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    Name:
    HpaII
    Description:
    HpaII 10 000 units
    Catalog Number:
    R0171L
    Price:
    269
    Category:
    Restriction Enzymes
    Size:
    10 000 units
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    New England Biolabs hpaii
    HpaII
    HpaII 10 000 units
    https://www.bioz.com/result/hpaii/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    hpaii - by Bioz Stars, 2021-06
    98/100 stars

    Images

    1) Product Images from "The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma"

    Article Title: The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

    Journal: JCI Insight

    doi: 10.1172/jci.insight.120422

    Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).
    Figure Legend Snippet: Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).

    Techniques Used: DNA Methylation Assay, Methylation, Marker

    2) Product Images from "Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma"

    Article Title: Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma

    Journal: The Journal of Clinical Investigation

    doi: 10.1172/JCI98747

    AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.
    Figure Legend Snippet: AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.

    Techniques Used: Activity Assay, Mass Spectrometry, In Vitro, Incubation, IF-P, Genome Wide, Methylation, HELP Assay

    3) Product Images from "SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation"

    Article Title: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation

    Journal: The EMBO Journal

    doi: 10.15252/embj.201899242

    SUMO transfer from XRCC1‐SUMO to TDG within the TDG‐BERosome Reconstitution SUMO‐conjugation/de‐conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2 364–489 ), free SUMO in equimolar amounts and 20‐fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP‐site containing synthetic DNA double‐strands as indicated (G•AP). TDG and TDG‐SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20‐fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1‐SUMO instead of free SUMO. SUMO transfer as depicted in (B) with additional APE1, POLβ, LIGIII and TDG. Immunoblot analysis of XRCC1, TDG, TDG‐SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null). Immunoblot analysis of TDG and TDG‐SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H 2 O 2 as indicated. Top: Schematic of the SUMO‐mediated TDG‐BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double‐strands in presence of dynamic SUMO‐conjugation/de‐conjugation. Left panels lanes 3–14: BER reconstitution with XRCC1 or XRCC1‐SUMO as indicated. Right panels lanes 15–21: BER reconstitution with free SUMO or XRCC1‐SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1–2 CG/CG HpaII methylation‐sensitive restriction site, cleavable by HpaII ; lane 3–6, 8–14, 15–17, 21 HpaII methylation‐sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII ; lane 7 and 20 repaired product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single‐stranded DNA. Source data are available online for this figure.
    Figure Legend Snippet: SUMO transfer from XRCC1‐SUMO to TDG within the TDG‐BERosome Reconstitution SUMO‐conjugation/de‐conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2 364–489 ), free SUMO in equimolar amounts and 20‐fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP‐site containing synthetic DNA double‐strands as indicated (G•AP). TDG and TDG‐SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20‐fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1‐SUMO instead of free SUMO. SUMO transfer as depicted in (B) with additional APE1, POLβ, LIGIII and TDG. Immunoblot analysis of XRCC1, TDG, TDG‐SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null). Immunoblot analysis of TDG and TDG‐SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H 2 O 2 as indicated. Top: Schematic of the SUMO‐mediated TDG‐BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double‐strands in presence of dynamic SUMO‐conjugation/de‐conjugation. Left panels lanes 3–14: BER reconstitution with XRCC1 or XRCC1‐SUMO as indicated. Right panels lanes 15–21: BER reconstitution with free SUMO or XRCC1‐SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1–2 CG/CG HpaII methylation‐sensitive restriction site, cleavable by HpaII ; lane 3–6, 8–14, 15–17, 21 HpaII methylation‐sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII ; lane 7 and 20 repaired product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single‐stranded DNA. Source data are available online for this figure.

    Techniques Used: Conjugation Assay, Purification, Methylation

    4) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

    Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).
    Figure Legend Snippet: Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    5) Product Images from "Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion"

    Article Title: Different Roles for Tet1 and Tet2 Proteins in Reprogramming-Mediated Erasure of Imprints Induced by EGC Fusion

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2013.01.032

    Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value
    Figure Legend Snippet: Evidence that 5hmC Levels Increase at ICRs in Somatic Cells after Fusion with EGCs (A) Detection of 5hmC ( I ) and unmodified cytosine ( II ) in human heterokaryon samples. Genomic DNA was divided and was either treated with T4-β-glucosyltransferase, which binds glucose groups selectively at 5hmC sites (red asterisk) and creates 5hgmC (open hexagon, left), or left untreated (H 2 O). Samples were digested with MspI (which does not digest 5hgmC) or left undigested (H 2 O), and the abundance of locus-specific DNA in each was compared by qPCR. In strategy II , unmodified (C) and modified CpG (5mC and 5hmC) levels were evaluated by HpaII digestion (right); DNA samples were treated with HpaII (which does not cut 5mC and 5hmC), left undigested (H 2 O), or treated with MspI (which cuts both and provides a positive control). The abundance of locus-specific DNA within each of these samples was estimated by qPCR and used to calculate the percentage of HpaII resistance. (B) Levels of 5hmC at OCT4 in hB cells before (0 hr), and 48 hr and 72 hr after fusion with mouse EGCs (black bars) or ESCs (white bars) are shown as the mean and SE of three to five independent experiments. (C) HpaII digestion analysis of OCT4 in hB lymphocytes before (0 hr) and 48 hr and 72 hr after fusion with EGCs (closed circles) or ESCs (open circles) are shown. Red bars mark the position of primer-amplified PCR products derived from the promoter (right) and downstream of the TSS (left), and values represent the mean and SE of three to five independent experiments. ∗∗ , p value

    Techniques Used: Real-time Polymerase Chain Reaction, Modification, Positive Control, Amplification, Polymerase Chain Reaction, Derivative Assay

    6) Product Images from "GADD45A Does Not Promote DNA Demethylation"

    Article Title: GADD45A Does Not Promote DNA Demethylation

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1000013

    Overexpression of GADD45A does not promote demethylation of a methylated pOct4-EGFP plasmid. A. Schematic diagram of the pOct4-EGFP plasmid (not to scale). The 2.4 kb Oct4 promoter sequences are upstream of the EGFP gene. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. This plasmid was methylated in vitro with HpaII or HpaII + HhaI. Black circles indicate HpaII sites and black triangles indicate HhaI sites. B. Confirmation of the methylation status of the six HpaII sites (black circles in panel A) by sodium bisulfite sequencing. Closed circles are methylated sites and open circles are unmethylated sites. C. Southern blot assay of plasmids recovered from HEK293 cells after transient transfection of the methylated and unmethylated pOct4-EGFP plasmid with and without GADD45A overexpression. The plasmids were digested with HincII and HpaII after in vitro methylation of the plasmid with HpaII (left panel) or HpaII and HhaI (right panel) prior to transfection. Digestion of the recovered methylated plasmid with HpaII does not lead to cleavage indicating that demethylation did not occur in HEK293 cells that overexpress GADD45A. D. Confirmation of GADD45A expression in transfected HEK293 cells by Western blotting with anti-GADD45A antibody. Beta-tubulin was probed as a loading control.
    Figure Legend Snippet: Overexpression of GADD45A does not promote demethylation of a methylated pOct4-EGFP plasmid. A. Schematic diagram of the pOct4-EGFP plasmid (not to scale). The 2.4 kb Oct4 promoter sequences are upstream of the EGFP gene. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. This plasmid was methylated in vitro with HpaII or HpaII + HhaI. Black circles indicate HpaII sites and black triangles indicate HhaI sites. B. Confirmation of the methylation status of the six HpaII sites (black circles in panel A) by sodium bisulfite sequencing. Closed circles are methylated sites and open circles are unmethylated sites. C. Southern blot assay of plasmids recovered from HEK293 cells after transient transfection of the methylated and unmethylated pOct4-EGFP plasmid with and without GADD45A overexpression. The plasmids were digested with HincII and HpaII after in vitro methylation of the plasmid with HpaII (left panel) or HpaII and HhaI (right panel) prior to transfection. Digestion of the recovered methylated plasmid with HpaII does not lead to cleavage indicating that demethylation did not occur in HEK293 cells that overexpress GADD45A. D. Confirmation of GADD45A expression in transfected HEK293 cells by Western blotting with anti-GADD45A antibody. Beta-tubulin was probed as a loading control.

    Techniques Used: Over Expression, Methylation, Plasmid Preparation, In Vitro, Methylation Sequencing, Southern Blot, Transfection, Expressing, Western Blot

    GADD45A does not promote demethylation of the endogenous pOct4 promoter in NIH3T3 cells. A. The transfection efficiency of NIH3T3 cells was analyzed with an EGFP expression vector and was calculated to be ∼60%. DIC, differential interference contrast image; GFP, green fluorescence. B. Schematic diagram of the mouse Oct4 promoter region. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. Black triangles indicate HpaII sites and the open triangle shows a HhaI site. C and D. Bisulfite sequence analysis of all CpGs in the Oct4 upstream region. Closed circles are methylated CpGs and open circles are unmethylated CpGs. C, control vector transfected cells; D, GADD45A expression vector transfected cells.
    Figure Legend Snippet: GADD45A does not promote demethylation of the endogenous pOct4 promoter in NIH3T3 cells. A. The transfection efficiency of NIH3T3 cells was analyzed with an EGFP expression vector and was calculated to be ∼60%. DIC, differential interference contrast image; GFP, green fluorescence. B. Schematic diagram of the mouse Oct4 promoter region. DE is the distal element, PE is the proximal element, and P is the minimal promoter of Oct4. Black triangles indicate HpaII sites and the open triangle shows a HhaI site. C and D. Bisulfite sequence analysis of all CpGs in the Oct4 upstream region. Closed circles are methylated CpGs and open circles are unmethylated CpGs. C, control vector transfected cells; D, GADD45A expression vector transfected cells.

    Techniques Used: Transfection, Expressing, Plasmid Preparation, Fluorescence, Sequencing, Methylation

    Bisulfite sequencing of the methylated pOct4-GFP plasmid recovered from HEK293 cells transfected in the absence or presence of the GADD45A expression vector. A. The plasmid was methylated with HpaII methylase only. B. The plasmid was methylated with HpaII and HhaI methylases. The recovered DNA was treated with sodium bisulfite and the pOct4-EGFP target sequences containing the six HpaII sites (see Figure 2A ) were amplified from bisulfite-treated DNA. The PCR products were cloned and between 22 and 25 individual molecules were sequenced. Closed circles mark methylated HpaII sites and open circles mark unmethylated HpaII sites. Overexpression of GADD45A (right panels) did not induce demethylation of the plasmid.
    Figure Legend Snippet: Bisulfite sequencing of the methylated pOct4-GFP plasmid recovered from HEK293 cells transfected in the absence or presence of the GADD45A expression vector. A. The plasmid was methylated with HpaII methylase only. B. The plasmid was methylated with HpaII and HhaI methylases. The recovered DNA was treated with sodium bisulfite and the pOct4-EGFP target sequences containing the six HpaII sites (see Figure 2A ) were amplified from bisulfite-treated DNA. The PCR products were cloned and between 22 and 25 individual molecules were sequenced. Closed circles mark methylated HpaII sites and open circles mark unmethylated HpaII sites. Overexpression of GADD45A (right panels) did not induce demethylation of the plasmid.

    Techniques Used: Methylation Sequencing, Methylation, Plasmid Preparation, Transfection, Expressing, Amplification, Polymerase Chain Reaction, Clone Assay, Over Expression

    7) Product Images from "DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells"

    Article Title: DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky947

    DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.
    Figure Legend Snippet: DNMT3L deficiency in mESCs results in hypomethylation at specific heterochromatin regions. ( A ) Mouse breeding strategies to generate mESC lines with different Dnmt3l genotypes. ( B and C ) Western blot analysis of the mESC lines (2 independent clones per genotype) for expression of DNMT3L (B) and the pluripotency factors OCT4 and SOX2 (C), with β-ACTIN serving as a loading control. ( D and E ) Southern blot analysis of the mESC lines (2-3 independent clones per genotype) for DNA methylation at the major satellite repeats (D) and the minor satellite repeats (E) after digestion of genomic DNA with methylation-sensitive restriction enzymes (MaeII for major satellite repeats and HpaII for minor satellite repeats). DKO, Dnmt3a/3b double KO mESC line.

    Techniques Used: Western Blot, Clone Assay, Expressing, Southern Blot, DNA Methylation Assay

    8) Product Images from "Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB"

    Article Title: Epigenetic Segregation of Microbial Genomes from Complex Samples Using Restriction Endonucleases HpaII and McrB

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0146064

    Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).
    Figure Legend Snippet: Enrichment workflow using HpaII. (A) Biotinylated HpaII enzyme is conjugated to streptavidin coated magnetic beads. A DNA mixture can then be added to the conjugated beads and following incubation the mixture is segregated into fractions that are bound (containing majority of Y . pestis ) or unbound (containing majority of human) to the beads. (B) Adding salt to the binding buffer enhances segregation of human (blue bars) from Y . pestis (green bars).

    Techniques Used: Magnetic Beads, Incubation, Binding Assay

    Genomic sequencing coverage of M . tuberculosis improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of M . tuberculosis .
    Figure Legend Snippet: Genomic sequencing coverage of M . tuberculosis improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of M . tuberculosis .

    Techniques Used: Genomic Sequencing

    HpaII mediated enrichment of DNA from a pooled sputum sample improves microbe sequencing detection and coverage. (A) The percent of microbial ID reads (blue) increases and human ID reads (red) decrease with enrichment. (B) Normalized microbial Order sequence Identification reads are plotted for bound and input samples. Greater than 95% of identified microbes have increased sequenced reads. Many microbes (red points) are only detectable after enrichment. (C) Comparison of ratio of microbial sequence ID reads in sputum input and sputum bound samples. (D) Genomic sequencing coverage of bacteria such as P . aeruginosa improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of P . aeruginosa .
    Figure Legend Snippet: HpaII mediated enrichment of DNA from a pooled sputum sample improves microbe sequencing detection and coverage. (A) The percent of microbial ID reads (blue) increases and human ID reads (red) decrease with enrichment. (B) Normalized microbial Order sequence Identification reads are plotted for bound and input samples. Greater than 95% of identified microbes have increased sequenced reads. Many microbes (red points) are only detectable after enrichment. (C) Comparison of ratio of microbial sequence ID reads in sputum input and sputum bound samples. (D) Genomic sequencing coverage of bacteria such as P . aeruginosa improves with enrichment. The input DNA sample coverage (red line) and HpaII bound coverage (blue line) are plotted across the genome position of P . aeruginosa .

    Techniques Used: Sequencing, Genomic Sequencing

    9) Product Images from "CpG-island promoters drive transcription of human telomeres"

    Article Title: CpG-island promoters drive transcription of human telomeres

    Journal: RNA

    doi: 10.1261/rna.1748309

    Cytosine methylation at 61-29-37 repeats. ( A,B ) Genomic DNA extracted from the indicated cell lines was digested with the methylation-sensitive HpaII restriction enzyme or with its methylation-insensitive isoschizomer MspI. Digested DNA was electrophoresed, blotted, and hybridized with radioactive DNA probes detecting 61-29-37 repeats. ( B ) DNMT1 −/− and DNMT3b − /− are HCT116-derived clonal cell lines knocked-out for DNA methyltransferases 1 and 3b, respectively. Double KO (DKO–) are HCT116-derived clonal cell lines knocked-out for both methyltransferases. Standard molecular weights are shown on the left of each blot in kilobases (kb).
    Figure Legend Snippet: Cytosine methylation at 61-29-37 repeats. ( A,B ) Genomic DNA extracted from the indicated cell lines was digested with the methylation-sensitive HpaII restriction enzyme or with its methylation-insensitive isoschizomer MspI. Digested DNA was electrophoresed, blotted, and hybridized with radioactive DNA probes detecting 61-29-37 repeats. ( B ) DNMT1 −/− and DNMT3b − /− are HCT116-derived clonal cell lines knocked-out for DNA methyltransferases 1 and 3b, respectively. Double KO (DKO–) are HCT116-derived clonal cell lines knocked-out for both methyltransferases. Standard molecular weights are shown on the left of each blot in kilobases (kb).

    Techniques Used: Methylation, Derivative Assay

    10) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.
    Figure Legend Snippet: Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.

    Techniques Used: Methylation, Mutagenesis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Isolation

    11) Product Images from "A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders"

    Article Title: A Set of Assays for the Comprehensive Analysis of FMR1 Alleles in the Fragile X–Related Disorders

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2016.06.001

    Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.
    Figure Legend Snippet: Analysis of the methylation status of the repeat region in DNA from carriers of normal (N), premutation (PM), and full mutation (FM) FMR1 . The resultant products were resolved on a 1% agarose gel. The + sign indicates those reactions that were predigested with HpaII in addition to HindIII. Molecular weight marker (MW)1 and MW2 are 100-bp and 1-Kb molecular weight ladders, respectively. A: Water (lane 1), DNA from a normal embryonic stem cell (ESC) line (H1) (lane 2), from SC120, a PM induced pluripotent stem cell (iPSC) line (lane 3), from SC128, a FM iPSC line (lane 4), and from two FM cell lines, GM04025 (lane 5) and GM09237 (lane 6). B: Analysis of DNA from a fragile X syndrome ESC WCMC37, MC37B, a subclone isolated from this cell line, and from two female (F) FM carriers F23856 and F23690. M, male.

    Techniques Used: Methylation, Mutagenesis, Agarose Gel Electrophoresis, Molecular Weight, Marker, Isolation

    Analysis of different ratios of methylated and unmethylated alleles using the qMS-PCR assay. DNA from a fully methylated full mutation carrier (GM09145) and a full unmethylated normal allele (GM06865) was mixed at different ratios before sonication. The DNA mixtures were then either mock digested or digested with HpaII and the amount of DNA corresponding to the 5′ end of the FMR1 . The percentage of uncut FMR1 DNA (ie, the percentage methylated) was then plotted as a function of the relative amount of the original methylated and unmethylated DNA (closed circles). Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ; open circles) was analyzed in parallel as a control for HpaII digestion.
    Figure Legend Snippet: Analysis of different ratios of methylated and unmethylated alleles using the qMS-PCR assay. DNA from a fully methylated full mutation carrier (GM09145) and a full unmethylated normal allele (GM06865) was mixed at different ratios before sonication. The DNA mixtures were then either mock digested or digested with HpaII and the amount of DNA corresponding to the 5′ end of the FMR1 . The percentage of uncut FMR1 DNA (ie, the percentage methylated) was then plotted as a function of the relative amount of the original methylated and unmethylated DNA (closed circles). Glyceraldehyde-3-phosphate dehydrogenase ( GAPDH ; open circles) was analyzed in parallel as a control for HpaII digestion.

    Techniques Used: Methylation, Polymerase Chain Reaction, Mutagenesis, Sonication

    12) Product Images from "Selective amplification of hypermethylated DNA from diverse tumor types via MSRE-PCR"

    Article Title: Selective amplification of hypermethylated DNA from diverse tumor types via MSRE-PCR

    Journal: Oncotarget

    doi: 10.18632/oncotarget.27825

    Methylation sensitive restriction sites in target cDMRs. Five MSREs are considered here: HhaI, HpaII, HpyCH4IV, AciI and BstUI. ( A ) Histogram of the number of MSRE sites in the target regions. ( B ) Scatterplot of the number of MSRE sites versus the number of CpGs in the target regions showing a linear relationship (R 2 = 0.89, p
    Figure Legend Snippet: Methylation sensitive restriction sites in target cDMRs. Five MSREs are considered here: HhaI, HpaII, HpyCH4IV, AciI and BstUI. ( A ) Histogram of the number of MSRE sites in the target regions. ( B ) Scatterplot of the number of MSRE sites versus the number of CpGs in the target regions showing a linear relationship (R 2 = 0.89, p

    Techniques Used: Methylation

    13) Product Images from "Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿"

    Article Title: Structure-Dependent Modulation of Alpha Interferon Production by Porcine Circovirus 2 Oligodeoxyribonucleotide and CpG DNAs in Porcine Peripheral Blood Mononuclear Cells ▿

    Journal: Journal of Virology

    doi: 10.1128/JVI.02797-06

    Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.
    Figure Legend Snippet: Southern blot analysis of low-molecular-weight DNA extracted from PCV2-infected PK15A cells. The methylation status of PCV2 DNA was studied using the RE isochizomer pairs HpaII/MspI and MboI/DpnI which differ in their sensitivity to CpG methylation as described in Materials and Methods. An asterisk indicates the specific RE of the pairs that is insensitive to methylation. Digestion with EcoRI was used as a control to linearize the PCV2 RF DNAs at a single site and provide a size reference. The positions of the linearized double-stranded RF of DNA (linearized dsDNA) and single-stranded covalently closed circular genomic DNA (circular ssDNA) are indicated by arrows.

    Techniques Used: Southern Blot, Molecular Weight, Infection, Methylation, CpG Methylation Assay

    14) Product Images from "High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers"

    Article Title: High-resolution genome-wide cytosine methylation profiling with simultaneous copy number analysis and optimization for limited cell numbers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp260

    In silico analysis of human HpaII/MspI fragments by length. The numbers of fragments computationally generated from the reference human genome sequence were plotted by length, demonstrating higher frequencies of shorter fragments. The three peaks observed (at 69, 135 and 204 bp) are due to the presence of Alu SINEs (mainly AluS and AluY), peaks that can also be observed in the ethidium bromide staining of the MspI reference representation in Figure 2 (b, c).
    Figure Legend Snippet: In silico analysis of human HpaII/MspI fragments by length. The numbers of fragments computationally generated from the reference human genome sequence were plotted by length, demonstrating higher frequencies of shorter fragments. The three peaks observed (at 69, 135 and 204 bp) are due to the presence of Alu SINEs (mainly AluS and AluY), peaks that can also be observed in the ethidium bromide staining of the MspI reference representation in Figure 2 (b, c).

    Techniques Used: In Silico, Generated, Sequencing, Staining

    HELP with limited amounts of DNA template: nanoHELP. To test whether HELP could be used with more limited quantities of DNA than previously attempted, we generated representations using 1 µg and 10 ng of material from the same DNA sample, using the PCR conditions shown ( a ). It is apparent that the conditions used generate representations from the 10-ng samples similar in quantity to those from the 1 µg sample. These representations were used for HELP microarray hybridization to test whether the smaller sample amounts reproduced the methylation profile of the 1 µg sample. HpaII/MspI log ratios were correlated for the 1 µg control and the three experimental replicates of 10 ng each. We found the limited sample quantities to generate profiles highly concordant with the 1 µg control, with R -values exceeding 0.96 in all cases ( b ). We conclude that HELP representations can be generated with DNA amounts equivalent to the yield from thousands of diploid mammalian cells.
    Figure Legend Snippet: HELP with limited amounts of DNA template: nanoHELP. To test whether HELP could be used with more limited quantities of DNA than previously attempted, we generated representations using 1 µg and 10 ng of material from the same DNA sample, using the PCR conditions shown ( a ). It is apparent that the conditions used generate representations from the 10-ng samples similar in quantity to those from the 1 µg sample. These representations were used for HELP microarray hybridization to test whether the smaller sample amounts reproduced the methylation profile of the 1 µg sample. HpaII/MspI log ratios were correlated for the 1 µg control and the three experimental replicates of 10 ng each. We found the limited sample quantities to generate profiles highly concordant with the 1 µg control, with R -values exceeding 0.96 in all cases ( b ). We conclude that HELP representations can be generated with DNA amounts equivalent to the yield from thousands of diploid mammalian cells.

    Techniques Used: Generated, Polymerase Chain Reaction, Microarray, Hybridization, Methylation

    The distribution of cytosine methylation by genomic compartment. The distribution of cytosine methylation using the new high-resolution genome-wide HELP microarray is categorized as hypomethylated (white), methylated (black) and intermediate (grey) based on thresholds categorising the bimodal distribution of HpaII/MspI ratios. The results show relative hypomethylation of CpG islands or CG clusters ( 18 ) whether at promoters, within gene bodies or intergenically compared with other sequences. This is consistent with expectations for the distribution of methylation and indicates that the assay is capable of defining methylation states in a range of genomic contexts.
    Figure Legend Snippet: The distribution of cytosine methylation by genomic compartment. The distribution of cytosine methylation using the new high-resolution genome-wide HELP microarray is categorized as hypomethylated (white), methylated (black) and intermediate (grey) based on thresholds categorising the bimodal distribution of HpaII/MspI ratios. The results show relative hypomethylation of CpG islands or CG clusters ( 18 ) whether at promoters, within gene bodies or intergenically compared with other sequences. This is consistent with expectations for the distribution of methylation and indicates that the assay is capable of defining methylation states in a range of genomic contexts.

    Techniques Used: Methylation, Genome Wide, Microarray

    Optimization of the HELP protocol for high-resolution representations. In ( a ) we show data representing the results of the two-adapter approach. Homologous ends using a single adapter can produce a hairpin structure in short fragments, an event that should be eliminated in 50% of fragments when using dual adapters to create heterologous ends. In ( b ) we demonstrate that the use of a single adapter (J, N) fails to create the smaller fragments seen when used together (JN, replicate experiments annotated as JN1, JN2). We also show that the type of buffer used, Mg 2+ concentration and betaine combine to improve representation and yield (b, c ). The original (red) and improved (blue) conditions for creating representations are compared in ( d ) as densitometric plots of gel electrophoreses, demonstrating the shift toward representation of PCR products of ≤100 bp. As the adapters contribute ∼50 bp to the size of the product, the representation of HpaII/MspI fragments in the genome includes those as small as 50 bp in this new protocol.
    Figure Legend Snippet: Optimization of the HELP protocol for high-resolution representations. In ( a ) we show data representing the results of the two-adapter approach. Homologous ends using a single adapter can produce a hairpin structure in short fragments, an event that should be eliminated in 50% of fragments when using dual adapters to create heterologous ends. In ( b ) we demonstrate that the use of a single adapter (J, N) fails to create the smaller fragments seen when used together (JN, replicate experiments annotated as JN1, JN2). We also show that the type of buffer used, Mg 2+ concentration and betaine combine to improve representation and yield (b, c ). The original (red) and improved (blue) conditions for creating representations are compared in ( d ) as densitometric plots of gel electrophoreses, demonstrating the shift toward representation of PCR products of ≤100 bp. As the adapters contribute ∼50 bp to the size of the product, the representation of HpaII/MspI fragments in the genome includes those as small as 50 bp in this new protocol.

    Techniques Used: Concentration Assay, Polymerase Chain Reaction

    Adaptation of the HELP assay to use massively parallel sequencing: HELP-seq. Instead of using microarrays to map the representations to their genomic source, we sequenced the ends of the fragments using massively parallel sequencing (Illumina). We show the promoter region of the KCNQ1 gene as an example of the concordant data obtained, with hypomethylated sites represented as an upward/positive peak and methylated as downward/negative, in both the microarray-based (HELP) and Illumina-based (HELP-seq) assays. The HELP data are represented by quantile-normalized HpaII/MspI log 2 ratios ( 13 ) while the HELP-seq data are represented by the number of HpaII reads per locus normalised to MspI reads for the same locus as described in the main text. Both embryonic stem (ES, red) and derived in vitro -differentiated erythroid progenitor (blue) cells are shown. The upstream (left) promoter of KCNQ1 is hypomethylated in both cell types, with a broader region of hypomethylation in erythroid compared with ES cells in both the HELP and HELP-seq studies. The downstream promoter appears to be methylated in ES cells in both assays, but microarray-based HELP weakly indicates hypomethylation in erythroid cells, whereas HELP-seq shows a strong hypomethylated signal in these cells. To determine which technique was more accurate, we performed validation using bisulphite MassArray (bottom), confirming the underlying hypomethylation of this locus in the erythroid and not ES cells, and demonstrating that HELP-seq offers improved capacity to identify sites of hypomethylated DNA in the genome.
    Figure Legend Snippet: Adaptation of the HELP assay to use massively parallel sequencing: HELP-seq. Instead of using microarrays to map the representations to their genomic source, we sequenced the ends of the fragments using massively parallel sequencing (Illumina). We show the promoter region of the KCNQ1 gene as an example of the concordant data obtained, with hypomethylated sites represented as an upward/positive peak and methylated as downward/negative, in both the microarray-based (HELP) and Illumina-based (HELP-seq) assays. The HELP data are represented by quantile-normalized HpaII/MspI log 2 ratios ( 13 ) while the HELP-seq data are represented by the number of HpaII reads per locus normalised to MspI reads for the same locus as described in the main text. Both embryonic stem (ES, red) and derived in vitro -differentiated erythroid progenitor (blue) cells are shown. The upstream (left) promoter of KCNQ1 is hypomethylated in both cell types, with a broader region of hypomethylation in erythroid compared with ES cells in both the HELP and HELP-seq studies. The downstream promoter appears to be methylated in ES cells in both assays, but microarray-based HELP weakly indicates hypomethylation in erythroid cells, whereas HELP-seq shows a strong hypomethylated signal in these cells. To determine which technique was more accurate, we performed validation using bisulphite MassArray (bottom), confirming the underlying hypomethylation of this locus in the erythroid and not ES cells, and demonstrating that HELP-seq offers improved capacity to identify sites of hypomethylated DNA in the genome.

    Techniques Used: HELP Assay, Sequencing, Methylation, Microarray, Derivative Assay, In Vitro

    15) Product Images from "High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model"

    Article Title: High-dose ascorbic acid synergizes with anti-PD1 in a lymphoma mouse model

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1908158117

    AA treatment leads to genomewide demethylation and enhanced endogenous retroviral expression in lymphoma cells. ( A ) Global methylation analysis was conducted using the HELP assay on OCI-Ly1 cells treated with control (Ctrl) or 1 mM AA for 6 h; cells harvested 24 h after treatment ( n = 2 replicates per group). Genomewide unsupervised clustering was performed on the resulting cytosine methylation data. Ward clustering shows global methylation changes are induced by AA treatment. The 2 AA-treated samples clustered separate from the 2 Ctrl-treated samples, with consistency between the 2 samples of each treatment. ( B ) Genomewide plot of HELP cytosine methylation (based on HpaII/MspI angle) in OCI-Ly1 Ctrl and AA samples. A reduction in the loci with cytosine methylation (shaded regions; HpaII/MSPI angle
    Figure Legend Snippet: AA treatment leads to genomewide demethylation and enhanced endogenous retroviral expression in lymphoma cells. ( A ) Global methylation analysis was conducted using the HELP assay on OCI-Ly1 cells treated with control (Ctrl) or 1 mM AA for 6 h; cells harvested 24 h after treatment ( n = 2 replicates per group). Genomewide unsupervised clustering was performed on the resulting cytosine methylation data. Ward clustering shows global methylation changes are induced by AA treatment. The 2 AA-treated samples clustered separate from the 2 Ctrl-treated samples, with consistency between the 2 samples of each treatment. ( B ) Genomewide plot of HELP cytosine methylation (based on HpaII/MspI angle) in OCI-Ly1 Ctrl and AA samples. A reduction in the loci with cytosine methylation (shaded regions; HpaII/MSPI angle

    Techniques Used: Expressing, Methylation, HELP Assay

    16) Product Images from "Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes"

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

    Journal: Journal of Virology

    doi: 10.1128/JVI.00736-17

    EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P
    Figure Legend Snippet: EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P

    Techniques Used: Expressing, DNA Methylation Assay, Binding Assay, Methylation, Negative Control, CpG Methylation Assay, Real-time Polymerase Chain Reaction

    17) Product Images from "Copper bis-Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove"

    Article Title: Copper bis-Dipyridoquinoxaline Is a Potent DNA Intercalator that Induces Superoxide-Mediated Cleavage via the Minor Groove

    Journal: Molecules

    doi: 10.3390/molecules24234301

    ( A ) Cartoon representation of enzyme restriction sites. ( B ) Control experiment with isoschizomers HpaII and MspI in the presence and absence of HpaII-MT. ( C ) A quantity of 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the absence of reductant. ( D ) 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the presence of reductant Na- L -asc.
    Figure Legend Snippet: ( A ) Cartoon representation of enzyme restriction sites. ( B ) Control experiment with isoschizomers HpaII and MspI in the presence and absence of HpaII-MT. ( C ) A quantity of 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the absence of reductant. ( D ) 400 ng of 798 bp linear sequence ( I non-methylated and II methylated) treated with Cu-DPQ in the presence of reductant Na- L -asc.

    Techniques Used: Sequencing, Methylation

    18) Product Images from "DNA Methylation Profile of β-1,3-Glucanase and Chitinase Genes in Flax Shows Specificity Towards Fusarium Oxysporum Strains Differing in Pathogenicity"

    Article Title: DNA Methylation Profile of β-1,3-Glucanase and Chitinase Genes in Flax Shows Specificity Towards Fusarium Oxysporum Strains Differing in Pathogenicity

    Journal: Microorganisms

    doi: 10.3390/microorganisms7120589

    Changes in DNA methylation pattern of β-glucanase 1 and chitinase in flax seedlings treated with non-pathogenic or pathogenic strains of Fusarium oxysporum and after sensitizing effects of the non-pathogenic strain treated with pathogenic Foln3 at 6, 12, 24, 36, and 48 h after inoculation presented as x-fold change in relation to control. The analysis of the third position in the exon, the first in the intron, and the third in the promoter of β-1,3-glucanase 1 and the first, seventh, and eighth positions in the exon of chitinase was performed by the digestion of genomic DNA by the restriction enzymes HpaII and MspI and then the real-time PCR reaction. The data represent the mean from three biological repetitions. Asterisks mark statistically significant differences ( p
    Figure Legend Snippet: Changes in DNA methylation pattern of β-glucanase 1 and chitinase in flax seedlings treated with non-pathogenic or pathogenic strains of Fusarium oxysporum and after sensitizing effects of the non-pathogenic strain treated with pathogenic Foln3 at 6, 12, 24, 36, and 48 h after inoculation presented as x-fold change in relation to control. The analysis of the third position in the exon, the first in the intron, and the third in the promoter of β-1,3-glucanase 1 and the first, seventh, and eighth positions in the exon of chitinase was performed by the digestion of genomic DNA by the restriction enzymes HpaII and MspI and then the real-time PCR reaction. The data represent the mean from three biological repetitions. Asterisks mark statistically significant differences ( p

    Techniques Used: DNA Methylation Assay, Real-time Polymerase Chain Reaction

    19) Product Images from "The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma"

    Article Title: The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

    Journal: JCI Insight

    doi: 10.1172/jci.insight.120422

    Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).
    Figure Legend Snippet: Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).

    Techniques Used: DNA Methylation Assay, Methylation, Marker

    20) Product Images from "CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons"

    Article Title: CGG-repeat dynamics and FMR1 gene silencing in fragile X syndrome stem cells and stem cell-derived neurons

    Journal: Molecular Autism

    doi: 10.1186/s13229-016-0105-9

    CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by
    Figure Legend Snippet: CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “ Methods ” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a . The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by

    Techniques Used: Methylation, Derivative Assay, Mass Spectrometry, Polymerase Chain Reaction, Agarose Gel Electrophoresis, DNA Methylation Assay

    Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells
    Figure Legend Snippet: Unmethylated FM alleles do not become silenced on differentiation into neurons. The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. A late passage culture of 37D ESCs containing little, if any methylated alleles, was differentiated into neurons as described in the “ Methods ” section. Methylation levels were measured by qMS-PCR on the indicated number of days after the initiation of neuronal differentiation. See Additional file 3 : Figure S3 for representative images during neuronal differentiation of 37D cells

    Techniques Used: Methylation, Polymerase Chain Reaction

    Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation
    Figure Legend Snippet: Selective growth advantage of cells carrying methylated FMR1 alleles with large CGG repeats. a – c The repeat size, methylation status, and FMR1 mRNA levels of the indicated cultures were monitored as described in the “ Methods ” section. The “+” and “−” signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. a , b Data for 37D and 37A lineages that were maintained in culture for extended periods of time. The DNA methylation status is indicated by the grey line and symbols in the right hand panel, and the mRNA level is indicated by the black line and symbols . c Growth of methylated 37A and unmethylated 37D cells. Late passage 37A cells that were completely methylated and late passage 37D cells that were unmethylated were either grown separately ( i ) or in a ~1:1 mixture ( ii ) for ~20 passages. S refers to the cells at the start of the experiment and E to the cells at the end of the experiment. Data for the mixed cultures are shown from two independent experiments (Rep 1 and Rep 2). Panel ( iii ) shows the DNA methylation for each set of cultures at the start and end of the experiment as an average from two experiments and the error bars indicate standard deviation

    Techniques Used: Methylation, DNA Methylation Assay, Standard Deviation

    21) Product Images from "How to Isolate a Plant's Hypomethylome in One Shot"

    Article Title: How to Isolate a Plant's Hypomethylome in One Shot

    Journal: BioMed Research International

    doi: 10.1155/2015/570568

    Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.
    Figure Legend Snippet: Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.

    Techniques Used:

    Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.
    Figure Legend Snippet: Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.

    Techniques Used:

    Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.
    Figure Legend Snippet: Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.

    Techniques Used: Filtration, Methylation

    22) Product Images from "Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function"

    Article Title: Distinct Roles of RNA Helicases MVH and TDRD9 in PIWI Slicing-Triggered Mammalian piRNA Biogenesis and Function

    Journal: Developmental Cell

    doi: 10.1016/j.devcel.2017.05.021

    Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.
    Figure Legend Snippet: Mouse TDRD9 Is an ATPase, and Its Activity Is Essential for Transposon Silencing, but Not for piRNA Biogenesis (A) Domain architecture of mouse TDRD9 with putative consensus amino acid residues responsible for ATP binding and ATP hydrolysis is shown. The point mutation E257Q that abolishes ATPase activity is indicated. (B) Quality of recombinant mouse TDRD9 protein used for ATPase assays. Wild-type and E257Q point mutant versions were produced. (C) Thin-layer chromatography of ATPase reactions revealing the faster-migrating free phosphate in the presence of the wild-type TDRD9 protein. (D) Creation of the catalytic-dead Tdrd9 knockin (KI) mouse carrying the E257Q mutation in the ATPase motif (DEVH → DQVH). The same mouse line also allows creation of the knockout (−/−) mutant, by using loxP sites flanking exons 3–5. See also Figure S6 . (E) Representative image of adult testes from indicated genotypes, showing atrophied testes in homozygous Tdrd9 knockout and knockin mutants. (F) H E staining of adult testes from homozygous Tdrd9 knockin mutant showing arrested germ cell development. Scale bar, 40 μm. See also Figure S7 A. (G) Northern analysis of transposons in total testicular RNA, showing derepression of LINE1 retrotransposons in homozygous Tdrd9 knockout and knockin mutants. Age of donor animals is indicated. (H) Western analysis of total testicular lysates for L1ORF1p expression. MILI (germ cell marker) and TUBULIN (loading control) expression was also examined. (I) Methylation-sensitive Southern blotting for LINE1 genomic loci. The red arrows point to fragments appearing under conditions of reduced DNA methylation in the homozygous Tdrd9 mutants. H, HpaII-digested DNA; M, MspI-digested DNA. (J) Immunoprecipitation of PIWI proteins and 5′ end labeling of associated small RNAs from neonatal (P0) testes. (K) Comparison of MILI-associated piRNAs mapping to individual repeats. There is a striking enrichment of the piRNAs produced from LINE and LTR repeats in Tdrd9 mutants ( Tdrd9 KI / KI and Tdrd9 − / − ). See also Figure S8 . (L) Graphs show the distribution of MIWI2-associated piRNAs mapped along B1Mus1.SINE consensus sequence, revealing a depletion of piRNAs in the Tdrd9 KI / KI and Tdrd9 − / − mutants. (M) Immunofluorescence analysis of indicated proteins in embryonic testes (embryonic day 16.5) of the different genotypes. Note the nucleo-cytoplasmic distribution of TDRD9 in wild-type germ cells, while it is restricted to the cytoplasm in the Tdrd9 KI / KI mutant.

    Techniques Used: Activity Assay, Binding Assay, Mutagenesis, Recombinant, Produced, Thin Layer Chromatography, Knock-In, Knock-Out, Staining, Northern Blot, Western Blot, Expressing, Marker, Methylation, Southern Blot, DNA Methylation Assay, Immunoprecipitation, End Labeling, Sequencing, Immunofluorescence

    Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.
    Figure Legend Snippet: Catalytic Activity of MVH Is Essential for Transposon Silencing and Biogenesis of MIWI2 piRNAs (A) Creation of the catalytic-dead Mvh mouse carrying a point mutation E446Q in the ATPase motif (DEAD → DQAD). See also Figure S2 . (B) Representative testes from adult animals (P80; 80 days old) of indicated Mvh genotypes. (C) Testes weight in different genotypes. (D and E) H E staining of adult mouse testes showing arrested germ cell development in the Mvh − / KI mutant (D), and (E) presence of sperm in the lumen of the wild-type epididymis, but not from that of the mutant. sp, spermatocytes; rs, round spermatids; es, elongated spermatids. Scale bars, 50 μm. (F) Staining for γ-H2AX in adult testes sections. Arrows point to the XY body. Scale bar, 10 μm. (G) Northern analysis for indicated transposon transcripts in total testicular RNA. The donor animals are numbered and their ages indicated. Total testicular DNA from the same animals were used for Southern blotting in (I). (H) Staining for L1ORF1p in mouse testes from animals of indicated ages. Scale bars, 38 μm (upper) and 48 μm (lower). (I) Methylation-sensitive Southern blotting examining L1 genomic loci. The donor animals are the same as those used for northern analysis (indicated by animal numbers). The red arrows point to the cleavage fragment seen under conditions of reduced DNA methylation, and only in the Mvh − / KI mutant. H, HpaII-digested DNA; M, MspI-digested DNA. (J and K) Immunoprecipitation of PIWI proteins from neonatal (P0) testes and 5′ end labeling of associated piRNAs. RNA size markers are 5′ end labeled (length in nucleotides). (L) Immunofluorescence detection of indicated proteins in neonatal testes Scale bar, 10 μm.

    Techniques Used: Activity Assay, Mutagenesis, Staining, Northern Blot, Southern Blot, Methylation, DNA Methylation Assay, Immunoprecipitation, End Labeling, Labeling, Immunofluorescence

    Related Articles

    Purification:

    Article Title: Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1
    Article Snippet: .. Genomic DNAs purified from testes were restriction digested with Eco RI and Bam HI (New England Biolabs) at 37°C for 4–6 h. DNA samples were then either restriction digested with Hpa II (New England Biolabs) for 4–6 h or methylated in vitro using Hpa II methyltransferase (New England Biolabs) and digested with Hpa II . .. Alternatively, the Eco RI - Bam HI -digested samples were further digested with Aci I , Fau I , or Hha I enzymes (New England Biolabs) for 4–6 h. Eight- to 10-μg aliquots of these processed samples were run on 1% agarose gels overnight and transferred overnight to Nytran N or Nytran N supercharged membranes (Whatman plc, Maidstone, Kent, U.K.) for Southern blot, as previously described [ ].

    Article Title: DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer
    Article Snippet: Inserts were then sub-cloned into the CpG-free vector, pCpGL-basic. .. Purified plasmids were methylated in vitro with M.Sss I, Hpa II, Hha I, or no enzyme (mock), in the presence of S-adenosyl methionine (all enzymes and co-factor obtained from NEB). .. Methylation efficiency was confirmed by digesting the methylated constructs using endonucleases Msp I, McrBC, Hha I, and Hpa II (NEB) (data not shown).

    Methylation:

    Article Title: Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1
    Article Snippet: .. Genomic DNAs purified from testes were restriction digested with Eco RI and Bam HI (New England Biolabs) at 37°C for 4–6 h. DNA samples were then either restriction digested with Hpa II (New England Biolabs) for 4–6 h or methylated in vitro using Hpa II methyltransferase (New England Biolabs) and digested with Hpa II . .. Alternatively, the Eco RI - Bam HI -digested samples were further digested with Aci I , Fau I , or Hha I enzymes (New England Biolabs) for 4–6 h. Eight- to 10-μg aliquots of these processed samples were run on 1% agarose gels overnight and transferred overnight to Nytran N or Nytran N supercharged membranes (Whatman plc, Maidstone, Kent, U.K.) for Southern blot, as previously described [ ].

    Article Title: DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer
    Article Snippet: Inserts were then sub-cloned into the CpG-free vector, pCpGL-basic. .. Purified plasmids were methylated in vitro with M.Sss I, Hpa II, Hha I, or no enzyme (mock), in the presence of S-adenosyl methionine (all enzymes and co-factor obtained from NEB). .. Methylation efficiency was confirmed by digesting the methylated constructs using endonucleases Msp I, McrBC, Hha I, and Hpa II (NEB) (data not shown).

    Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease
    Article Snippet: Genomic DNA and total RNA were copurified from isolated Tem using the AllPrep DNA/RNA mini kit (QIAGEN, Hilden, Germany), according to the manufacturer’s instructions. .. DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ]. .. A cocktail consisting of 1 μL each of three MSREs was diluted by 5 μL CutSmart Buffer (New England BioLabs) and RNase-free water in a total reaction volume of 50 μL, and 1 μg of DNA was digested at 37°C.

    In Vitro:

    Article Title: Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1
    Article Snippet: .. Genomic DNAs purified from testes were restriction digested with Eco RI and Bam HI (New England Biolabs) at 37°C for 4–6 h. DNA samples were then either restriction digested with Hpa II (New England Biolabs) for 4–6 h or methylated in vitro using Hpa II methyltransferase (New England Biolabs) and digested with Hpa II . .. Alternatively, the Eco RI - Bam HI -digested samples were further digested with Aci I , Fau I , or Hha I enzymes (New England Biolabs) for 4–6 h. Eight- to 10-μg aliquots of these processed samples were run on 1% agarose gels overnight and transferred overnight to Nytran N or Nytran N supercharged membranes (Whatman plc, Maidstone, Kent, U.K.) for Southern blot, as previously described [ ].

    Article Title: DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer
    Article Snippet: Inserts were then sub-cloned into the CpG-free vector, pCpGL-basic. .. Purified plasmids were methylated in vitro with M.Sss I, Hpa II, Hha I, or no enzyme (mock), in the presence of S-adenosyl methionine (all enzymes and co-factor obtained from NEB). .. Methylation efficiency was confirmed by digesting the methylated constructs using endonucleases Msp I, McrBC, Hha I, and Hpa II (NEB) (data not shown).

    Incubation:

    Article Title: 6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells
    Article Snippet: This results in a change in intensity of the bands on DNA gel electrophoresis optical analysis. .. DNA (200 ng) was incubated with Hpa II (New England BioLabs) or Msp I (New England BioLabs) along with supplied NE Buffer and nuclease-free water for one hour at 37°C. ..

    Article Title: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation
    Article Snippet: Reactions were incubated at 37°C for 2 h, and the DNA ethanol precipitated O/N at −20°C. .. DNA was resuspended and incubated with HpaII (1 U, NEB) in 1× CutSmart buffer (NEB) at 37°C for 1 h. DNA was ethanol precipitated O/N at −20°C, resuspended on glycerol loading buffer (0.5× TBE, 50% glycerol) and separated on a 8% native PAGE, and labelled DNA was detected using the blue fluorescence mode of the Typhoon 9400 (GE Healthcare) and analysed quantitatively by ImageQuant TL software (v7.0, GE Healthcare). .. PB generated XRCC1‐knockout cell lines, performed clonogenic and differentiation assays and immunoblot analyses of TDG and TDG‐SUMO in XRCC1null mESCs.

    Article Title: Highly sensitive detection of sentinel lymph node metastasis of breast cancer by digital PCR for RASSF1A methylation
    Article Snippet: Detection of RASSF1A methylation using RE-dMSP DNA was extracted from 100 µl SN lysate using the QIAamp Circulating Nucleic Acid Kit (Qiagen GmbH) and eluted in 50 µl desalted water. .. DNA solution (6.6 µl) was incubated for 16 h at 37°C in a final volume of 20 µl, containing 1X ddPCR Supermix for probes (Bio-Rad Laboratories, Inc.), 900 nM each primer, 250 nM probe and 10 U Hha I, Hpa II (New England BioLabs, Inc.) and Bst UI (Thermo Fisher Scientific, Inc.) each. .. These three methylation-sensitive restriction enzymes were selected since they can be used at the same incubation temperature (37°C).

    Clear Native PAGE:

    Article Title: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation
    Article Snippet: Reactions were incubated at 37°C for 2 h, and the DNA ethanol precipitated O/N at −20°C. .. DNA was resuspended and incubated with HpaII (1 U, NEB) in 1× CutSmart buffer (NEB) at 37°C for 1 h. DNA was ethanol precipitated O/N at −20°C, resuspended on glycerol loading buffer (0.5× TBE, 50% glycerol) and separated on a 8% native PAGE, and labelled DNA was detected using the blue fluorescence mode of the Typhoon 9400 (GE Healthcare) and analysed quantitatively by ImageQuant TL software (v7.0, GE Healthcare). .. PB generated XRCC1‐knockout cell lines, performed clonogenic and differentiation assays and immunoblot analyses of TDG and TDG‐SUMO in XRCC1null mESCs.

    Fluorescence:

    Article Title: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation
    Article Snippet: Reactions were incubated at 37°C for 2 h, and the DNA ethanol precipitated O/N at −20°C. .. DNA was resuspended and incubated with HpaII (1 U, NEB) in 1× CutSmart buffer (NEB) at 37°C for 1 h. DNA was ethanol precipitated O/N at −20°C, resuspended on glycerol loading buffer (0.5× TBE, 50% glycerol) and separated on a 8% native PAGE, and labelled DNA was detected using the blue fluorescence mode of the Typhoon 9400 (GE Healthcare) and analysed quantitatively by ImageQuant TL software (v7.0, GE Healthcare). .. PB generated XRCC1‐knockout cell lines, performed clonogenic and differentiation assays and immunoblot analyses of TDG and TDG‐SUMO in XRCC1null mESCs.

    Software:

    Article Title: SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation
    Article Snippet: Reactions were incubated at 37°C for 2 h, and the DNA ethanol precipitated O/N at −20°C. .. DNA was resuspended and incubated with HpaII (1 U, NEB) in 1× CutSmart buffer (NEB) at 37°C for 1 h. DNA was ethanol precipitated O/N at −20°C, resuspended on glycerol loading buffer (0.5× TBE, 50% glycerol) and separated on a 8% native PAGE, and labelled DNA was detected using the blue fluorescence mode of the Typhoon 9400 (GE Healthcare) and analysed quantitatively by ImageQuant TL software (v7.0, GE Healthcare). .. PB generated XRCC1‐knockout cell lines, performed clonogenic and differentiation assays and immunoblot analyses of TDG and TDG‐SUMO in XRCC1null mESCs.

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    New England Biolabs hpaii
    Melanoma is characterized by widespread changes in <t>DNA</t> methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every <t>HpaII-amplifiable</t> fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).
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    Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).

    Journal: JCI Insight

    Article Title: The RUNX1/IL-34/CSF-1R axis is an autocrinally regulated modulator of resistance to BRAF-V600E inhibition in melanoma

    doi: 10.1172/jci.insight.120422

    Figure Lengend Snippet: Melanoma is characterized by widespread changes in DNA methylation. ( A . ( B ) Volcano plot showing difference of mean methylation and statistical significance of the difference, with the number of differentially methylated loci indicated. Numbers to the left indicate hypermethylated loci, and numbers to the right indicate Differentially methylated regions (DMRs) in melanoma. An FDR of less than 5% is used as a marker of significant differences. ( C ) Global methylation levels tested by the LUMA assay indicate loss of methylation in malignant melanoma samples ( n = 36) (unpaired t test, 2-tailed). ( D and E ) Predominantly hypomethylated loci are seen in primary tumors and nodal and distant metastasis. The single blue dot (located in the right upper quadrant) in the volcano plots indicate the CSF1R locus. ( F ) The genomic position of every HpaII-amplifiable fragment on the HELP array was compared with the location of known CpG islands, demonstrating demethylated DMRs enriched outside of CpG-islands (proportions test). ( G ) Box plots of mean DNA copy number alterations (gains + losses) for melanoma sample clusters based on methylation (M1, M2, and M3) (1-way ANOVA). ( H ) Global methylation (%5mC) and hydroxymethylation (%5HmC) in melanocytes ( n = 3), melanoma tumors ( n = 4), and cells ( n = 3) (1-way ANOVA).

    Article Snippet: Genomic DNA (1 μg)was digested overnight with either HpaII or MspI (New England Biolabs).

    Techniques: DNA Methylation Assay, Methylation, Marker

    AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.

    Journal: The Journal of Clinical Investigation

    Article Title: Ascorbic acid–induced TET activation mitigates adverse hydroxymethylcytosine loss in renal cell carcinoma

    doi: 10.1172/JCI98747

    Figure Lengend Snippet: AA leads to increased TET activity and 5hmC levels in ccRCC cells. ( A ) Schematic showing the role of AA as an essential cofactor for TET enzymatic activity. ( B ) Intracellular L2HG levels measured by MS in ccRCC cell line 786-O are much higher than in the immortalized normal kidney cell line HKC8 ( n = 2). ( C and D ) TET activity was measured in vitro with AA-treated RCC cells (769-P and 786-O) and was increased after treatment. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). Exposure time was 4 hours, mimicking bioavailability curves with i.v. AA, followed by 24-hour incubation with fresh media prior to harvesting the cells for nuclear extraction and TET activity analysis. We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.025 (0.05/2 to account for multiple comparisons). ( E ) 5hmC was measured by LC-ESI-MS/MS and was significantly increased after AA treatment of RCC cells 769-P. Addition of catalase did not change the percentage of 5hmC. t test, P values as indicated. Data are shown as mean ± SEM with individual data points overlaid ( n = 2). We adjusted for multiple comparisons by dividing the significance level by the number of comparisons performed via Bonferroni’s correction. Hypotheses were deemed significant if P values were lower than 0.0125 (0.05/4 to account for multiple variations). ( F ) Unsupervised clustering based on genome-wide methylation analysis conducted by HELP assay. Ward clustering shows global methylation changes are induced by AA treatment. ( G ) Histograms based on methylation (log [HpaII/MspI]) show increased hypomethylation after AA treatment. ( H ) Smad6 promoter becomes demethylated after AA treatment in both 786-O and 769-P ccRCC cells.

    Article Snippet: One microgram of genomic DNA was digested overnight with either HpaII or MspI (NEB).

    Techniques: Activity Assay, Mass Spectrometry, In Vitro, Incubation, IF-P, Genome Wide, Methylation, HELP Assay

    Effect of DNA methylation on CT45 promoter activity. (A) Diagram of the CT45A2 promoter region and 2 constructs used for promoter activity assay. Black hash marks represent CpG sites. NCBI-predicted and RLM-RACE mapped TSS are indicated by the right broken arrows as in Fig. 3A . The two regions analyzed by pyrosequencing are indicated by black lines. The different methyltransferase acceptor sites are labeled in the legend. (B-C) Luciferase activity data for CT45 promoter constructs in (B) OVCAR3 and (C) A2780 EOC cell lines. Constructs were either mock-methylated or methylated with Hha I, Hpa II, or M.Sss I prior to transfection as described in Materials and Methods. CT45 firefly luciferase/renilla luciferase (control) enzymatic activity data are shown. Data represent mean ± SD.

    Journal: Epigenetics

    Article Title: DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer

    doi: 10.1080/15592294.2015.1062206

    Figure Lengend Snippet: Effect of DNA methylation on CT45 promoter activity. (A) Diagram of the CT45A2 promoter region and 2 constructs used for promoter activity assay. Black hash marks represent CpG sites. NCBI-predicted and RLM-RACE mapped TSS are indicated by the right broken arrows as in Fig. 3A . The two regions analyzed by pyrosequencing are indicated by black lines. The different methyltransferase acceptor sites are labeled in the legend. (B-C) Luciferase activity data for CT45 promoter constructs in (B) OVCAR3 and (C) A2780 EOC cell lines. Constructs were either mock-methylated or methylated with Hha I, Hpa II, or M.Sss I prior to transfection as described in Materials and Methods. CT45 firefly luciferase/renilla luciferase (control) enzymatic activity data are shown. Data represent mean ± SD.

    Article Snippet: Purified plasmids were methylated in vitro with M.Sss I, Hpa II, Hha I, or no enzyme (mock), in the presence of S-adenosyl methionine (all enzymes and co-factor obtained from NEB).

    Techniques: DNA Methylation Assay, Activity Assay, Construct, Labeling, Luciferase, Methylation, Transfection

    The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with Hpa II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II -methyltransferase then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.

    Journal: Biology of Reproduction

    Article Title: Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1

    doi: 10.1095/biolreprod.112.099879

    Figure Lengend Snippet: The Klf1 exon 2 CGI is unmethylated during development. A ) Schematic of the mouse Klf1 gene across exon 2 and the flanking introns shows salient restriction enzyme sites within the region and a 1.1-kbp cDNA probe used for Southern blot. B ) Genomic DNA samples from whole testis between P5 and P45 (lanes 1–7, left and center panels) and P45 spleen (lane 8). DNAs were purified and double-digested with Eco RI and Bam HI restriction enzymes to liberate the 1.85-kb genomic fragment containing the Klf1 exon 2 CGI. Thereafter, the DNA samples were further digested with Hpa II (left panel) or not digested (center panel) and then Southern blotted using the 1.1-kb probe shown in A . In addition, several samples were methylated using Hpa II -methyltransferase then digested with Hpa II (right panel) and Southern blotted. Because of the total number of samples in this experiment, three Southern blots were generated, and the relevant lanes were used to form composite panels. Data for the P5–P15 lanes (break in Southern blots) were obtained from separate Southern blots. Arrowheads in A , locations of Hpa II restriction sites; FL in B , full-length 1.85-kb Eco RI - Bam HI fragments that are visible in the center and right panels; Spl, spleen.

    Article Snippet: Genomic DNAs purified from testes were restriction digested with Eco RI and Bam HI (New England Biolabs) at 37°C for 4–6 h. DNA samples were then either restriction digested with Hpa II (New England Biolabs) for 4–6 h or methylated in vitro using Hpa II methyltransferase (New England Biolabs) and digested with Hpa II .

    Techniques: Southern Blot, Purification, Methylation, Generated