L1 significantly reduces global levels of 5-methylcytosine. Genomic DNA was digested to single nucleosides for LC-MS analysis. Methylation changes were normalized to total levels of corresponding nucleotides. Total levels of 5-methylcytosine were significantly lower ( p = 0.029) after transfection of PH20L1 siRNA compared with control siRNA. Data represent two experimental replicates and four technical replicates. Error bars , S.D.

    Techniques Used: Over Expression, Western Blot, Liquid Chromatography with Mass Spectroscopy, Methylation, Transfection

    Related Articles

    Clone Assay:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. DNMT1 was cloned into pVIC1 (New England Biolabs) and purified as described previously ( ).

    Amplification:

    Article Title: Virome and bacteriome characterization of children with pneumonia and asthma in Mexico City during winter seasons 2014 and 2015
    Article Snippet: Paragraph title: Viral enrichment and random amplification ... To reduce the amount of human DNA, the aliquot for DNA viruses was treated with NEBNext Microbiome DNA Enrichment Kit, according to the manufacturer’s instructions (New England BioLabs Inc.).

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: TrueStart Hot Start Taq DNA Polymerase from Fermentas (Burlington, Canada) was used for the amplification of RAR- β and ATM , whereas FastStart Taq DNA Polymerase (Roche Diagnostics, Germany) was used for the amplification of CDH1 , FHIT , and CDKN2A . .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions.

    Filtration:

    Article Title: Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum
    Article Snippet: Thick and thin blood films were prepared before and after cellulose filtration to confirm complete removal of leucocytes. .. To further deplete human DNA extracted with the parasite DNA, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, MA, USA) was used.

    Positive Control:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions. .. Amplification products were separated on 2.5 % agarose gels and visualized in UV light after ethidium bromide staining.

    Article Title: Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis
    Article Snippet: Human DNA was removed from E . histolytica positive samples using a Microbiome DNA Enrichment Kit used by the manufacturer’s direction (NEB). .. As a positive control, DNA extracted from the HM-782D Mock Bacteria Community (ATCC through BEI Resources) was added, and as a control for reagent and laboratory contamination a no-template control reaction was added.

    Real-time Polymerase Chain Reaction:

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: The percent of S. aureus DNA was then determined by measuring the amount of S. aureus DNA by real-time PCR in relation to the total DNA concentration. .. Removal of human DNA with the NEBNext microbial DNA enrichment kit improved the relative amount of S. aureus DNA to 6.1%, representing a 5.7-fold enrichment of bacterial DNA.

    Article Title: Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum
    Article Snippet: Real-time PCR was carried out to determine the quantity of parasite DNA relative to human DNA contained in each sample using the procedures described by Veron et al. [ ]. .. To further deplete human DNA extracted with the parasite DNA, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, MA, USA) was used.

    Incubation:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: .. GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. DNMT1 was cloned into pVIC1 (New England Biolabs) and purified as described previously ( ).

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: .. To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA). .. The enriched microbial DNA samples were purified by ethanol precipitation.

    Modification:

    Article Title: Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis
    Article Snippet: Microbiome diversity analysis from human stool samples DNA was extracted from fecal material using a modified QiaAmp stool DNA extraction protocol which incorporates a 3 min “bead-beating” step as per standard study protocols [ ]. .. Human DNA was removed from E . histolytica positive samples using a Microbiome DNA Enrichment Kit used by the manufacturer’s direction (NEB).

    Western Blot:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: Immunoprecipitations were loaded into SDS-polyacrylamide gels for subsequent Western blot analyses. .. GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg).

    Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine
    Article Snippet: Paragraph title: Western blot ... Proteins were separated by SDS–PAGE, transferred to PVDF membrane (Millipore) and probed with the following primary antibodies: anti-IDH1 R132H (Dianova, DIA-H09), anti-DNMT1 (New England Biolabs, M0230S), anti-GFAP (Cell Signaling Tech, 2118S) and anti-β-actin (Sigma, A5316).

    DNA Methylation Assay:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: DNA was converted in the presence of sodium bisulfite using the EZ DNA Methylation Kit from ZymoResearch (Orange, CA, USA). .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions.

    Southern Blot:

    Article Title: Both hypomethylation and hypermethylation in a 0.2-kb region of a DNA repeat in cancer
    Article Snippet: .. For Southern blot analysis, 1.5 μg of human DNA was digested with 15–30 U of restriction endonuclease overnight according to the manufacturer’s procedures (New England Biolabs), all with parallel internal controls as previously ( ). ..

    Concentration Assay:

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: The percent of S. aureus DNA was then determined by measuring the amount of S. aureus DNA by real-time PCR in relation to the total DNA concentration. .. Removal of human DNA with the NEBNext microbial DNA enrichment kit improved the relative amount of S. aureus DNA to 6.1%, representing a 5.7-fold enrichment of bacterial DNA.

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA). .. DNA concentration and sizes were determined using NanoDrop and agarose gel electrophoresis.

    Infection:

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: .. The very low relative abundance of bacterial to human DNA in many clinical specimens deriving from patients with infection presents a unique challenge when using WGS to detect and identify pathogens. ..

    Generated:

    Article Title: Virome and bacteriome characterization of children with pneumonia and asthma in Mexico City during winter seasons 2014 and 2015
    Article Snippet: To reduce the amount of human DNA, the aliquot for DNA viruses was treated with NEBNext Microbiome DNA Enrichment Kit, according to the manufacturer’s instructions (New England BioLabs Inc.). .. For RNA viruses, reverse transcription was performed on 10 μl de RNA using a m13-random hexamer primer with Transcriptor First Strand cDNA Synthesis Kit (Roche diagnostics) Next double strand cDNA (dscDNA) was generated by two rounds of synthesis using m13-random hexamer primer with Klenow fragment (Thermo Scientific).

    Polymerase Chain Reaction:

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: .. Preanalytic removal of human DNA eliminates false signals in general 16S rDNA PCR monitoring of bacterial pathogens in blood. ..

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: The methylation status of RAR- β, CDH1 , FHIT , CDKN2A , and ATM was assessed using the methylation-specific polymerase chain reaction (MSP). .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions.

    Article Title: The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine-based adjuvant chemotherapy
    Article Snippet: Reaction specificity for methylated DNA was confirmed separately using human DNA treated with CpG methyltransferase SssI (New England Biolabs). .. In addition to CIMP markers, methylation of O6 -methylguanine-DNA methyltransferase (MGMT ) gene was also analyzed by methylation-specific polymerase chain reaction due to its importance in KRAS mutations [ - ] and clinical significance in survival [ ].

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: .. A novel method of selective removal of human DNA improves PCR sensitivity for detection of Salmonella Typhi in blood samples. ..

    Binding Assay:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. In vitro pull-downs of MBP-PHF20L1a with DNMT1K142me1 peptide were carried out by first incubating 10 μm peptide (biotin-LSKPRTPRRSK(me1)SDGEAKPE) with agarose-streptavidin beads (25 μl of bead slurry, Thermo Scientific, catalog no. 2359) for 1 h at 4 °C with rotation in peptide binding buffer containing 10% glycerol (v/v), 0.1 mm DTT, 1 mm EDTA, 20 mm HEPES, and 100 mm KCl.

    Article Title: Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum
    Article Snippet: To further deplete human DNA extracted with the parasite DNA, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, MA, USA) was used. .. Methylated human DNA was removed from the DNA mixture by binding to the methyl-CpG binding domain of human MBD2-Fc protein [ ].

    Immunofluorescence:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: Paragraph title: Cell Treatments, Cell Cycle Synchronization, Protein Stability, Immunoprecipitation, GST Pull-down, Far-Western Blot, and Immunofluorescence ... GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg).

    DNA Extraction:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: Paragraph title: DNA extraction and gene methylation analysis ... DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions.

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: DNA extraction of spiked sonicate fluid without enrichment yielded 1.1% of the total DNA being from S. aureus ( ). .. Removal of human DNA with the NEBNext microbial DNA enrichment kit improved the relative amount of S. aureus DNA to 6.1%, representing a 5.7-fold enrichment of bacterial DNA.

    Article Title: Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis
    Article Snippet: Microbiome diversity analysis from human stool samples DNA was extracted from fecal material using a modified QiaAmp stool DNA extraction protocol which incorporates a 3 min “bead-beating” step as per standard study protocols [ ]. .. Human DNA was removed from E . histolytica positive samples using a Microbiome DNA Enrichment Kit used by the manufacturer’s direction (NEB).

    Far Western Blot:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: Paragraph title: Cell Treatments, Cell Cycle Synchronization, Protein Stability, Immunoprecipitation, GST Pull-down, Far-Western Blot, and Immunofluorescence ... GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg).

    Methylation:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions. .. Amplification products were separated on 2.5 % agarose gels and visualized in UV light after ethidium bromide staining.

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: .. GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. DNMT1 was cloned into pVIC1 (New England Biolabs) and purified as described previously ( ).

    Article Title: The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine-based adjuvant chemotherapy
    Article Snippet: .. Reaction specificity for methylated DNA was confirmed separately using human DNA treated with CpG methyltransferase SssI (New England Biolabs). .. In addition to CIMP markers, methylation of O6 -methylguanine-DNA methyltransferase (MGMT ) gene was also analyzed by methylation-specific polymerase chain reaction due to its importance in KRAS mutations [ - ] and clinical significance in survival [ ].

    Article Title: Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum
    Article Snippet: To further deplete human DNA extracted with the parasite DNA, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, MA, USA) was used. .. Methylated human DNA was removed from the DNA mixture by binding to the methyl-CpG binding domain of human MBD2-Fc protein [ ].

    Isolation:

    Article Title: Virome and bacteriome characterization of children with pneumonia and asthma in Mexico City during winter seasons 2014 and 2015
    Article Snippet: Nucleic acids were isolated from the concentrated sample pool using the PureLink Viral RNA/DNA kit according to the manufacturer’s instructions (Invitrogen, Waltham, MA). .. To reduce the amount of human DNA, the aliquot for DNA viruses was treated with NEBNext Microbiome DNA Enrichment Kit, according to the manufacturer’s instructions (New England BioLabs Inc.).

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: Paragraph title: Saliva sampling and isolation of bacterial genomic DNA. ... To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA).

    Size-exclusion Chromatography:

    Article Title: Virome and bacteriome characterization of children with pneumonia and asthma in Mexico City during winter seasons 2014 and 2015
    Article Snippet: To reduce the amount of human DNA, the aliquot for DNA viruses was treated with NEBNext Microbiome DNA Enrichment Kit, according to the manufacturer’s instructions (New England BioLabs Inc.). .. DNA from each process was amplified with Platinum Taq DNA Polymerase High Fidelity (Thermo Scientific) using m13 primers and the following program: 2 min 95°C, 30 cycles of 15 sec at 95°C, 30 sec at 50°C, 3 min at 68°C and a final step of 5 min at 68°C.

    Labeling:

    Article Title: Virome and bacteriome characterization of children with pneumonia and asthma in Mexico City during winter seasons 2014 and 2015
    Article Snippet: To reduce the amount of human DNA, the aliquot for DNA viruses was treated with NEBNext Microbiome DNA Enrichment Kit, according to the manufacturer’s instructions (New England BioLabs Inc.). .. For DNA viruses and Bacteria, DNA previously enriched was labeled using m13-random hexamer primer.

    Purification:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: .. GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. DNMT1 was cloned into pVIC1 (New England Biolabs) and purified as described previously ( ).

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA). .. The enriched microbial DNA samples were purified by ethanol precipitation.

    Sequencing:

    Article Title: Microbiome-mediated neutrophil recruitment via CXCR2 and protection from amebic colitis
    Article Snippet: Human DNA was removed from E . histolytica positive samples using a Microbiome DNA Enrichment Kit used by the manufacturer’s direction (NEB). .. The sequencing library was prepared using phased Illumina-eubacteria primers to both amplify the V4 16S region rDNA (515–806), add the adaptors necessary for illumina sequencing and the GOLAY index necessary for de-multiplexing after parallel sequencing [ , ].

    Article Title: Pooled-DNA sequencing identifies genomic regions of selection in Nigerian isolates of Plasmodium falciparum
    Article Snippet: Paragraph title: Parasite examination and pooled sequencing ... To further deplete human DNA extracted with the parasite DNA, NEBNext Microbiome DNA Enrichment Kit (New England Biolabs, MA, USA) was used.

    SDS Page:

    Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine
    Article Snippet: .. Proteins were separated by SDS–PAGE, transferred to PVDF membrane (Millipore) and probed with the following primary antibodies: anti-IDH1 R132H (Dianova, DIA-H09), anti-DNMT1 (New England Biolabs, M0230S), anti-GFAP (Cell Signaling Tech, 2118S) and anti-β-actin (Sigma, A5316). .. Flow Cytometry For single-color flow cytometry, 106 cells were washed with ice-cold PBS, permeabilized and fixed using BD Cytoperm/Cytofix solution (BD, PharMingen), and incubated with anti-GFAP (1:200, BD Pharmingen) for 30 min at room temperature.

    Plasmid Preparation:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. MBP-SET7 and MBP-PHF20L1 were cloned into pMAL-C5x vector (New England Biolabs, catalog no. N8108S) and purified using amylose resin (New England Biolabs, catalog no. E8021).

    Negative Control:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: .. DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions. .. Amplification products were separated on 2.5 % agarose gels and visualized in UV light after ethidium bromide staining.

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: 5 μg of purified normal rabbit IgG (Cell Signaling Technology, catalog no. 2729) was used as a negative control. .. GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg).

    Agarose Gel Electrophoresis:

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA). .. DNA concentration and sizes were determined using NanoDrop and agarose gel electrophoresis.

    In Vitro:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg). .. In vitro pull-downs of MBP-PHF20L1a with DNMT1K142me1 peptide were carried out by first incubating 10 μm peptide (biotin-LSKPRTPRRSK(me1)SDGEAKPE) with agarose-streptavidin beads (25 μl of bead slurry, Thermo Scientific, catalog no. 2359) for 1 h at 4 °C with rotation in peptide binding buffer containing 10% glycerol (v/v), 0.1 mm DTT, 1 mm EDTA, 20 mm HEPES, and 100 mm KCl.

    Ethanol Precipitation:

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA). .. The enriched microbial DNA samples were purified by ethanol precipitation.

    Next-Generation Sequencing:

    Article Title: Comparison of microbial DNA enrichment tools for metagenomic whole genome sequencing
    Article Snippet: .. [ ] [ ] [ ] Hasan MR, Rawat A, Tang P, Jithesh PV, Thomas E, Tan R, Tilley P. Depletion of human DNA in spiked clinical specimens to improve the sensitivity of pathogen detection by next generation sequencing. ..

    Sampling:

    Article Title: Oral Microbiome Alterations Associated with Early Childhood Caries Highlight the Importance of Carbohydrate Metabolic Activities
    Article Snippet: Paragraph title: Saliva sampling and isolation of bacterial genomic DNA. ... To reduce contamination by human DNA, every 4 μg DNA was incubated with 160 μl MBD-Fc-bound beads from a NEBNext microbiome DNA enrichment kit (New England Biolabs, Inc., Ipswich, MA, USA).

    Immunoprecipitation:

    Article Title: Methyllysine Reader Plant Homeodomain (PHD) Finger Protein 20-like 1 (PHF20L1) Antagonizes DNA (Cytosine-5) Methyltransferase 1 (DNMT1) Proteasomal Degradation *
    Article Snippet: Paragraph title: Cell Treatments, Cell Cycle Synchronization, Protein Stability, Immunoprecipitation, GST Pull-down, Far-Western Blot, and Immunofluorescence ... GST pull-downs were performed with increasing amounts (1, 2, and 4 μg) of baculovirus purified full-length DNMT1 (New England Biolabs, catalog no. M0230S) that were methylated overnight by MBP-SET7 (New England Biolabs, catalog no. M0233S) and then incubated with purified MBT-GST (10 μg).

    Marker:

    Article Title: The CpG island methylator phenotype may confer a survival benefit in patients with stage II or III colorectal carcinomas receiving fluoropyrimidine-based adjuvant chemotherapy
    Article Snippet: Reaction specificity for methylated DNA was confirmed separately using human DNA treated with CpG methyltransferase SssI (New England Biolabs). .. Tumors were classified as CIMP-high if three or more markers were methylated, CIMP-low if one or two markers were methylated, and CIMP-negative if a methylated marker was not observed [ , ].

    Staining:

    Article Title: Prognostic factors in oral and oropharyngeal cancer based on ultrastructural analysis and DNA methylation of the tumor and surgical margin
    Article Snippet: DNA extracted from the lymphocytes of healthy blood donors was used as a negative control, and completely methylated human DNA (New England Biolabs, Ipswich, MA, USA) was used as a positive control in the MSP reactions. .. Amplification products were separated on 2.5 % agarose gels and visualized in UV light after ethidium bromide staining.

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    New England Biolabs human genomic dna
    TRD and <t>DNA</t> binding activity of CXXC3 in MBD1. (A) The reporter construct contains five copies of the GAL4 DNA binding site (5 × GAL) upstream of the <t>SNRPN</t> promoter. Effector constructs express the regions of MBD1 fused to the GAL4 DNA binding domain. CMV, cytomegalovirus promoter. (B) Transcriptional repression domain in MBD1 isoforms. Eleven GAL4 fusion proteins (termed GAL4-MBD1Δ1 to GAL4-MBD1Δ11) were constructed: Δ1 (amino acids 62 to 605 of MBD1v1), Δ2 (amino acids 62 to 549 of MBD1v3), Δ3 (amino acids 62 to 173 of MBD1v1), Δ4 (amino acids 62 to 327 of MBD1v3), Δ5 (amino acids 62 to 379 of MBD1v1), Δ6 (amino acids 380 to 605 of MBD1v1), Δ7 (amino acids 1 to 61 of MBD1v1), Δ8 (amino acids 361 to 586 of MBD1v2), Δ9 (amino acids 361 to 523 of MBD1v2), Δ10 (amino acids 361 to 459 of MBD1v2), and Δ11 (amino acids 460 to 523 of MBD1v2). +, shown; −, not shown. (C) Relative transcription levels under the expression of GAL4-MBD1Δ4, GAL4-MBD1Δ5, GAL4-MBD1Δ7, and GAL4-MBD1Δ11. GAL4-MBD1Δ11 specifically repressed transcription from the reporter. (D) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of GST-fused MBD1v1 and MBD1v3 stained with Coomassie blue: full-length (full) and with deletion of the MBD (amino acids 1 to 61) (ΔN). (E) Band shift analysis of methylated and unmethylated DNAs complexed with recombinant MBD1. Unmethylated (M−) and Sss I-methylated (M+) DNAs of the SNRPN promoter were incubated with one of the GST-MBD1 proteins. The CXXC3 of MBD1v1 has a DNA binding activity. Numbers to the right are molecular masses (in kilodaltons).
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    TRD and DNA binding activity of CXXC3 in MBD1. (A) The reporter construct contains five copies of the GAL4 DNA binding site (5 × GAL) upstream of the SNRPN promoter. Effector constructs express the regions of MBD1 fused to the GAL4 DNA binding domain. CMV, cytomegalovirus promoter. (B) Transcriptional repression domain in MBD1 isoforms. Eleven GAL4 fusion proteins (termed GAL4-MBD1Δ1 to GAL4-MBD1Δ11) were constructed: Δ1 (amino acids 62 to 605 of MBD1v1), Δ2 (amino acids 62 to 549 of MBD1v3), Δ3 (amino acids 62 to 173 of MBD1v1), Δ4 (amino acids 62 to 327 of MBD1v3), Δ5 (amino acids 62 to 379 of MBD1v1), Δ6 (amino acids 380 to 605 of MBD1v1), Δ7 (amino acids 1 to 61 of MBD1v1), Δ8 (amino acids 361 to 586 of MBD1v2), Δ9 (amino acids 361 to 523 of MBD1v2), Δ10 (amino acids 361 to 459 of MBD1v2), and Δ11 (amino acids 460 to 523 of MBD1v2). +, shown; −, not shown. (C) Relative transcription levels under the expression of GAL4-MBD1Δ4, GAL4-MBD1Δ5, GAL4-MBD1Δ7, and GAL4-MBD1Δ11. GAL4-MBD1Δ11 specifically repressed transcription from the reporter. (D) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of GST-fused MBD1v1 and MBD1v3 stained with Coomassie blue: full-length (full) and with deletion of the MBD (amino acids 1 to 61) (ΔN). (E) Band shift analysis of methylated and unmethylated DNAs complexed with recombinant MBD1. Unmethylated (M−) and Sss I-methylated (M+) DNAs of the SNRPN promoter were incubated with one of the GST-MBD1 proteins. The CXXC3 of MBD1v1 has a DNA binding activity. Numbers to the right are molecular masses (in kilodaltons).

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: TRD and DNA binding activity of CXXC3 in MBD1. (A) The reporter construct contains five copies of the GAL4 DNA binding site (5 × GAL) upstream of the SNRPN promoter. Effector constructs express the regions of MBD1 fused to the GAL4 DNA binding domain. CMV, cytomegalovirus promoter. (B) Transcriptional repression domain in MBD1 isoforms. Eleven GAL4 fusion proteins (termed GAL4-MBD1Δ1 to GAL4-MBD1Δ11) were constructed: Δ1 (amino acids 62 to 605 of MBD1v1), Δ2 (amino acids 62 to 549 of MBD1v3), Δ3 (amino acids 62 to 173 of MBD1v1), Δ4 (amino acids 62 to 327 of MBD1v3), Δ5 (amino acids 62 to 379 of MBD1v1), Δ6 (amino acids 380 to 605 of MBD1v1), Δ7 (amino acids 1 to 61 of MBD1v1), Δ8 (amino acids 361 to 586 of MBD1v2), Δ9 (amino acids 361 to 523 of MBD1v2), Δ10 (amino acids 361 to 459 of MBD1v2), and Δ11 (amino acids 460 to 523 of MBD1v2). +, shown; −, not shown. (C) Relative transcription levels under the expression of GAL4-MBD1Δ4, GAL4-MBD1Δ5, GAL4-MBD1Δ7, and GAL4-MBD1Δ11. GAL4-MBD1Δ11 specifically repressed transcription from the reporter. (D) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of GST-fused MBD1v1 and MBD1v3 stained with Coomassie blue: full-length (full) and with deletion of the MBD (amino acids 1 to 61) (ΔN). (E) Band shift analysis of methylated and unmethylated DNAs complexed with recombinant MBD1. Unmethylated (M−) and Sss I-methylated (M+) DNAs of the SNRPN promoter were incubated with one of the GST-MBD1 proteins. The CXXC3 of MBD1v1 has a DNA binding activity. Numbers to the right are molecular masses (in kilodaltons).

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Binding Assay, Activity Assay, Construct, Expressing, Polyacrylamide Gel Electrophoresis, Staining, Electrophoretic Mobility Shift Assay, Methylation, Recombinant, Incubation

    Effect of MBD1 isoforms on methylated and unmethylated promoters. (A) PCR-amplified DNA fragments from human imprinted SNRPN and tumor suppressor p16 genes were used for a band shift analysis and subcloned upstream of a luciferase cDNA in a pGL3-Basic vector. The PCR fragments and pGL3 constructs were methylated in vitro using Hpa II, Hha I, and Sss I (CpG) methyltransferases. The methyl-CpG sites modified by these enzymes are shown by vertical lines. (B) Band shift of methylated DNA complexed with the methyl-CpG binding domain of MBD1. Unmethylated (−) and methylated fragments containing SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. In the upper and lower panels, the amount of the protein incubated with DNA fragments was 0.5 and 1.0 μg, respectively. (C and D) Regulation of Sp1-activated transcription by MBD1v1 and -v3 in Drosophila SL2 cells ( SNRPN [C] or p16 [D]). Unmethylated (M-) or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with Sp1-expressing plasmid pPacSp1 (0.5 μg), MBD1-expressing plasmids (pAc5.1-MBD1v1 and pAc5.1-MBD1v3) (0 to 1.0 μg), and insertless plasmid pAc5.1/V5-His (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pAc5.1-pRL (0.1 μg). (E) Detection of endogenous MBD1 by an antibody raised against the recombinant MBD1. MBD1 was found to be approximately 80 kDa in HeLa and A549 cells but not in SL2 and CHO-K1 cells.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: Effect of MBD1 isoforms on methylated and unmethylated promoters. (A) PCR-amplified DNA fragments from human imprinted SNRPN and tumor suppressor p16 genes were used for a band shift analysis and subcloned upstream of a luciferase cDNA in a pGL3-Basic vector. The PCR fragments and pGL3 constructs were methylated in vitro using Hpa II, Hha I, and Sss I (CpG) methyltransferases. The methyl-CpG sites modified by these enzymes are shown by vertical lines. (B) Band shift of methylated DNA complexed with the methyl-CpG binding domain of MBD1. Unmethylated (−) and methylated fragments containing SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. In the upper and lower panels, the amount of the protein incubated with DNA fragments was 0.5 and 1.0 μg, respectively. (C and D) Regulation of Sp1-activated transcription by MBD1v1 and -v3 in Drosophila SL2 cells ( SNRPN [C] or p16 [D]). Unmethylated (M-) or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with Sp1-expressing plasmid pPacSp1 (0.5 μg), MBD1-expressing plasmids (pAc5.1-MBD1v1 and pAc5.1-MBD1v3) (0 to 1.0 μg), and insertless plasmid pAc5.1/V5-His (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pAc5.1-pRL (0.1 μg). (E) Detection of endogenous MBD1 by an antibody raised against the recombinant MBD1. MBD1 was found to be approximately 80 kDa in HeLa and A549 cells but not in SL2 and CHO-K1 cells.

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Methylation, Polymerase Chain Reaction, Amplification, Electrophoretic Mobility Shift Assay, Luciferase, Plasmid Preparation, Construct, In Vitro, Modification, Binding Assay, Incubation, Expressing, Activity Assay, Transfection, Recombinant

    Residues within the methyl-CpG binding domain of MBD1 required for the methylated DNA binding, intranuclear localization, and transcriptional repression of methylated promoter. (A) Band shift of methylated DNA complexed with wild-type (wt) and mutant MBD1. Unmethylated (−) and Sss I-methylated (+) DNA fragments of the SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. (B) Intranuclear localization of GFP-fused MBD1 (full-length and N-terminal deletion ΔN) and MBD1 (residues 1 to 89) with the above-mentioned point mutations. (C) Hha I- or Sss I-methylated pGL3-SNRPN (0.5 μg) was cotransfected with pPacSp1 (0.5 μg) and one of the MBD1-expressing plasmids [pAc5.1-MBD1v1, pAc5.1-MBD1v1ΔN, pAc5.1-wt. MBD1v1, pAc5.1-MBD1v1(R22A) to pAc5.1-MBD1v1(Y52A), pAc5.1-MBD1v3, pAc5.1-MBD1v3ΔN, pAc5.1-wt. MBD1v3, and pAc5.1-MBD1v3(R22A) to pAc5.1-MBD1v3(Y52A)] (1.0 μg) or insertless plasmid pAc5.1/V5-His (mock) (1.0 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and pAc5.1/V5-His (mock) was normalized to 100 (data not shown). Relative luciferase activities (means + standard deviations) are given. The numbers 22, 30, 32, 34, 44, 45, and 52 correspond to R22A, R30A, D32A, Y34A, R44A, S45A, and Y52A, respectively.

    Journal: Molecular and Cellular Biology

    Article Title: Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1

    doi:

    Figure Lengend Snippet: Residues within the methyl-CpG binding domain of MBD1 required for the methylated DNA binding, intranuclear localization, and transcriptional repression of methylated promoter. (A) Band shift of methylated DNA complexed with wild-type (wt) and mutant MBD1. Unmethylated (−) and Sss I-methylated (+) DNA fragments of the SNRPN promoter were incubated with MBD1 (residues 1 to 75) or GST. (B) Intranuclear localization of GFP-fused MBD1 (full-length and N-terminal deletion ΔN) and MBD1 (residues 1 to 89) with the above-mentioned point mutations. (C) Hha I- or Sss I-methylated pGL3-SNRPN (0.5 μg) was cotransfected with pPacSp1 (0.5 μg) and one of the MBD1-expressing plasmids [pAc5.1-MBD1v1, pAc5.1-MBD1v1ΔN, pAc5.1-wt. MBD1v1, pAc5.1-MBD1v1(R22A) to pAc5.1-MBD1v1(Y52A), pAc5.1-MBD1v3, pAc5.1-MBD1v3ΔN, pAc5.1-wt. MBD1v3, and pAc5.1-MBD1v3(R22A) to pAc5.1-MBD1v3(Y52A)] (1.0 μg) or insertless plasmid pAc5.1/V5-His (mock) (1.0 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and pAc5.1/V5-His (mock) was normalized to 100 (data not shown). Relative luciferase activities (means + standard deviations) are given. The numbers 22, 30, 32, 34, 44, 45, and 52 correspond to R22A, R30A, D32A, Y34A, R44A, S45A, and Y52A, respectively.

    Article Snippet: DNA fragments (516 bp long) for the promoter region of the human SNRPN gene were amplified from human genomic DNA ( ) and were methylated with Hpa II, Hha I, or Sss I methyltransferases as indicated by the manufacturer (New England Biolabs, Beverly, Mass.).

    Techniques: Binding Assay, Methylation, Electrophoretic Mobility Shift Assay, Mutagenesis, Incubation, Expressing, Plasmid Preparation, Luciferase, Activity Assay

    A 40 kb region is frequently mutated in HGSOCs and consists of a distal SOX2 repressor. a. To determine whether any of the six mutations (BB1 to BB6) mapping near the SOX2 gene locus marked regions that were sites of frequent occurrence of private variants or mutations in HGSOCs, we performed deep-targeted sequencing of the 2 Mb region flanking SOX2 on 33 HGSOCs ( Supplementary Table 2 ). A total of 861 single nucleotide substitutions ( Supplementary Table 5 ) were identified that were not previously reported in the 1000 Genomes Project (median = 21, range = 11 to 97). Because functionally important genomic regions tend to be significantly less susceptible to genomic variation within a population, we determined whether the identified rare variants accumulated in specific areas within the 2 Mb region that were less susceptible to genomic alterations on a population scale. To test this hypothesis, we constructed overlapping moving windows of 40 kb size and compared the observed frequency of rare mutations (not previously described in the 1000 Genomes Project) in our group of patients. The expected frequency of SNPs in the same windows was based on 1000 sets of simulated cohorts of 33 individuals from the previously reported 1000 Genomes Project data. Upper panel: shown is the ratio of the observed number of variants in 40 kb “moving” windows in the cancer set to the expected number of variants in the equivalent windows based on 1000 permutations of simulated 1000 Genomes Project data. Based on this analysis, a peak observed/expected ratio (enrichment statistic) was identified in a 40 kb region flanking the BB5 nucleotide referred to as the BB5 region. Lower panel: To test whether this observation was higher than what would be expected by chance, we sequenced germline DNA from 597 healthy elderly volunteers and sequenced germline DNA from 436 individuals from the 1000 Genomes Project at higher depth. We then identified rare variants in the elderly set and repeated the above analysis. Comparing the enrichment statistic in the BB5 region in the cancer set to that obtained from 100 permutations of 33 individuals from the elderly set confirmed the significant enrichment of rare variants in the ovarian cancer set ( p

    Journal: EBioMedicine

    Article Title: Premalignant SOX2 overexpression in the fallopian tubes of ovarian cancer patients: Discovery and validation studies

    doi: 10.1016/j.ebiom.2016.06.048

    Figure Lengend Snippet: A 40 kb region is frequently mutated in HGSOCs and consists of a distal SOX2 repressor. a. To determine whether any of the six mutations (BB1 to BB6) mapping near the SOX2 gene locus marked regions that were sites of frequent occurrence of private variants or mutations in HGSOCs, we performed deep-targeted sequencing of the 2 Mb region flanking SOX2 on 33 HGSOCs ( Supplementary Table 2 ). A total of 861 single nucleotide substitutions ( Supplementary Table 5 ) were identified that were not previously reported in the 1000 Genomes Project (median = 21, range = 11 to 97). Because functionally important genomic regions tend to be significantly less susceptible to genomic variation within a population, we determined whether the identified rare variants accumulated in specific areas within the 2 Mb region that were less susceptible to genomic alterations on a population scale. To test this hypothesis, we constructed overlapping moving windows of 40 kb size and compared the observed frequency of rare mutations (not previously described in the 1000 Genomes Project) in our group of patients. The expected frequency of SNPs in the same windows was based on 1000 sets of simulated cohorts of 33 individuals from the previously reported 1000 Genomes Project data. Upper panel: shown is the ratio of the observed number of variants in 40 kb “moving” windows in the cancer set to the expected number of variants in the equivalent windows based on 1000 permutations of simulated 1000 Genomes Project data. Based on this analysis, a peak observed/expected ratio (enrichment statistic) was identified in a 40 kb region flanking the BB5 nucleotide referred to as the BB5 region. Lower panel: To test whether this observation was higher than what would be expected by chance, we sequenced germline DNA from 597 healthy elderly volunteers and sequenced germline DNA from 436 individuals from the 1000 Genomes Project at higher depth. We then identified rare variants in the elderly set and repeated the above analysis. Comparing the enrichment statistic in the BB5 region in the cancer set to that obtained from 100 permutations of 33 individuals from the elderly set confirmed the significant enrichment of rare variants in the ovarian cancer set ( p

    Article Snippet: 2.8 Cloning, Mutant Generation and Chicken Embryo Transfection The 1 kb regions flanking the BB5 SNP was cloned from Human genomic blood DNA by PCR amplification using Phusion high fidelity polymerase (NEB) according to the manufacturer's instructions using the following primers BB5_F; ‘TTTTTTCGTCTCgccaggTTACTCCAATATGAGAGATAAGAGCA’ and BB5_R; ‘TTTTTTCGTCTCcaacagCGCTCACACGGTGATTAGAA’, Test1_F; ‘TTTTTTCGTCTCgccaggCATTACTGGCAGCTGAGGGG’ and Test1_R; ‘TTTTTTCGTCTCcaacagTGATTTTCCCTGGGCAGACA’, Test2_F; ‘TTTTTTCGTCTCgccaggCTACTAGACCCCAGGCAAGG’ and Test2_R; ‘TTTTTTCGTCTCcaacagTGATTTTCCCTGGGCAGACA’, Test3_F; ‘TTTTTTCGTCTCgccaggTCCCTGTTCCTCACTCCTCT’ and Test3_R; ‘TTTTTTCGTCTCcaacagTGATTTTCCCTGGGCAGACA’.

    Techniques: Sequencing, Construct

    Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.

    Journal: Oncotarget

    Article Title: Efficient induction of differentiation and growth inhibition in IDH1 mutant glioma cells by the DNMT Inhibitor Decitabine

    doi:

    Figure Lengend Snippet: Decitabine reverses genome-wide DNA methylation and induces expression of genes associated with differentiation in IDH mutant glioma cells A, Low dose DAC inhibits DNMT1. Results from western blot shown. B, DAC treatment results in loss of DNA methylation. DNA methylome analysis of TS603 and TS667 cells following DAC treatment (200nM) is shown. Results from the Illumina HumanMethylation450 array. C, Gene expression changes following DAC treatment (200nM). Results from Affymetrix gene expression arrays. Most significantly altered genes following 200nM DAC are shown. D, Significant concordance between demethylated and upregulated genes and polycomb targets. Venn diagram showing overlap between the gene sets. P value (hypergeometric) is shown.

    Article Snippet: Proteins were separated by SDS–PAGE, transferred to PVDF membrane (Millipore) and probed with the following primary antibodies: anti-IDH1 R132H (Dianova, DIA-H09), anti-DNMT1 (New England Biolabs, M0230S), anti-GFAP (Cell Signaling Tech, 2118S) and anti-β-actin (Sigma, A5316).

    Techniques: Genome Wide, DNA Methylation Assay, Expressing, Mutagenesis, Western Blot

    Scheme for creation of epitope-tagging vectors. Step I: left and right homology arms (LHA and RHA) are created by PCR from a human genomic DNA template using primers tailed with restriction sites needed for subsequent cloning. Step II: the PCR products are digested and cloned simultaneously into either pAAV-SEPT-Acceptor or pAAV-TK-Acceptor that had been digested with the same enzymes and treated with calf intestinal alkaline phosphatase. Step III: the desired epitope tag is added to the LHA by site-directed mutagenesis.

    Journal: Nucleic Acids Research

    Article Title: Epitope tagging of endogenous genes in diverse human cell lines

    doi: 10.1093/nar/gkn566

    Figure Lengend Snippet: Scheme for creation of epitope-tagging vectors. Step I: left and right homology arms (LHA and RHA) are created by PCR from a human genomic DNA template using primers tailed with restriction sites needed for subsequent cloning. Step II: the PCR products are digested and cloned simultaneously into either pAAV-SEPT-Acceptor or pAAV-TK-Acceptor that had been digested with the same enzymes and treated with calf intestinal alkaline phosphatase. Step III: the desired epitope tag is added to the LHA by site-directed mutagenesis.

    Article Snippet: PCR-based creation and assembly of epitope-tagging vectors Homology arms for creation of both PTEN and p53 epitope-tagging vectors were created by PCR from a human genomic DNA template using VENT Polymerase (New England Biolabs) as described by the manufacturer.

    Techniques: Polymerase Chain Reaction, Clone Assay, Mutagenesis