hpa ii  (New England Biolabs)


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    HpaII
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    HpaII 10 000 units
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    r0171l
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    10 000 units
    Category:
    Restriction Enzymes
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    New England Biolabs hpa ii
    HpaII
    HpaII 10 000 units
    https://www.bioz.com/result/hpa ii/product/New England Biolabs
    Average 95 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
    hpa ii - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs"

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs

    Journal: Archives Animal Breeding

    doi: 10.5194/aab-62-69-2019

    Methylation analysis of the ZPBP gene in whole blood samples of Berkshire sows by PMP assay. gDNAs were cut with the methylation-sensitive restriction enzymes Hpa II/ Msp I. M: size marker; U: undigested DNA; SLG: smaller litter size group; LLG: larger litter size group.
    Figure Legend Snippet: Methylation analysis of the ZPBP gene in whole blood samples of Berkshire sows by PMP assay. gDNAs were cut with the methylation-sensitive restriction enzymes Hpa II/ Msp I. M: size marker; U: undigested DNA; SLG: smaller litter size group; LLG: larger litter size group.

    Techniques Used: Methylation, Marker

    2) Product Images from "Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila"

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    Journal: Disease Models & Mechanisms

    doi: 10.1242/dmm.022350

    P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P
    Figure Legend Snippet: P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P

    Techniques Used: Expressing, Inhibition, LDH Cytotoxicity Assay, Concentration Assay, Activity Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Amplification, Western Blot, Mutagenesis

    Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P
    Figure Legend Snippet: Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P

    Techniques Used: Expressing, Sequencing, In Vitro, Binding Assay, Polymerase Chain Reaction, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, Construct, Chromatin Immunoprecipitation, DNA Methylation Assay, Plasmid Preparation, Methylation, Amplification, Activity Assay

    3) Product Images from "Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter"

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.21.22.7682-7695.2001

    Effects of DNA methylation on the translational positioning of nucleosomes on the human HPRT promoter in vitro. Nucleosomes were assembled in vitro onto methylated and unmethylated DNA templates containing the human HPRT promoter. Hpa II methylase, Hha I methylase, and Sss I methylase, DNA methyltransferases used to methylate each template; uncut, reconstituted chromatin that was digested with MNase but not Bam HI; Bam HI, reconstituted chromatin that was digested with MNase, purified, and then digested with Bam HI. All samples were probed with Bam HINuc1Probe, an 18-mer oligonucleotide immediately upstream of the Bam HI site in the first intron of the HPRT gene. Triangles indicate increasing MNase digestion times used to cleave the reconstituted chromatin. Numbers to the left, approximate sizes of the bands.
    Figure Legend Snippet: Effects of DNA methylation on the translational positioning of nucleosomes on the human HPRT promoter in vitro. Nucleosomes were assembled in vitro onto methylated and unmethylated DNA templates containing the human HPRT promoter. Hpa II methylase, Hha I methylase, and Sss I methylase, DNA methyltransferases used to methylate each template; uncut, reconstituted chromatin that was digested with MNase but not Bam HI; Bam HI, reconstituted chromatin that was digested with MNase, purified, and then digested with Bam HI. All samples were probed with Bam HINuc1Probe, an 18-mer oligonucleotide immediately upstream of the Bam HI site in the first intron of the HPRT gene. Triangles indicate increasing MNase digestion times used to cleave the reconstituted chromatin. Numbers to the left, approximate sizes of the bands.

    Techniques Used: DNA Methylation Assay, In Vitro, Methylation, Purification

    4) Product Images from "Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis"

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0009831

    Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.
    Figure Legend Snippet: Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.

    Techniques Used: Methylation, Expressing, Southern Blot, Hybridization, Molecular Weight, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Amplification, Genomic Sequencing

    5) Product Images from "Immediate Genetic and Epigenetic Changes in F1 Hybrids Parented by Species with Divergent Genomes in the Rice Genus (Oryza)"

    Article Title: Immediate Genetic and Epigenetic Changes in F1 Hybrids Parented by Species with Divergent Genomes in the Rice Genus (Oryza)

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0132911

    Examples of TE mobility, DNA methylation alterations, and transcriptional activation in the three F1 hybrids. (A) Southern blot hybridization patterns of three TEs in the three hybrids, respectively, along with the corresponding parents and their mix. The DNA samples were digested with Xba I and a pair of cytosine methylation-sensitive isoschizomers, Hpa II and Msp I, respectively. (a) Osr7 , which showed transpositional activation in several individuals of Hybrid 1; (b) Lullaby , which showed CG hypomethylation in several individuals of Hybrid 2; (c) Tos17 , which showed immobility in all individuals of Hybrid 3. Red arrow indicates novel bands, blue arrow indicates loss of bands. (B) Transcriptional activation of TEs in the three F1 hybrids based on semi-quentitative RT-PCR analysis. A cDNA sample from callus of Nipponbare (six-month old) was included as a positive control, red arrows indicate transcriptional activation in the hybrids relative to their parents and mix (the invitro
    Figure Legend Snippet: Examples of TE mobility, DNA methylation alterations, and transcriptional activation in the three F1 hybrids. (A) Southern blot hybridization patterns of three TEs in the three hybrids, respectively, along with the corresponding parents and their mix. The DNA samples were digested with Xba I and a pair of cytosine methylation-sensitive isoschizomers, Hpa II and Msp I, respectively. (a) Osr7 , which showed transpositional activation in several individuals of Hybrid 1; (b) Lullaby , which showed CG hypomethylation in several individuals of Hybrid 2; (c) Tos17 , which showed immobility in all individuals of Hybrid 3. Red arrow indicates novel bands, blue arrow indicates loss of bands. (B) Transcriptional activation of TEs in the three F1 hybrids based on semi-quentitative RT-PCR analysis. A cDNA sample from callus of Nipponbare (six-month old) was included as a positive control, red arrows indicate transcriptional activation in the hybrids relative to their parents and mix (the invitro "hybrids").

    Techniques Used: DNA Methylation Assay, Activation Assay, Southern Blot, Hybridization, Methylation, Reverse Transcription Polymerase Chain Reaction, Positive Control

    6) Product Images from "Lack of Association between the Serotonin Transporter (5-HTT) and Serotonin Receptor (5-HT2A) Gene Polymorphisms with Smoking Behavior among Malaysian Malays"

    Article Title: Lack of Association between the Serotonin Transporter (5-HTT) and Serotonin Receptor (5-HT2A) Gene Polymorphisms with Smoking Behavior among Malaysian Malays

    Journal: Scientia Pharmaceutica

    doi: 10.3797/scipharm.1406-01

    a: PCR products for amplification using 5-HT2A primers, b: PCR-RFLP result after digestion with Hpa II restriction enzyme. Lane 1, 2, 5, 6, 7, 8 show mutant allele with 342 bp. Lane 9 shows wild type allele with 126 and 216 bp fragments and Lane 3 and 4 show wild type-mutant alleles
    Figure Legend Snippet: a: PCR products for amplification using 5-HT2A primers, b: PCR-RFLP result after digestion with Hpa II restriction enzyme. Lane 1, 2, 5, 6, 7, 8 show mutant allele with 342 bp. Lane 9 shows wild type allele with 126 and 216 bp fragments and Lane 3 and 4 show wild type-mutant alleles

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis

    7) Product Images from "Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm"

    Article Title: Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

    Journal: Frontiers in Genetics

    doi: 10.3389/fgene.2017.00207

    Analyze the differentially amplified loci using polyacrylamide gel electrophoresis and silver staining. M, Msp I/ EcoR I digestion lanes; H, Hpa II/ EcoR I digestion lanes. A: Type I (1,1) bands; B: Type II (1,0) bands; C: Type III (0,1) bands; D: A hypermethylated DAL that the band type tended from Type I to type II. E: another hypermethylated DAL; F: A DNA methylation level unchanged DAL that the band type tended from type III to type II. Arrows indicated corresponding band types.
    Figure Legend Snippet: Analyze the differentially amplified loci using polyacrylamide gel electrophoresis and silver staining. M, Msp I/ EcoR I digestion lanes; H, Hpa II/ EcoR I digestion lanes. A: Type I (1,1) bands; B: Type II (1,0) bands; C: Type III (0,1) bands; D: A hypermethylated DAL that the band type tended from Type I to type II. E: another hypermethylated DAL; F: A DNA methylation level unchanged DAL that the band type tended from type III to type II. Arrows indicated corresponding band types.

    Techniques Used: Amplification, Polyacrylamide Gel Electrophoresis, Silver Staining, DNA Methylation Assay

    8) Product Images from "Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development"

    Article Title: Closely related proteins MBD2 and MBD3 play distinctive but interacting roles in mouse development

    Journal: Genes & Development

    doi: 10.1101/gad.194101

    Normal levels of DNA methylation in Mbd2 (−/−) animals. ( a ) The level of CpG methylation at Mbo I cut sites was determined in liver and spleen DNA derived from heterozygous (+/−) and Mbd2 -deficient (−/−) animals and expressed as a percentage of total CpG (±s.e.). ( b ) Tail DNA from Mbd2 -mutant (−/−) or wild-type (+/+) mice was digested with either Msp I or Hpa II, end-labeled with 32 P, and fractionated on an agarose gel. The HTF island fraction is indicated, and the region of each lane used for quantitation is boxed. A portion of each digest was Southern blotted and probed with a mitochondrial DNA sequence as a digestion control.
    Figure Legend Snippet: Normal levels of DNA methylation in Mbd2 (−/−) animals. ( a ) The level of CpG methylation at Mbo I cut sites was determined in liver and spleen DNA derived from heterozygous (+/−) and Mbd2 -deficient (−/−) animals and expressed as a percentage of total CpG (±s.e.). ( b ) Tail DNA from Mbd2 -mutant (−/−) or wild-type (+/+) mice was digested with either Msp I or Hpa II, end-labeled with 32 P, and fractionated on an agarose gel. The HTF island fraction is indicated, and the region of each lane used for quantitation is boxed. A portion of each digest was Southern blotted and probed with a mitochondrial DNA sequence as a digestion control.

    Techniques Used: DNA Methylation Assay, CpG Methylation Assay, Derivative Assay, Mutagenesis, Mouse Assay, Labeling, Agarose Gel Electrophoresis, Quantitation Assay, Sequencing

    9) Product Images from "C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD"

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-014-1286-y

    Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative
    Figure Legend Snippet: Hypermethylation of the C9orf72 promoter. a Cerebellar DNA from control ( n = 8, left ) and repeat-expanded cases ( n = 8, right ) were mock digested (no enzyme) or digested with Msp I, Hpa II or MspJ I. DNA was subject to repeat primed PCR and representative

    Techniques Used: Polymerase Chain Reaction

    10) Product Images from "DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation"

    Article Title: DNA Methylation Density Influences the Stability of an Epigenetic Imprint and Dnmt3a/b-Independent De Novo Methylation

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.21.7572-7580.2002

    Opposing influences of the 5′LTR enhancer and CpG methylation density on the probability of proviral silencing at a defined genomic site. (a) Principle of Cre-RMCE-mediated targeting of proviral DNA. A MEL cell line containing a stably integrated HYTK fusion gene flanked by inverted loxP sites (solid triangles) is transfected with a construct (also flanked by inverted loxP sites) together with a Cre recombinase expression plasmid. Recombination between the loxP sites in the two constructs results in exchange of the cassettes and thus loss of the TK selectable marker. Alternatively, recombination between the inverted loxP sites on the same DNA molecule occurs, resulting in the inversion of the intervening DNA. Cells that have undergone the latter recombination event still express the HYTK gene and thus can be selected against with ganciclovir, allowing isolation of cells that have undergone the targeting reaction. (b) GFP expression analysis of wild-type or Δenh cassettes introduced at the RL5 integration site by RMCE. RL5 MEL cells were transfected with wild-type (WT) or Δenh constructs, either unmethylated (−), methylated at low density (with Hpa II and Hha I MTases), or fully methylated (with Sss I MTase). Ganciclovir-resistant cells were pooled, and GFP expression was analyzed by flow cytometry. Results of a representative experiment are shown for each of the constructs introduced. The percentage of GFP-positive cells, as determined by electronic gating, is shown for each sample. Histograms of control untransfected MEL cells are also shown (grey lines). (c) The percentage (mean ± standard deviation) of cells expressing GFP was determined for three independent transfections of the low-density-methylated (gray histograms) or unmethylated (white histograms) wild-type and Δenh constructs.
    Figure Legend Snippet: Opposing influences of the 5′LTR enhancer and CpG methylation density on the probability of proviral silencing at a defined genomic site. (a) Principle of Cre-RMCE-mediated targeting of proviral DNA. A MEL cell line containing a stably integrated HYTK fusion gene flanked by inverted loxP sites (solid triangles) is transfected with a construct (also flanked by inverted loxP sites) together with a Cre recombinase expression plasmid. Recombination between the loxP sites in the two constructs results in exchange of the cassettes and thus loss of the TK selectable marker. Alternatively, recombination between the inverted loxP sites on the same DNA molecule occurs, resulting in the inversion of the intervening DNA. Cells that have undergone the latter recombination event still express the HYTK gene and thus can be selected against with ganciclovir, allowing isolation of cells that have undergone the targeting reaction. (b) GFP expression analysis of wild-type or Δenh cassettes introduced at the RL5 integration site by RMCE. RL5 MEL cells were transfected with wild-type (WT) or Δenh constructs, either unmethylated (−), methylated at low density (with Hpa II and Hha I MTases), or fully methylated (with Sss I MTase). Ganciclovir-resistant cells were pooled, and GFP expression was analyzed by flow cytometry. Results of a representative experiment are shown for each of the constructs introduced. The percentage of GFP-positive cells, as determined by electronic gating, is shown for each sample. Histograms of control untransfected MEL cells are also shown (grey lines). (c) The percentage (mean ± standard deviation) of cells expressing GFP was determined for three independent transfections of the low-density-methylated (gray histograms) or unmethylated (white histograms) wild-type and Δenh constructs.

    Techniques Used: CpG Methylation Assay, Stable Transfection, Transfection, Construct, Expressing, Plasmid Preparation, Marker, Isolation, Methylation, Flow Cytometry, Cytometry, Standard Deviation

    In vitro methylation and targeting of L1-MFGhGFP-1L. (a) Map of the L1-MFGhGFP-1L cassette, including the proviral LTRs, splice donor (SD), splice acceptor (SA), GFP gene, loxP sites (L1 and 1L), and flanking mouse genomic DNA. Primers specific for the 5′LTR (open arows ) and GFP coding sequence (solid arrows) used for bisulfite sequencing, and the GFP probe (black line) used for Southern analysis are also shown. Flanking plasmid DNA includes vector sequence flanking the L1-HyTK-1L vector at the RL5 integration site. CpG sites methylated in vitro with Sss I (194 sites) or Hpa II and Hha I (34 sites) methylases are also shown. (b) Map of the 5′LTRs of the wild-type and 5′ LTR enhancer deletion (Δenh) L1-MFGhGFP-1L constructs. The transcription start site (arrow), U3, R, and U5 regions are shown, along with the Nhe I and Xba I restriction sites used to generate the deletion. The Hpa II sites within the deleted tandem enhancer (solid squares) are also shown. (c) Methylation state of the unmethylated (−) or Hpa II and Hha I methylated (+) wild-type and Δenh constructs was determined by digestion with the methylation-sensitive restriction enzyme Hpa II (H) and the methylation-insensitive restriction enzyme Msp I (M). Digestion products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.
    Figure Legend Snippet: In vitro methylation and targeting of L1-MFGhGFP-1L. (a) Map of the L1-MFGhGFP-1L cassette, including the proviral LTRs, splice donor (SD), splice acceptor (SA), GFP gene, loxP sites (L1 and 1L), and flanking mouse genomic DNA. Primers specific for the 5′LTR (open arows ) and GFP coding sequence (solid arrows) used for bisulfite sequencing, and the GFP probe (black line) used for Southern analysis are also shown. Flanking plasmid DNA includes vector sequence flanking the L1-HyTK-1L vector at the RL5 integration site. CpG sites methylated in vitro with Sss I (194 sites) or Hpa II and Hha I (34 sites) methylases are also shown. (b) Map of the 5′LTRs of the wild-type and 5′ LTR enhancer deletion (Δenh) L1-MFGhGFP-1L constructs. The transcription start site (arrow), U3, R, and U5 regions are shown, along with the Nhe I and Xba I restriction sites used to generate the deletion. The Hpa II sites within the deleted tandem enhancer (solid squares) are also shown. (c) Methylation state of the unmethylated (−) or Hpa II and Hha I methylated (+) wild-type and Δenh constructs was determined by digestion with the methylation-sensitive restriction enzyme Hpa II (H) and the methylation-insensitive restriction enzyme Msp I (M). Digestion products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining.

    Techniques Used: In Vitro, Methylation, Sequencing, Methylation Sequencing, Plasmid Preparation, Construct, Agarose Gel Electrophoresis, Staining

    11) Product Images from "Tissue-specific variation in DNA methylation levels along human chromosome 1"

    Article Title: Tissue-specific variation in DNA methylation levels along human chromosome 1

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-2-7

    Fragment yield simulations . ( A ). The same methylation ratio (1:2) can give different measured ratios for BACs of different GC content. Horizontal lines represent the genomic region of a BAC in a single hybridizing sample. Sample 2 is twice as methylated as sample 1. The upper two rows represent a BAC of higher GC content than the lower rows. Vertical ticks represent unmethylated (digestable) Hpa II sites; check marks denote Hpa II fragments passing the size filter. Hpa II sites methylated in sample 2 but not sample 1 are marked with letters (see text). ( B ) Simulations based on BAC RP11-47A4. The amount of hybridizing material decreases with increasing methylation. ( C ) Simulation results for all array BACs. Contour plots summarize the amount of hybridizing material for all BACs at 40% methylation (black contours) and 80% (gray). ( D ) Predicted ratios when samples of 40% (test) and 80% (reference) methylation are co-hybridized. ( E ) A more complex scenario results in an opposite relationship between GC content and ratio. In this scenario, each BAC is simulated to report cross-hybridizing material in both test and reference channels equal to 5% of the number of bases of repetitive DNA it contains. ( F ) Real data for a selected array (male liver:spleen) show that, as predicted, the amount of hybridizing material is higher for higher GC content BACs (liver intensities, black contours; spleen, gray). ( G ) Measured ratios from the same array (liver:spleen) also have a strong relationship with GC content, as predicted if samples have different overall methylation levels. ( H ) GC-content-normalized ratios for the same array.
    Figure Legend Snippet: Fragment yield simulations . ( A ). The same methylation ratio (1:2) can give different measured ratios for BACs of different GC content. Horizontal lines represent the genomic region of a BAC in a single hybridizing sample. Sample 2 is twice as methylated as sample 1. The upper two rows represent a BAC of higher GC content than the lower rows. Vertical ticks represent unmethylated (digestable) Hpa II sites; check marks denote Hpa II fragments passing the size filter. Hpa II sites methylated in sample 2 but not sample 1 are marked with letters (see text). ( B ) Simulations based on BAC RP11-47A4. The amount of hybridizing material decreases with increasing methylation. ( C ) Simulation results for all array BACs. Contour plots summarize the amount of hybridizing material for all BACs at 40% methylation (black contours) and 80% (gray). ( D ) Predicted ratios when samples of 40% (test) and 80% (reference) methylation are co-hybridized. ( E ) A more complex scenario results in an opposite relationship between GC content and ratio. In this scenario, each BAC is simulated to report cross-hybridizing material in both test and reference channels equal to 5% of the number of bases of repetitive DNA it contains. ( F ) Real data for a selected array (male liver:spleen) show that, as predicted, the amount of hybridizing material is higher for higher GC content BACs (liver intensities, black contours; spleen, gray). ( G ) Measured ratios from the same array (liver:spleen) also have a strong relationship with GC content, as predicted if samples have different overall methylation levels. ( H ) GC-content-normalized ratios for the same array.

    Techniques Used: Methylation, BAC Assay

    Summary of bisulfite sequencing analyses of male heart (H) and spleen (S) DNA in the RYR2 gene sequence represented by BAC clone RP11-47A4 (AL391809) . More striking methylation differences between these tissues are observed at dispersed CpGs outside of the CpG island (top) than in the island (bottom). Each CpG analyzed by bisulfite sequencing is represented by a fraction in which the denominator is the total number of sequenced clones of PCR products amplified from bisulfite-converted DNA, and the numerator is the number of times methylation was detected at that site. Data for CpGs contained within Hpa II sites (CCGG) are shaded gray. The results for distributed CpG sites not included in CpG islands are indicated along the top, with their relative positions in the AL391809 sequence indicated. Vertical bars separate the sites on different PCR amplicons. The CpG island is highlighted by the gray vertical bar in the schematic of the AL391809 sequence and expanded below to indicate the position of exon 1 of RYR2 gene (white) and the GC boxes (dark gray). Two PCR products (P1 and P2) containing multiple CpGs within this CpG island were analyzed by bisulfite sequencing, and the results are indicated at the bottom of the figure. Boxes contain overall average methylation levels for heart (H) and spleen (S) for CpGs sampled outside of the CpG island (top) and for CpGs sampled within the CpG island (bottom).
    Figure Legend Snippet: Summary of bisulfite sequencing analyses of male heart (H) and spleen (S) DNA in the RYR2 gene sequence represented by BAC clone RP11-47A4 (AL391809) . More striking methylation differences between these tissues are observed at dispersed CpGs outside of the CpG island (top) than in the island (bottom). Each CpG analyzed by bisulfite sequencing is represented by a fraction in which the denominator is the total number of sequenced clones of PCR products amplified from bisulfite-converted DNA, and the numerator is the number of times methylation was detected at that site. Data for CpGs contained within Hpa II sites (CCGG) are shaded gray. The results for distributed CpG sites not included in CpG islands are indicated along the top, with their relative positions in the AL391809 sequence indicated. Vertical bars separate the sites on different PCR amplicons. The CpG island is highlighted by the gray vertical bar in the schematic of the AL391809 sequence and expanded below to indicate the position of exon 1 of RYR2 gene (white) and the GC boxes (dark gray). Two PCR products (P1 and P2) containing multiple CpGs within this CpG island were analyzed by bisulfite sequencing, and the results are indicated at the bottom of the figure. Boxes contain overall average methylation levels for heart (H) and spleen (S) for CpGs sampled outside of the CpG island (top) and for CpGs sampled within the CpG island (bottom).

    Techniques Used: Methylation Sequencing, Sequencing, BAC Assay, Methylation, Clone Assay, Polymerase Chain Reaction, Amplification

    Schematic of methylation profiling technique . DNA is harvested from samples ( 1 ) and digested with methylation-sensitive enzyme, Hpa II, which cuts at its CCGG target site only if the CpG is not methylated ( 2 ). The DNA is then size-fractionated on a sucrose gradient, followed by determination of fragment size by agarose gel electrophoresis in order to choose fractions containing fragments > approximately 80 bp and
    Figure Legend Snippet: Schematic of methylation profiling technique . DNA is harvested from samples ( 1 ) and digested with methylation-sensitive enzyme, Hpa II, which cuts at its CCGG target site only if the CpG is not methylated ( 2 ). The DNA is then size-fractionated on a sucrose gradient, followed by determination of fragment size by agarose gel electrophoresis in order to choose fractions containing fragments > approximately 80 bp and

    Techniques Used: Methylation, Agarose Gel Electrophoresis

    12) Product Images from "nc886, a non-coding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer"

    Article Title: nc886, a non-coding RNA of anti-proliferative role, is suppressed by CpG DNA methylation in human gastric cancer

    Journal: Oncotarget

    doi:

    nc886 expression suppression in gastric cancer cell lines through CpG DNA methylation A. Northern hybridization of nc886, vtRNA1-1, and 5S rRNA (control for equal loading) in indicated gastric cell lines. Molecular sizes from 5′- 32 P-labeled Decade markers (10 nt ladder) are indicated on the right. B. Diagram depicting a genomic region encoding nc886. A 649 nt DNA segment (also used in panel E-G) is illustrated in a double helix and nc886 RNA in a wavy line with an arrowhead indicating the direction of transcription. All nt coordinates are numbered referring to the 5'-end nt of nc886 RNA as +1. In the region for pyrosequencing, CpG dinucleotides are highlighted in grey and numbered consistently with Fig S3. A and B boxes (Pol III recognition elements) are also indicated. C. nc886 RNA expression and CpG DNA methylation levels. nc886RNA expression was measured by qRT-PCR. Ct values of each gene were converted to relative quantity (2 -Ct ) and normalized to 2 -Ct values from 18S rRNA. The value of HFE-145 was set as 1 (grey bars on the left y-axis). Percent methylation of cytosine at seven CpG sites (shown in Fig 2B , except for CpG #37) was measured by pyrosequencing. The seven values (shown in Fig S3) were averaged and plotted (black bars on the right y-axis). For both measurements, an average and the standard deviation were calculated from triplicate samples. D. Northern hybridization after treatment with 10 μM of AzadC. All other descriptions are the same as in panel A. E-G. Transfection of an in vitro methylated nc886 DNA fragment. The experimental scheme is illustrated in panel E. After M.SssI enzyme treatment (or no enzyme control), methylation of the 649-mer DNA (see panel B and E) was assured by digestion with methylation-sensitive restriction endonucleases Hha I/ Hpa II (panel F). “M” (lane 1) indicates molecular size markers whose sizes in nts are indicated on the left. “(-) ctrl DNA” (in panel G) denotes a 597-mer DNA fragment from an irrelevant gene, MKRN1. At 24 hrs after transfection of indicated DNA into MKN-01 cells (lane 2-4), nc886 and 5S rRNA were measured by Northern hybridization (panel G). Comparable transfection efficiencies were confirmed by a tRNA-Ser(mut) signal from the co-transfected plasmid “pCR-tRF1001_338(mut1)”. This signal was almost absent in the untransfected sample (lane 1).
    Figure Legend Snippet: nc886 expression suppression in gastric cancer cell lines through CpG DNA methylation A. Northern hybridization of nc886, vtRNA1-1, and 5S rRNA (control for equal loading) in indicated gastric cell lines. Molecular sizes from 5′- 32 P-labeled Decade markers (10 nt ladder) are indicated on the right. B. Diagram depicting a genomic region encoding nc886. A 649 nt DNA segment (also used in panel E-G) is illustrated in a double helix and nc886 RNA in a wavy line with an arrowhead indicating the direction of transcription. All nt coordinates are numbered referring to the 5'-end nt of nc886 RNA as +1. In the region for pyrosequencing, CpG dinucleotides are highlighted in grey and numbered consistently with Fig S3. A and B boxes (Pol III recognition elements) are also indicated. C. nc886 RNA expression and CpG DNA methylation levels. nc886RNA expression was measured by qRT-PCR. Ct values of each gene were converted to relative quantity (2 -Ct ) and normalized to 2 -Ct values from 18S rRNA. The value of HFE-145 was set as 1 (grey bars on the left y-axis). Percent methylation of cytosine at seven CpG sites (shown in Fig 2B , except for CpG #37) was measured by pyrosequencing. The seven values (shown in Fig S3) were averaged and plotted (black bars on the right y-axis). For both measurements, an average and the standard deviation were calculated from triplicate samples. D. Northern hybridization after treatment with 10 μM of AzadC. All other descriptions are the same as in panel A. E-G. Transfection of an in vitro methylated nc886 DNA fragment. The experimental scheme is illustrated in panel E. After M.SssI enzyme treatment (or no enzyme control), methylation of the 649-mer DNA (see panel B and E) was assured by digestion with methylation-sensitive restriction endonucleases Hha I/ Hpa II (panel F). “M” (lane 1) indicates molecular size markers whose sizes in nts are indicated on the left. “(-) ctrl DNA” (in panel G) denotes a 597-mer DNA fragment from an irrelevant gene, MKRN1. At 24 hrs after transfection of indicated DNA into MKN-01 cells (lane 2-4), nc886 and 5S rRNA were measured by Northern hybridization (panel G). Comparable transfection efficiencies were confirmed by a tRNA-Ser(mut) signal from the co-transfected plasmid “pCR-tRF1001_338(mut1)”. This signal was almost absent in the untransfected sample (lane 1).

    Techniques Used: Expressing, DNA Methylation Assay, Northern Blot, Hybridization, Labeling, RNA Expression, Quantitative RT-PCR, Methylation, Standard Deviation, Transfection, In Vitro, Plasmid Preparation

    13) Product Images from "DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer"

    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

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1062206

    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.
    Figure Legend 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.

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

    14) Product Images from "Genotyping of Porcine Endogenous Retroviruses from a Family of Miniature Swine"

    Article Title: Genotyping of Porcine Endogenous Retroviruses from a Family of Miniature Swine

    Journal: Journal of Virology

    doi: 10.1128/JVI.78.1.314-319.2004

    Genotyping of a miniature swine family by RFLP analysis of gag mRNA. (A) RT-PCR of PERV gag gene from PBMC of family members. (B) Hae III RFLP analysis of 35 S-labeled PERV gag RT-PCR products. (C) Hae III/ Hpa II RFLP analysis of gag RT-PCR products. (D) Nucleotide basis for Hpa II and Hae III RFLPs; clone 1.1, nonhumantropic PERV; clone 1.3, humantropic PERV with Hae III and Hpa II RFLP; clone 1.4, humantropic PERV with Hae III RFLP only. Restriction sites are indicated by the open box. Hae III site, GGCC; Hpa II site, CCGG.
    Figure Legend Snippet: Genotyping of a miniature swine family by RFLP analysis of gag mRNA. (A) RT-PCR of PERV gag gene from PBMC of family members. (B) Hae III RFLP analysis of 35 S-labeled PERV gag RT-PCR products. (C) Hae III/ Hpa II RFLP analysis of gag RT-PCR products. (D) Nucleotide basis for Hpa II and Hae III RFLPs; clone 1.1, nonhumantropic PERV; clone 1.3, humantropic PERV with Hae III and Hpa II RFLP; clone 1.4, humantropic PERV with Hae III RFLP only. Restriction sites are indicated by the open box. Hae III site, GGCC; Hpa II site, CCGG.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Labeling

    15) Product Images from "DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato"

    Article Title: DNA Methylation and Transcriptomic Changes in Response to Different Lights and Stresses in 7B-1 Male-Sterile Tomato

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0121864

    A representative MSAP gel using E2/H4 primer combination. W, B, R, and D correspond to white, blue, and red lights and dark, respectively. Letters H and M show Eco RI/ Hpa II and Eco RI/ Msp I enzymes combinations, respectively. Type I, II and III methylation profiles are indicated by arrows.
    Figure Legend Snippet: A representative MSAP gel using E2/H4 primer combination. W, B, R, and D correspond to white, blue, and red lights and dark, respectively. Letters H and M show Eco RI/ Hpa II and Eco RI/ Msp I enzymes combinations, respectively. Type I, II and III methylation profiles are indicated by arrows.

    Techniques Used: Methylation

    16) Product Images from "Genetic and epigenetic analyses of MBD3 in colon and lung cancer"

    Article Title: Genetic and epigenetic analyses of MBD3 in colon and lung cancer

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601776

    ( A ) MBD3 gene structure. ( B ) MBD3 CpG island Hpa II /Msp I restriction sites (CCGG), from Genbank AC005943.
    Figure Legend Snippet: ( A ) MBD3 gene structure. ( B ) MBD3 CpG island Hpa II /Msp I restriction sites (CCGG), from Genbank AC005943.

    Techniques Used:

    17) Product Images from "Tissue-Restricted Transcription from a Conserved Intragenic CpG Island in the Klf1 Gene in Mice 1"

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

    Journal: Biology of Reproduction

    doi: 10.1095/biolreprod.112.099879

    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.
    Figure Legend 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.

    Techniques Used: Southern Blot, Purification, Methylation, Generated

    18) Product Images from "Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination"

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp814

    Visualization of DNA circles from high molecular weight telomere-enriched DNA from stn1-M1 . ( A–E ) Electron micrographs of DNA circles observed in the telomere-enriched fractions from stn1-M1 . Circle lengths for the molecules in A–E, respectively, are estimated at 15.7, 12.1, 3.1, 1.2 and 1.2, and 0.9 kb for fractions A–E, respectively. Note that there is also a ∼0.8 kb linear fragment in the lower right portion of panel A. Samples were prepared by coating the DNA in denatured cytochrome c and are shown in negative contrast. Bar is equivalent to 1.5 kb. ( F ) Relative DNA abundance and telomeric DNA content of eluted fractions from gel chromatography separation of Alu I, Hpa II and Nla III dirgest of stn1-M1 genomic DNA. Molecules shown in A–E are from fractions 26 to 28 (F). Size distribution of the measured circles from fractions 26 to 27 ( n = 29) and 28 ( n = 32).
    Figure Legend Snippet: Visualization of DNA circles from high molecular weight telomere-enriched DNA from stn1-M1 . ( A–E ) Electron micrographs of DNA circles observed in the telomere-enriched fractions from stn1-M1 . Circle lengths for the molecules in A–E, respectively, are estimated at 15.7, 12.1, 3.1, 1.2 and 1.2, and 0.9 kb for fractions A–E, respectively. Note that there is also a ∼0.8 kb linear fragment in the lower right portion of panel A. Samples were prepared by coating the DNA in denatured cytochrome c and are shown in negative contrast. Bar is equivalent to 1.5 kb. ( F ) Relative DNA abundance and telomeric DNA content of eluted fractions from gel chromatography separation of Alu I, Hpa II and Nla III dirgest of stn1-M1 genomic DNA. Molecules shown in A–E are from fractions 26 to 28 (F). Size distribution of the measured circles from fractions 26 to 27 ( n = 29) and 28 ( n = 32).

    Techniques Used: Molecular Weight, Chromatography

    19) Product Images from "Three New Loci for Determining X Chromosome Inactivation Patterns"

    Article Title: Three New Loci for Determining X Chromosome Inactivation Patterns

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2011.05.003

    Fragment analysis results for the ZDHHC15 , SLITRK4 , and PCSK1N loci. Undigested (− Hpa II) or digested (+ Hpa II) DNA was amplified with PCR, using the respective primer sets for the three loci. The fragment analysis was performed with GeneMapper
    Figure Legend Snippet: Fragment analysis results for the ZDHHC15 , SLITRK4 , and PCSK1N loci. Undigested (− Hpa II) or digested (+ Hpa II) DNA was amplified with PCR, using the respective primer sets for the three loci. The fragment analysis was performed with GeneMapper

    Techniques Used: Amplification, Polymerase Chain Reaction

    20) Product Images from "CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression"

    Article Title: CpG Methylation of Human Papillomavirus Type 16 DNA in Cervical Cancer Cell Lines and in Clinical Specimens: Genomic Hypomethylation Correlates with Carcinogenic Progression

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.11.6227-6234.2003

    Hpa II/ Msp I cleavage identifies CpG methylation of SiHa and CaSki DNA as a likely source of transcriptional repression. (A) Distribution of CpGs in the LCR (positions 7154 to 7906 and 1 to 96) and in the E6 gene of HPV-16 (positions 97 to 559) and positions of two Hpa II/ Msp I sites, one (position 57) overlapping with elements of the E6 promoter P97 and the other located in the 3′ part of the E6 gene (position 502). (B) Reverse transcription-PCR confirms that similar amounts of E6 and E7 transcripts are generated by SiHa and CaSki cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, served as the cellular control transcript. (C) Genomic PCR confirms the large excess of HPV-16 DNA in CaSki cells. (D) Chromosomal DNA of SiHa and CaSki cells was not cut (CT, control) or was cleaved with one of the two enzymes (H, Hpa II; M, Msp I) and PCR amplified to generate the amplicons P2, P5, and P11. None of the three amplicons could be generated after cleavage with either of the two enzymes in the case of SiHa cells, indicating lack of any methylated CCGG sequences. In the case of CaSki cells, most of the DNA was resistant to Hpa II digestion but was readily cleaved by Msp I, indicating methylation of these two sites in most of the 500 HPV-16 copies in CaSki cells.
    Figure Legend Snippet: Hpa II/ Msp I cleavage identifies CpG methylation of SiHa and CaSki DNA as a likely source of transcriptional repression. (A) Distribution of CpGs in the LCR (positions 7154 to 7906 and 1 to 96) and in the E6 gene of HPV-16 (positions 97 to 559) and positions of two Hpa II/ Msp I sites, one (position 57) overlapping with elements of the E6 promoter P97 and the other located in the 3′ part of the E6 gene (position 502). (B) Reverse transcription-PCR confirms that similar amounts of E6 and E7 transcripts are generated by SiHa and CaSki cells. GAPDH, glyceraldehyde-3-phosphate dehydrogenase, served as the cellular control transcript. (C) Genomic PCR confirms the large excess of HPV-16 DNA in CaSki cells. (D) Chromosomal DNA of SiHa and CaSki cells was not cut (CT, control) or was cleaved with one of the two enzymes (H, Hpa II; M, Msp I) and PCR amplified to generate the amplicons P2, P5, and P11. None of the three amplicons could be generated after cleavage with either of the two enzymes in the case of SiHa cells, indicating lack of any methylated CCGG sequences. In the case of CaSki cells, most of the DNA was resistant to Hpa II digestion but was readily cleaved by Msp I, indicating methylation of these two sites in most of the 500 HPV-16 copies in CaSki cells.

    Techniques Used: CpG Methylation Assay, Polymerase Chain Reaction, Generated, Amplification, Methylation

    21) Product Images from "A Hypomethylated population of Brassica rapa for forward and reverse Epi-genetics"

    Article Title: A Hypomethylated population of Brassica rapa for forward and reverse Epi-genetics

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-12-193

    Effect of 5-Azac on epigenetic instability . Principal coordinate analysis (PCoA) based on the Euclidean analysis of methylation-sensitive amplified polymorphism (MSAP) assays using primer combinations H2/E1 and H3/E3. Plant lines are colour coded as shown in key, with square symbols ( Hpa II) and circles ( Msp I). a ) Main graphic, PCoA showing mean epigenetic distances for each of the groups; b ) Nested graphic, epigenetic distances for all the samples in lines BraRoAZ_12447e2 and BraRoAZ_12845s2. Euclidian space within circles represent intra-population variation (i.e. SSWP) for control line BraRoAZ_12845s2 using Hpa II (dashed) and Msp I (dotted). Arrow indicates the epigenetic distance between BraRoAZ_12845e2 samples restricted with Hpa II or Msp I (PhiPT).
    Figure Legend Snippet: Effect of 5-Azac on epigenetic instability . Principal coordinate analysis (PCoA) based on the Euclidean analysis of methylation-sensitive amplified polymorphism (MSAP) assays using primer combinations H2/E1 and H3/E3. Plant lines are colour coded as shown in key, with square symbols ( Hpa II) and circles ( Msp I). a ) Main graphic, PCoA showing mean epigenetic distances for each of the groups; b ) Nested graphic, epigenetic distances for all the samples in lines BraRoAZ_12447e2 and BraRoAZ_12845s2. Euclidian space within circles represent intra-population variation (i.e. SSWP) for control line BraRoAZ_12845s2 using Hpa II (dashed) and Msp I (dotted). Arrow indicates the epigenetic distance between BraRoAZ_12845e2 samples restricted with Hpa II or Msp I (PhiPT).

    Techniques Used: Methylation, Amplification

    22) Product Images from "Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii"

    Article Title: Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-9-21

    MSAP analysis of eight S. ruiz-lealii plants . A, representative MSAP profiles of two EcoR I/ Hpa II (H) and EcoR I/ Msp I (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as
    Figure Legend Snippet: MSAP analysis of eight S. ruiz-lealii plants . A, representative MSAP profiles of two EcoR I/ Hpa II (H) and EcoR I/ Msp I (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the Hpa II- Msp I isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. a Methylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.

    Techniques Used: Methylation

    23) Product Images from "Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients"

    Article Title: Hypermethylation of p16INK4a in Korean Non-small Cell Lung Cancer Patients

    Journal: Journal of Korean Medical Science

    doi: 10.3346/jkms.2007.22.S.S32

    Results of p16 INK4a gene promoter methylation of normal ( A ) and tumor ( B ) lung tissues by real-time PCR. 2, 3, 5: Normal lung tissues ( A ); 2 * , 3 * , 5 * : tumor lung tissues ( B ); 1, 4, 6: positive control (wi-38); 7: negative control (water); a: no-cut DNA amplification; b: Hpa II-cut DNA amplification; c: Msp I-cut DNA amplification. Delta Rn: the magnitude of the fluorescence signal generated during the PCR at each time point.
    Figure Legend Snippet: Results of p16 INK4a gene promoter methylation of normal ( A ) and tumor ( B ) lung tissues by real-time PCR. 2, 3, 5: Normal lung tissues ( A ); 2 * , 3 * , 5 * : tumor lung tissues ( B ); 1, 4, 6: positive control (wi-38); 7: negative control (water); a: no-cut DNA amplification; b: Hpa II-cut DNA amplification; c: Msp I-cut DNA amplification. Delta Rn: the magnitude of the fluorescence signal generated during the PCR at each time point.

    Techniques Used: Methylation, Real-time Polymerase Chain Reaction, Positive Control, Negative Control, Amplification, Fluorescence, Generated, Polymerase Chain Reaction

    24) Product Images from "Methylation analysis of the glypican 3 gene in embryonal tumours"

    Article Title: Methylation analysis of the glypican 3 gene in embryonal tumours

    Journal: British Journal of Cancer

    doi: 10.1038/sj.bjc.6601716

    Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in nontumoural samples. ( A ) CpG dinucleotides positions in the GPC3 promoter region. The methylation status of 11 of these CpG sites was determined either by the PCR-based methylation assay (#) or by the Southern blot-based methylation assay (+) using methyl-sensitive restriction endonucleases Hpa II (H), Sac II (S), Eag I (E), and Bss HII (B). Hpa II contains one CpG site, whereas Sac II, Eag I and Bss HII contain two CpG sites each. The distal and proximal sites were amplified in distinct PCRs. ( B and C ) Representative results of the methylation analysis in normal peripheral blood and placental DNA samples obtained by the PCR-based ( B ) and Southern blot-based ( C ) methylation assays. See Table 1 for details concerning the samples. ( D ) PCR-based methylation assay performed on DNA samples from two individuals (AC-1 and DC) affected by the Turner syndrome. Digestions: ( B and D ) H, Hpa II; M, Msp I; U, undigested; ( C ) H, Hind III; B, Bss HII; S, Sac II and E, Eag I. GPC3+, expression of GPC3; GPC3−, no expression of GPC3.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, Amplification, Expressing

    Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.
    Figure Legend Snippet: Methylation analysis of the GPC3 promoter in tumour cell DNA samples. PCR- ( A ) and Southern blot- ( B ) based methylation assays were performed on tumour cell DNA samples from NB cell lines (SK-N-AS, SK-N-SH), primary NBs (N4, N5) and primary WTs (WT51, WT116, WT158, WT177). Only results for samples with abnormal DNA methylation patterns are shown. Digestions: ( A ) H: Hpa II; M: Msp I; U: undigested; ( B ) H: Hind III; B: Bss HII; S: Sac II and E: Eag I.

    Techniques Used: Methylation, Polymerase Chain Reaction, Southern Blot, DNA Methylation Assay

    25) Product Images from "The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos"

    Article Title: The HIST1 Locus Escapes Reprogramming in Cloned Bovine Embryos

    Journal: G3: Genes|Genomes|Genetics

    doi: 10.1534/g3.115.026666

    Epigenetic regulation of HIST1 histone gene expression. (A) MSRE-PCR for analysis of DNA methylation at HIST1 histone gene promoters. Genomic DNAs obtained from blastocysts ( n = 10) of each group were first digested with either Hpa II or Msp I. This was followed by PCR using gene-specific primers. Both enzymes equally recognize the 5′-CCGG-3′ sequence, but Hpa II cannot function when the second cytosine is methylated. Stronger band intensity in the gel electrophoresis image indicates higher methylation level at the corresponding promoter. PCR was repeated twice for each gene promoter. Uncut, positive control; Msp I, negative control. (B) Trichostatin A (TSA)-mediated global gene expression increase in IVF and fSCNT blastocysts. Differentially expressed genes (DEGs; P
    Figure Legend Snippet: Epigenetic regulation of HIST1 histone gene expression. (A) MSRE-PCR for analysis of DNA methylation at HIST1 histone gene promoters. Genomic DNAs obtained from blastocysts ( n = 10) of each group were first digested with either Hpa II or Msp I. This was followed by PCR using gene-specific primers. Both enzymes equally recognize the 5′-CCGG-3′ sequence, but Hpa II cannot function when the second cytosine is methylated. Stronger band intensity in the gel electrophoresis image indicates higher methylation level at the corresponding promoter. PCR was repeated twice for each gene promoter. Uncut, positive control; Msp I, negative control. (B) Trichostatin A (TSA)-mediated global gene expression increase in IVF and fSCNT blastocysts. Differentially expressed genes (DEGs; P

    Techniques Used: Expressing, Polymerase Chain Reaction, DNA Methylation Assay, Sequencing, Methylation, Nucleic Acid Electrophoresis, Positive Control, Negative Control

    26) Product Images from "DNA from Protozoan Parasites Babesia bovis, Trypanosoma cruzi, and T. brucei Is Mitogenic for B Lymphocytes and Stimulates Macrophage Expression of Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide"

    Article Title: DNA from Protozoan Parasites Babesia bovis, Trypanosoma cruzi, and T. brucei Is Mitogenic for B Lymphocytes and Stimulates Macrophage Expression of Interleukin-12, Tumor Necrosis Factor Alpha, and Nitric Oxide

    Journal: Infection and Immunity

    doi: 10.1128/IAI.69.4.2162-2171.2001

    Unmethylated DNA is susceptible but methylated DNA is resistant to digestion with Hpa II. DNAs from E. coli, T. cruzi, T. brucei , and B. bovis were treated with Sss I methylase and tested for sensitivity to Hpa II. DNA was visualized following electrophoresis on ethidium bromide-agarose gels. Lanes: a and b, unmethylated DNA; c and d, methylated DNA. DNA in lanes a and c was not subjected to Hpa II digestion, whereas DNA in lanes b and d was subjected to Hpa II digestion.
    Figure Legend Snippet: Unmethylated DNA is susceptible but methylated DNA is resistant to digestion with Hpa II. DNAs from E. coli, T. cruzi, T. brucei , and B. bovis were treated with Sss I methylase and tested for sensitivity to Hpa II. DNA was visualized following electrophoresis on ethidium bromide-agarose gels. Lanes: a and b, unmethylated DNA; c and d, methylated DNA. DNA in lanes a and c was not subjected to Hpa II digestion, whereas DNA in lanes b and d was subjected to Hpa II digestion.

    Techniques Used: Methylation, Electrophoresis

    27) Product Images from "DNA hypomethylation-mediated activation of Cancer/Testis Antigen 45 (CT45) genes is associated with disease progression and reduced survival in epithelial ovarian cancer"

    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

    Journal: Epigenetics

    doi: 10.1080/15592294.2015.1062206

    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.
    Figure Legend 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.

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

    28) Product Images from "DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta"

    Article Title: DNA methylation patterns and gene expression associated with litter size in Berkshire pig placenta

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0184539

    Analysis of PRKG2 , CLCA4 , and PCK1 genes methylation in blood by PCR-based methylation assay. The y-axis shows the relative methylation levels of genes. The band shows DNA digested with Hpa II or Msp I. * Significant difference at p
    Figure Legend Snippet: Analysis of PRKG2 , CLCA4 , and PCK1 genes methylation in blood by PCR-based methylation assay. The y-axis shows the relative methylation levels of genes. The band shows DNA digested with Hpa II or Msp I. * Significant difference at p

    Techniques Used: Methylation, Polymerase Chain Reaction

    29) Product Images from "Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes"

    Article Title: Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    Journal: Frontiers in Plant Science

    doi: 10.3389/fpls.2017.02056

    Detailed MSAP-Seq assay overview (A) Genomic DNA is cleaved using rare cutter ( Eco RI) and methylation sensitive restriction enzyme ( Hpa II); only unmethylated recognition sites are digested by Hpa II. Then adapters specific to sticky ends are ligated and obtained fragments are amplified in PCR using selective primers; only fragments generated from unmethylated regions are amplified as they contain ends complementary to adapters. Products are then purified and fragmented by sonication to create shorter tags. (B) Purified fragments are used for standard library preparation involving following steps: end repair, adenylation, barcoded adapters ligation and purification, PCR amplification and purification. Then libraries quality and quantity is estimated. (C) Prepared libraries are pooled and processed thru cluster generation and high-throughput sequencing. (D) Sequencing data are analyzed using dedicated automatic pipeline—MSEQER. Firstly, reads are filtered for presence of Hpa II adapter and adapters are clipped. Then only reads containing CGG tags on the ends are mapped to the reference genome and functionally annotated. Obtained counts at each of the CCGG sites are normalized and differential methylation analysis among sets of samples is performed.
    Figure Legend Snippet: Detailed MSAP-Seq assay overview (A) Genomic DNA is cleaved using rare cutter ( Eco RI) and methylation sensitive restriction enzyme ( Hpa II); only unmethylated recognition sites are digested by Hpa II. Then adapters specific to sticky ends are ligated and obtained fragments are amplified in PCR using selective primers; only fragments generated from unmethylated regions are amplified as they contain ends complementary to adapters. Products are then purified and fragmented by sonication to create shorter tags. (B) Purified fragments are used for standard library preparation involving following steps: end repair, adenylation, barcoded adapters ligation and purification, PCR amplification and purification. Then libraries quality and quantity is estimated. (C) Prepared libraries are pooled and processed thru cluster generation and high-throughput sequencing. (D) Sequencing data are analyzed using dedicated automatic pipeline—MSEQER. Firstly, reads are filtered for presence of Hpa II adapter and adapters are clipped. Then only reads containing CGG tags on the ends are mapped to the reference genome and functionally annotated. Obtained counts at each of the CCGG sites are normalized and differential methylation analysis among sets of samples is performed.

    Techniques Used: Methylation, Amplification, Polymerase Chain Reaction, Generated, Purification, Sonication, Ligation, Next-Generation Sequencing, Sequencing

    30) Product Images from "Genome-Wide Demethylation Promotes Triplet Repeat Instability Independently of Homologous Recombination"

    Article Title: Genome-Wide Demethylation Promotes Triplet Repeat Instability Independently of Homologous Recombination

    Journal:

    doi: 10.1016/j.dnarep.2007.11.002

    Methylation analysis of the Aprt locus. A. Analysis of global methylation in GSAA5 cells. Genomic DNA was isolated from untreated cells and from cells treated with 5-aza-CdR, digested with Msp I (M) or its methyl-sensitive isoschizomer Hpa II (H), and displayed
    Figure Legend Snippet: Methylation analysis of the Aprt locus. A. Analysis of global methylation in GSAA5 cells. Genomic DNA was isolated from untreated cells and from cells treated with 5-aza-CdR, digested with Msp I (M) or its methyl-sensitive isoschizomer Hpa II (H), and displayed

    Techniques Used: Methylation, Isolation

    31) Product Images from "Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination"

    Article Title: Arabidopsis thaliana siRNA biogenesis mutants have the lower frequency of homologous recombination

    Journal: Plant Signaling & Behavior

    doi: 10.1080/15592324.2016.1151599

    DNA methylation levels in wt and mutant plants in response to MMS. Global genome methylation was analyzed using the cytosine extension assay. Genomic DNA was digested with methylation-sensitive enzymes, Hpa II (A) and Msp I (B), and the average 3 H incorporation
    Figure Legend Snippet: DNA methylation levels in wt and mutant plants in response to MMS. Global genome methylation was analyzed using the cytosine extension assay. Genomic DNA was digested with methylation-sensitive enzymes, Hpa II (A) and Msp I (B), and the average 3 H incorporation

    Techniques Used: DNA Methylation Assay, Mutagenesis, Methylation

    32) Product Images from "Mechanism of Transcriptional Regulation by Methyl-CpG Binding Protein MBD1"

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

    Journal: Molecular and Cellular Biology

    doi:

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

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

    Transcriptional regulation by MBD1 isoforms and their mutants with deletions of the methyl-CpG binding domain in mammalian CHO-K1 cells. Unmethylated or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with MBD1-expressing plasmids (pCGN-MBD1v1, pCGN-MBD1v3, pCGN-MBD1v1ΔN, and pCGN-MBD1v3ΔN) (0 to 1.0 μg) and insertless plasmid pCGN (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with 1.0 μg of pCGN (mock) was normalized to 10,000, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pRL-SV40 (0.1 μg). The combinations of pGL3-SNRPN and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (A), pGL3-SNRPN and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (B), pGL3-p16 and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (C), and pGL3-p16 and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (D) are shown.
    Figure Legend Snippet: Transcriptional regulation by MBD1 isoforms and their mutants with deletions of the methyl-CpG binding domain in mammalian CHO-K1 cells. Unmethylated or Hpa II-, Hha I-, or Sss I-methylated promoter-inserted pGL3 vector (0.5 μg) was cotransfected with MBD1-expressing plasmids (pCGN-MBD1v1, pCGN-MBD1v3, pCGN-MBD1v1ΔN, and pCGN-MBD1v3ΔN) (0 to 1.0 μg) and insertless plasmid pCGN (mock) (1.0 to 0 μg). The luciferase activity of unmethylated pGL3 in combination with 1.0 μg of pCGN (mock) was normalized to 10,000, and the relative luciferase activities (means + standard deviations [error bars]) were determined after correcting the transfection efficiency by pRL-SV40 (0.1 μg). The combinations of pGL3-SNRPN and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (A), pGL3-SNRPN and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (B), pGL3-p16 and pCGN-MBD1v1 or pCGN-MBD1v1ΔN (C), and pGL3-p16 and pCGN-MBD1v3 or pCGN-MBD1v3ΔN (D) are shown.

    Techniques Used: Binding Assay, Methylation, Plasmid Preparation, Expressing, Luciferase, Activity Assay, Transfection

    Effect of MBD1v1 mutants deleted of the CXXC domains on Sp1-activated transcription in Drosophila SL2 cells. (A) Four pAc5.1/V5-His plasmids expressing deletion mutants of MBD1v1 were designated MBD1v1Δ1 to MBD1v1Δ4. (B) Each of the plasmids for MBD1v1 deletion mutants (1.0 μg) was transfected into Drosophila cells together with pPacSp1 (0.5 μg), and either unmethylated (black bars) or Hpa II (gray bars)-, Hha I (white bars)-, or Sss I (hatched bars)-methylated pGL3-SNRPN (0.5 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100. Relative luciferase activities (means + standard deviations [error bars]) are given.
    Figure Legend Snippet: Effect of MBD1v1 mutants deleted of the CXXC domains on Sp1-activated transcription in Drosophila SL2 cells. (A) Four pAc5.1/V5-His plasmids expressing deletion mutants of MBD1v1 were designated MBD1v1Δ1 to MBD1v1Δ4. (B) Each of the plasmids for MBD1v1 deletion mutants (1.0 μg) was transfected into Drosophila cells together with pPacSp1 (0.5 μg), and either unmethylated (black bars) or Hpa II (gray bars)-, Hha I (white bars)-, or Sss I (hatched bars)-methylated pGL3-SNRPN (0.5 μg). The luciferase activity of unmethylated pGL3-SNRPN in combination with pPacSp1 and 1.0 μg of pAc5.1/V5-His (mock) was normalized to 100. Relative luciferase activities (means + standard deviations [error bars]) are given.

    Techniques Used: Expressing, Transfection, Methylation, Luciferase, Activity Assay

    33) Product Images from "Epigenetic rather than genetic factors may explain phenotypic divergence between coastal populations of diploid and tetraploid Limonium spp. (Plumbaginaceae) in Portugal"

    Article Title: Epigenetic rather than genetic factors may explain phenotypic divergence between coastal populations of diploid and tetraploid Limonium spp. (Plumbaginaceae) in Portugal

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-13-205

    Principal Coordinate Analysis (PCoA) representing genetic variability in Limonium nydeggeri populations. PCoA was based on presence/absence scores of 488 (primer I, E1/H1) (A) and 347 (primer II, E1/H3) (B) polymorphic loci obtained from MSAP profiles using isoschizomers Msp I (methylation insensitive) or Hpa II (methylation sensitive) as frequent cutters. The first two coordinates were extracted and plotted against each other.
    Figure Legend Snippet: Principal Coordinate Analysis (PCoA) representing genetic variability in Limonium nydeggeri populations. PCoA was based on presence/absence scores of 488 (primer I, E1/H1) (A) and 347 (primer II, E1/H3) (B) polymorphic loci obtained from MSAP profiles using isoschizomers Msp I (methylation insensitive) or Hpa II (methylation sensitive) as frequent cutters. The first two coordinates were extracted and plotted against each other.

    Techniques Used: Methylation

    Principal Coordinate Analysis (PCoA) representing genetic and epigenetic variability in diploid and tetraploid Limonium species. PCoA was based on presence/absence scores of 488 polymorphic loci obtained from MSAP profiles using isoschizomers Msp I (methylation insensitive - red symbols in A and B) or Hpa II (methylation sensitive - blue symbols in A and C) as frequent cutters and amplified with primers (E1/H3). The first two coordinates were extracted and plotted against each other. Percentage of the variability shown by each coordinate is indicated between parentheses. Diploid species are represented by solid symbols ( L. lanceolatum , triangles; L. nydeggeri , rhomboids; L. ovalifolium , rectangles) and tetraploid species are represented by empty symbols ( L. dodartii , triangles; L. multiflorum , rectangles; L. vulgare , circles).
    Figure Legend Snippet: Principal Coordinate Analysis (PCoA) representing genetic and epigenetic variability in diploid and tetraploid Limonium species. PCoA was based on presence/absence scores of 488 polymorphic loci obtained from MSAP profiles using isoschizomers Msp I (methylation insensitive - red symbols in A and B) or Hpa II (methylation sensitive - blue symbols in A and C) as frequent cutters and amplified with primers (E1/H3). The first two coordinates were extracted and plotted against each other. Percentage of the variability shown by each coordinate is indicated between parentheses. Diploid species are represented by solid symbols ( L. lanceolatum , triangles; L. nydeggeri , rhomboids; L. ovalifolium , rectangles) and tetraploid species are represented by empty symbols ( L. dodartii , triangles; L. multiflorum , rectangles; L. vulgare , circles).

    Techniques Used: Methylation, Amplification

    34) Product Images from "6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells"

    Article Title: 6-Thioguanine and zebularine down-regulate DNMT1 and globally demethylate canine malignant lymphoid cells

    Journal: BMC Veterinary Research

    doi: 10.1186/s12917-014-0290-8

    DNA gel electrophoresis showing endonuclease activity in restriction landmark genomic scanning. Lanes: Lad = ladder, 1 = Control, 2 = Control + Hpa, 3 = Control + Msp, 4 = 6TG, 5 = 6TG + Hpa, 6 = Hpa + Msp, 7 = Zeb, 8 = Zeb + Hpa, 9 = Zeb + Msp. 1 kb pair DNA ladders were loaded into lane 1, followed by control, Hpa II, and Msp I incubated extracted DNA from the lymphoma cell line for low and high doses of 6-TG and Zeb respectively. The MSV equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$ \left(\mathrm{M}\mathrm{S}\mathrm{V}=\frac{\mathrm{Densit}{\mathrm{y}}_{\mathrm{HpaII}}\hbox{-}\ \mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}{\mathrm{Densit}{\mathrm{y}}_{\mathrm{MspI}}\hbox{-}\ \mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}\right) $$ \end{document} M S V = Densit y HpaII ‐ Densit y Background Densit y MspI ‐ Densit y Background was used for each treatment subset. Densities were calculated at the 2.0-2.3 kb pair region. Boxes shown in the first three lanes demonstrate the region of analysis.
    Figure Legend Snippet: DNA gel electrophoresis showing endonuclease activity in restriction landmark genomic scanning. Lanes: Lad = ladder, 1 = Control, 2 = Control + Hpa, 3 = Control + Msp, 4 = 6TG, 5 = 6TG + Hpa, 6 = Hpa + Msp, 7 = Zeb, 8 = Zeb + Hpa, 9 = Zeb + Msp. 1 kb pair DNA ladders were loaded into lane 1, followed by control, Hpa II, and Msp I incubated extracted DNA from the lymphoma cell line for low and high doses of 6-TG and Zeb respectively. The MSV equation \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$ \left(\mathrm{M}\mathrm{S}\mathrm{V}=\frac{\mathrm{Densit}{\mathrm{y}}_{\mathrm{HpaII}}\hbox{-}\ \mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}{\mathrm{Densit}{\mathrm{y}}_{\mathrm{MspI}}\hbox{-}\ \mathrm{Densit}{\mathrm{y}}_{\mathrm{Background}}}\right) $$ \end{document} M S V = Densit y HpaII ‐ Densit y Background Densit y MspI ‐ Densit y Background was used for each treatment subset. Densities were calculated at the 2.0-2.3 kb pair region. Boxes shown in the first three lanes demonstrate the region of analysis.

    Techniques Used: DNA Gel Electrophoresis, Activity Assay, Restriction Landmark Genomic Scanning, Incubation

    35) Product Images from "Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino"

    Article Title: Morphological, Genome and Gene Expression Changes in Newly Induced Autopolyploid Chrysanthemum lavandulifolium (Fisch. ex Trautv.) Makino

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17101690

    Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.
    Figure Legend Snippet: Representative variation in MSAP profiles. “→” to red arrows represents variation in DNA methylation between diploid and tetraploid plants; “+” represents fragments obtained after digestion with EcoR I or Hpa II/ Msp I; “−” represents fragments not digested by EcoR I or Hpa II/ Msp I; Type I fragments are nonmethylated and were presented in both the H ( EcoR I or Hpa II digest) and M ( EcoR I or Msp I digest) lanes; Type II are fully methylated and only appeared in the M lanes; Type III are hemimethylated and appeared in the H lanes; Type IV were fragments absent from both H and M lanes in diploid but present in either H or M lane of tetraploid, and vice versa.

    Techniques Used: DNA Methylation Assay, Methylation

    36) Product Images from "Angiogenin stimulates ribosomal RNA transcription by epigenetic activation of the ribosomal DNA promoter"

    Article Title: Angiogenin stimulates ribosomal RNA transcription by epigenetic activation of the ribosomal DNA promoter

    Journal: Journal of cellular physiology

    doi: 10.1002/jcp.24477

    ANG knockdown decreases the ratio of active to silent rDNA genes. ChIP input DNA from the control and ANG knockdown cells were digested with Hpa II. qPCR reactions were carried out on both mock- and Hpa II-digested DNA. The value from Hpa II-digested DNA represents the level of silent chromatin ( Hpa II resistant, black bar). The value from mock-digested DNA represents the total input DNA. The difference between mock- and Hpa II-digested DNA represents the level of active chromatinn ( Hpa II sensitive, light bar).
    Figure Legend Snippet: ANG knockdown decreases the ratio of active to silent rDNA genes. ChIP input DNA from the control and ANG knockdown cells were digested with Hpa II. qPCR reactions were carried out on both mock- and Hpa II-digested DNA. The value from Hpa II-digested DNA represents the level of silent chromatin ( Hpa II resistant, black bar). The value from mock-digested DNA represents the total input DNA. The difference between mock- and Hpa II-digested DNA represents the level of active chromatinn ( Hpa II sensitive, light bar).

    Techniques Used: Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction

    37) Product Images from "Kaiso mainly locates in the nucleus in vivo and binds to methylated, but not hydroxymethylated DNA"

    Article Title: Kaiso mainly locates in the nucleus in vivo and binds to methylated, but not hydroxymethylated DNA

    Journal: Chinese Journal of Cancer Research

    doi: 10.3978/j.issn.1000-9604.2015.04.03

    Kaiso cannot bind to the hydroxymethylated CGCG-containing CDKN2A sequence in cultured HCT116 cells. (A) Illustration of the strategy to detect methylation and hydroxymethylation in the CGCG-containing DNA sequence in the combined glucosylation- Msp I/ Hpa II-PCR assay. PCR products can be successfully amplified from the Msp I-digested hmC-containing DNA sequences after the glucosylation treatment, but not before the treatment. PCR products could be successfully amplified from the Hpa II-digested mC- or hmC-containing DNA sequences before and after the glucosylation treatment; (B) The CDKN2A promoter templates present in the Kasio-IPed chromatin in the CDKN2A -hemimethylated HCT116 cells. The unmethylated-KBS-containing CCND1 templates are also detectable. However, the unmethylated-CGCG-containing GAPDH promoter is not detectable; (C) Hpa II digests only part of CDKN2A templates in the Kaiso-IP chromatin in HCT116 cells, but it digests all of CDKN2A templates in the input control chromatin in the CDKN2A -unmethylated MGC803 cells; (D) The CDKN2A template is amplified in the glucosylated and Msp I-digested input control chromatin. However, the CDKN2A template is not amplified in the glucosylated and Msp I-digested Kaiso-IPed chromatin (bottom image), indicating that Kaiso cannot bind to hmC in the CDKN2A promoter.
    Figure Legend Snippet: Kaiso cannot bind to the hydroxymethylated CGCG-containing CDKN2A sequence in cultured HCT116 cells. (A) Illustration of the strategy to detect methylation and hydroxymethylation in the CGCG-containing DNA sequence in the combined glucosylation- Msp I/ Hpa II-PCR assay. PCR products can be successfully amplified from the Msp I-digested hmC-containing DNA sequences after the glucosylation treatment, but not before the treatment. PCR products could be successfully amplified from the Hpa II-digested mC- or hmC-containing DNA sequences before and after the glucosylation treatment; (B) The CDKN2A promoter templates present in the Kasio-IPed chromatin in the CDKN2A -hemimethylated HCT116 cells. The unmethylated-KBS-containing CCND1 templates are also detectable. However, the unmethylated-CGCG-containing GAPDH promoter is not detectable; (C) Hpa II digests only part of CDKN2A templates in the Kaiso-IP chromatin in HCT116 cells, but it digests all of CDKN2A templates in the input control chromatin in the CDKN2A -unmethylated MGC803 cells; (D) The CDKN2A template is amplified in the glucosylated and Msp I-digested input control chromatin. However, the CDKN2A template is not amplified in the glucosylated and Msp I-digested Kaiso-IPed chromatin (bottom image), indicating that Kaiso cannot bind to hmC in the CDKN2A promoter.

    Techniques Used: Sequencing, Cell Culture, Methylation, Polymerase Chain Reaction, Amplification

    38) Product Images from "Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii"

    Article Title: Phenotypic instability and epigenetic variability in a diploid potato of hybrid origin, Solanum ruiz-lealii

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-9-21

    MSAP analysis of eight S. ruiz-lealii plants . A, representative MSAP profiles of two EcoR I/ Hpa II (H) and EcoR I/ Msp I (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as
    Figure Legend Snippet: MSAP analysis of eight S. ruiz-lealii plants . A, representative MSAP profiles of two EcoR I/ Hpa II (H) and EcoR I/ Msp I (M) digest of DNA extracted from eight S. ruiz-lealii plants. The primer combinations used were E-AGA/HM-TCAA (left panel) and E-AGA/HM-TCCA (right panel). The arrows indicate positions of size markers. B, detail of the primer combination E-AGA/HM-TCCA. Arrows heads, fragments analyzed as "methylation sensitive polymorphism". Arrow, fragment analyzed as "methylation insensitive polymorphism". C, graphical interpretation of methylation sensitive fragments. The boxes represent the double-stranded recognition site (CCGG) of the Hpa II- Msp I isoschizomer. Black boxes indicate methylated cytosine. Fragments 1 and 2 epialleles present in plants with normal and intermediate flower phenotype. Fragments 3 and 4, specific epialleles of plants with abnormal flower phenotype. Fragments 5 and 6, methylated epialleles present in three plants with abnormal flower phenotype and in plant 6, with intermediate flower phenotype; and demethylated epialleles specific of plants with normal flower phenotype. a Methylation patterns not determined, because the absence of a MSAP fragment can result from either a full methylation of cytosines on both strands or the absence of the restriction sites.

    Techniques Used: Methylation

    39) Product Images from "Highly sensitive detection of sentinel lymph node metastasis of breast cancer by digital PCR for RASSF1A methylation"

    Article Title: Highly sensitive detection of sentinel lymph node metastasis of breast cancer by digital PCR for RASSF1A methylation

    Journal: Oncology Reports

    doi: 10.3892/or.2019.7363

    Schematic of the primers and probe used for RE-dMSP of the RASSF1A promoter. The location of the primers and probe, and the recognition sites of three methylation-sensitive restriction enzymes ( Hpa II, Hha I and Bst UI) are presented. RE-dMSP, PCR with methylation-specific restriction enzymes; RASSF1A, Ras association domain-containing protein 1.
    Figure Legend Snippet: Schematic of the primers and probe used for RE-dMSP of the RASSF1A promoter. The location of the primers and probe, and the recognition sites of three methylation-sensitive restriction enzymes ( Hpa II, Hha I and Bst UI) are presented. RE-dMSP, PCR with methylation-specific restriction enzymes; RASSF1A, Ras association domain-containing protein 1.

    Techniques Used: Methylation, Polymerase Chain Reaction

    40) Product Images from "Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region"

    Article Title: Prolonged transcriptional silencing and CpG methylation induced by siRNAs targeted to the HIV-1 promoter region

    Journal: Journal of RNAi and Gene Silencing : An International Journal of RNA and Gene Targeting Research

    doi:

    HIV-prom-A siRNA induces CpG methylation of the HIV-1 LTR and transcriptional suppression. (A) The DNA from HIV-1 provirus in cells transfected with HIV-prom-A, B, and D siRNA is protected from digestion with the methylation-sensitive restriction enzyme Hpa II. DNA was extracted from HIV-1 infected cells at 14, 21, and 38 day after infection (day 7, 14, and 31 post-transfection respectively), digested with Hpa II and subjected to PCR using primers with binding sites denoted by arrows above the map. Nucleotide numbering is relative to the transcription initiation site. The downstream primer is located outside the LTR, so that this primer set specifically amplifies only the 5′ LTR. The restriction enzyme Hpa II is methylation sensitive such that it will not cut if the CpG in the CCGG recognition site is methylated. Hpa II cuts this region of HIV-1 at the single CpG site at position −146 (labeled Hpa II); PCR amplification will occur only if the DNA between the primer binding sites has not been cut. W/O indicates that DNA has not been subjected to Hpa II digestion. Amplification of proviral DNA from mock-transfected cells was abrogated by pre-digestion with Hpa II, indicating that it was unmethylated. By contrast amplification from Hpa II-digested DNA is successful from the cultures transfected with HIV-prom-A, B, and D siRNA, indicating that the Hpa II sites were methylated. (B) Bisulfite allelic sequencing of the 5′ LTR of the HIV-1 provirus at 38 day after infection. Bisulfite-modified genomic DNA was PCR amplified with nested primers specific for the bisulfite-converted sequence (see Methods) and ligated into a plasmid vector; at least 10 individual plasmid clones were sequenced. Methylated cytosines are spared from bisulfite conversion, while unmethylated cytosines are converted to uracils. The figure shows sequences of individual clones as horizontal lines, with the CpGs numbered as in Figure 6 A; unfilled boxes are unmethylated CpGs, and filled boxes are methylated CpGs. (C) The demethylating agent 5-azacytidine (5-aza-C) partially restores HIV transcription in cells previously transfected with HIV-prom-A siRNA. Real-time PCR amplification of the gag region from RNA was used to detect productive infection in MAGIC-5 cells. Six days after transfection with siRNA cells were treated with 5-aza-C at the indicated concentrations for 30 hours. The positive control (PC) was a culture in which no siRNA was transfected, and the negative control (NC) was uninfected cells. The results are shown as HIV RNA copy number per 1000 copies of beta-actin.
    Figure Legend Snippet: HIV-prom-A siRNA induces CpG methylation of the HIV-1 LTR and transcriptional suppression. (A) The DNA from HIV-1 provirus in cells transfected with HIV-prom-A, B, and D siRNA is protected from digestion with the methylation-sensitive restriction enzyme Hpa II. DNA was extracted from HIV-1 infected cells at 14, 21, and 38 day after infection (day 7, 14, and 31 post-transfection respectively), digested with Hpa II and subjected to PCR using primers with binding sites denoted by arrows above the map. Nucleotide numbering is relative to the transcription initiation site. The downstream primer is located outside the LTR, so that this primer set specifically amplifies only the 5′ LTR. The restriction enzyme Hpa II is methylation sensitive such that it will not cut if the CpG in the CCGG recognition site is methylated. Hpa II cuts this region of HIV-1 at the single CpG site at position −146 (labeled Hpa II); PCR amplification will occur only if the DNA between the primer binding sites has not been cut. W/O indicates that DNA has not been subjected to Hpa II digestion. Amplification of proviral DNA from mock-transfected cells was abrogated by pre-digestion with Hpa II, indicating that it was unmethylated. By contrast amplification from Hpa II-digested DNA is successful from the cultures transfected with HIV-prom-A, B, and D siRNA, indicating that the Hpa II sites were methylated. (B) Bisulfite allelic sequencing of the 5′ LTR of the HIV-1 provirus at 38 day after infection. Bisulfite-modified genomic DNA was PCR amplified with nested primers specific for the bisulfite-converted sequence (see Methods) and ligated into a plasmid vector; at least 10 individual plasmid clones were sequenced. Methylated cytosines are spared from bisulfite conversion, while unmethylated cytosines are converted to uracils. The figure shows sequences of individual clones as horizontal lines, with the CpGs numbered as in Figure 6 A; unfilled boxes are unmethylated CpGs, and filled boxes are methylated CpGs. (C) The demethylating agent 5-azacytidine (5-aza-C) partially restores HIV transcription in cells previously transfected with HIV-prom-A siRNA. Real-time PCR amplification of the gag region from RNA was used to detect productive infection in MAGIC-5 cells. Six days after transfection with siRNA cells were treated with 5-aza-C at the indicated concentrations for 30 hours. The positive control (PC) was a culture in which no siRNA was transfected, and the negative control (NC) was uninfected cells. The results are shown as HIV RNA copy number per 1000 copies of beta-actin.

    Techniques Used: CpG Methylation Assay, Transfection, Methylation, Infection, Polymerase Chain Reaction, Binding Assay, Labeling, Amplification, Sequencing, Modification, Plasmid Preparation, Clone Assay, Real-time Polymerase Chain Reaction, Positive Control, Negative Control

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    Diagnostic Assay:

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    Clone Assay:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Amplification:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Synthesized:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Construct:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Real-time Polymerase Chain Reaction:

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    Incubation:

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    Luciferase:

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    Expressing:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Modification:

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    Western Blot:

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    DNA Methylation Assay:

    Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4
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    Derivative Assay:

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    Hybridization:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
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    Transfection:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
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    Ligation:

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. A ligation mixture in a volume of 5 μl contained 5 pmol of Eco RI adaptor and 50 pmol of Hpa II/Msp I adaptor, and 30 U of T4 DNA ligase (New England Biolabs) was added to digestion products and incubated at 37°C for 3 hr, followed by 16°C for 9 hr, and then 65°C for 20 min. Preselective polymerase chain reaction (PCR) was carried out in a total volume of 20 μl containing 2 μl of the ligation product, 0.4 μM each of Eco RI_ADAPTER (F)+A and Hpa II/Msp I_ADAPTER (F)+T preselective primers, 0.5 U of Taq DNA polymerase (Takara), 2 mM of MgCl2 , and 0.2 mM of each nucleotide.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. After ligation of linkers H12/H24 (New England Biolabs), fragments were digested with methylation-sensitive restriction endonucleases Bst UI and Hpa II and amplified for 20 cycles with H24 as a primer.

    Cell Culture:

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    Article Snippet: Cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), Lipofectamine-2000, and Superscript one-step RT-PCR kit were obtained from Invitrogen (Carlsbad, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: For 16S rRNA products amplified from cultured bacteria, a 1.5 kbp 16S product was amplified from 48 bacterial isolates using 20 ng DNA each and Taq polymerase (NEB) in a 50 μl reaction. .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Generated:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: A set of reporter constructs containing truncated CHD5 promoter sequences generated by progressively deleting 200 or 100 bp from the 5' or 3' end were PCR amplified from pGL4.10-CHD5-P-WT and self-ligated. .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions.

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Polymerase Chain Reaction:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions. .. Sss I methylates all 5′-CpG-3′ sites, Hpa II methylates only the CpG within the sequence 5′-CCGG-3′ , and Hha I methylates only the CpG within the sequence 5′-GCGC-3′ .

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: .. Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). ..

    Article Title: A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples
    Article Snippet: .. Multiple Restriction Enzyme Digestion Eighty microliters (~2 μg) of PCR products were digested with 10–20 units each, Alu I, Ava I, Dde I, Hha I, and Hpa II (New England BioLabs) as per the supplier's directives for 2.5 to 4 h. .. Gel Extraction and Subcloning MRED digestions were ethanol precipitated, resuspended in TE buffer pH 8.0, and electrophoresed on 3% agarose gels.

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB). ..

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. A ligation mixture in a volume of 5 μl contained 5 pmol of Eco RI adaptor and 50 pmol of Hpa II/Msp I adaptor, and 30 U of T4 DNA ligase (New England Biolabs) was added to digestion products and incubated at 37°C for 3 hr, followed by 16°C for 9 hr, and then 65°C for 20 min. Preselective polymerase chain reaction (PCR) was carried out in a total volume of 20 μl containing 2 μl of the ligation product, 0.4 μM each of Eco RI_ADAPTER (F)+A and Hpa II/Msp I_ADAPTER (F)+T preselective primers, 0.5 U of Taq DNA polymerase (Takara), 2 mM of MgCl2 , and 0.2 mM of each nucleotide.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs). .. Amplification of DNA fragments for MSAP followed the same PCR cycling conditions used for AFLP.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions. .. Subsequently, regions of the AR and JARID1C (formerly SMCX ) loci were amplified by PCR in separate 25 μl reactions with 2.5 μl of the digested DNA by adding Taq Buffer (5× Taq Buffer is 83 mM Tris-HCl [pH 8.8], 850 mg/mL BSA, 83 mM (NH4 )2 SO4 , 33.5 mM MgCl2 , 34 mM EDTA, and 50mM β-mercaptoethanol), 5% dimethyl sulfoxide, 12.5 pmol of each primer, 1.5 mM dNTPs, and 0.625 U AmpliTaq (Applied Biosystems).

    Article Title: (Epi)Genetic Analyses of Age-Related Macular Degeneration: Case-control and Discordant Twin Studies
    Article Snippet: For methylation analysis, 3 μg of genomic DNA was digested at 37 degrees Celsius for16 hours with a methyl sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 μL reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. .. After this pre-treatment, the DNA enters the mapping array procedure; it is further digested with the Nsp I and Sty I restriction enzymes, and fragments of 100–1,100 bp (containing the polymorphic sites to be assessed) are PCR amplified, with the resulting amplicons then end-labeled and hybridized to the array.

    Sequencing:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: We purchased sequence-specific oligonucleotides from Midland Certified Reagent Company (Midland, TX). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: Transient transfection and luciferase assay The CHD5 promoter was obtained by whole gene synthesis using the 2000 bp sequence upstream of the TSS. .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions.

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Directing sequencing of MYH6 was conducted on at least 2 individuals from each sampling locality (n = 64; Additional file ; GenBank FJ638345 – FJ638410 ).

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl. .. Msp I and Hpa II can recognize and cleave the same sequence 5′‐CCGG‐3′, but their susceptibility is different depending on the methylation state of cytosines at restriction sites (Schulz et al., ).

    Antiviral Assay:

    Article Title: A Modified Protocol for Bisulfite Genomic Sequencing of Difficult Samples
    Article Snippet: .. Multiple Restriction Enzyme Digestion Eighty microliters (~2 μg) of PCR products were digested with 10–20 units each, Alu I, Ava I, Dde I, Hha I, and Hpa II (New England BioLabs) as per the supplier's directives for 2.5 to 4 h. .. Gel Extraction and Subcloning MRED digestions were ethanol precipitated, resuspended in TE buffer pH 8.0, and electrophoresed on 3% agarose gels.

    Binding Assay:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: Constructs pGL4.10-CHD5-2000 to −370 and pGL4.10-CHD5-356 to −1, in which the AP2 binding site (located at –369 to –357 bp from the TSS) is deleted, were PCR amplified from pGL4.10-CHD5-2000 to −1. .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions.

    Methylation:

    Article Title: Evidence for Widespread Genomic Methylation in the Migratory Locust, Locusta migratoria (Orthoptera: Acrididae)
    Article Snippet: Paragraph title: Methylation-specific restriction enzyme assays ... Restriction digests with Hpa II and Msp I (New England Biolabs) were carried out for 4 hr at 37°C with 2 µg of genomic DNA and 10 U of enzyme in a final volume of 50 µl.

    Article Title: A role for the Werner syndrome protein in epigenetic inactivation of the pluripotency factor Oct4
    Article Snippet: .. Cells were stimulated with 10 µM RA (Sigma, St. Louis, MO, USA) for 7 days, then harvested, DNA extracted and digested (600ng) with the methylation-sensitive enzymes HpaII or HhaI [R0171 and R0139 respectively (New England Biolabs, Ipswich, MA, USA)], see for location of restriction sites]. .. The digested DNA was then subjected to real-time PCR with primers targeting regions of the Oct4 promoter that flank the restriction sites.

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions. .. Sss I methylates all 5′-CpG-3′ sites, Hpa II methylates only the CpG within the sequence 5′-CCGG-3′ , and Hha I methylates only the CpG within the sequence 5′-GCGC-3′ .

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Paragraph title: Methylation‐sensitive amplification polymorphism assay ... Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Article Title: Quantitative trait variation is revealed in a novel hypomethylated population of woodland strawberry (Fragaria vesca)
    Article Snippet: Paragraph title: Assessment of DNA methylation polymorphism using Methylation Sensitive Amplified Polymorphisms (MSAP) ... Briefly, genomic DNA from each sample analyzed was digested separately with 10 units Eco RI/5 units Hpa II (New England Biolabs) and 10 units Eco RI/10units Msp I (New England Biolabs).

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: Paragraph title: Differential Methylation Hybridization Analysis and CGI Identification ... CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites.

    Article Title: A Large X-Chromosomal Deletion is Associated with Microphthalmia with Linear Skin Defects (MLS) and Amelogenesis Imperfecta (XAI)
    Article Snippet: The X-inactivation pattern was evaluated by examining the methylation pattern at the AR (androgen receptor) locus [ ]. .. DNA aliquots of 250 ng were incubated in a total volume of 12.5 μl with no enzyme, Dde I only, Hpa II only, or Dde I and Hpa II for 18 h at 37 °C (New England Biolabs) using the manufacturer's instructions.

    Article Title: (Epi)Genetic Analyses of Age-Related Macular Degeneration: Case-control and Discordant Twin Studies
    Article Snippet: .. For methylation analysis, 3 μg of genomic DNA was digested at 37 degrees Celsius for16 hours with a methyl sensitive restriction enzyme (MSRE) cocktail including Aci I (60 units), BsaH I (3.9 units), Hha I (7.5 units), Hpa II (7.5 units), and HpyCH4 IV (30 units) (New England Biolabs), in a 200 μL reaction volume with 1% BSA and 10% NEB buffer #4 (New England Biolabs) and heat inactivated for 20 minutes at 60°C. ..

    Isolation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The plasmid isolation kit and RNeasy mini-kit for total RNA isolation were obtained from Qiagen (Valencia, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: 2.2 Methylation‐sensitive amplification polymorphism assay Total genomic DNA was isolated from approximately 50 mg of siphon tissues following the proteinase K method (Waters, Dijkstra, & Wallis, ). .. Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: .. CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Purification:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: Cell culture media, fetal calf serum, ITS (insulin, transferrin, and sodium selenite), Lipofectamine-2000, and Superscript one-step RT-PCR kit were obtained from Invitrogen (Carlsbad, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Polymorphism Assay:

    Article Title: Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence. Methylation divergence of invasive Ciona ascidians: Significant population structure and local environmental influence
    Article Snippet: Paragraph title: Methylation‐sensitive amplification polymorphism assay ... Total genomic DNA (300 ng) was digested at 37°C for 3 hr in two parallel reactions using 5 U of Eco RI‐HF and 5 U of either Msp I or Hpa II (New England Biolabs) in a final volume of 10 μl.

    Chromatin Immunoprecipitation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA). .. We obtained the LightShift Chemiluminescent EMSA kit from Pierce (Rockford, IL) and the EZ ChIP kit from Upstate Biotechnology (Temecula, CA).

    Plasmid Preparation:

    Article Title: Regulation of Natriuretic Peptide Receptor-A gene expression and stimulation of its guanylyl cyclase activity by transcription factor Ets-1
    Article Snippet: The plasmid isolation kit and RNeasy mini-kit for total RNA isolation were obtained from Qiagen (Valencia, CA). .. The Hha I, Hpa II, and Sss I methylases and restriction enzymes Hha I, Hpa II, and Bst UI were purchased from New England Biolabs (Ipswich, MA).

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: The synthesized promoter was cloned into the pGL4.10 (Promega, Madison, WI, USA), a promoterless luciferase expression vector, to produce the pGL4.10-CHD5-2000 to −1 recombinant vector. .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions.

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: For PKS products amplified from sorted bacteria, two of fifty microliters of each plasmid preparation were digested with each of seven restriction enzymes: Kpn I, Bam HI, Xba I, Xho I, Hpa II, Rsa I and Sph I (NEB). .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: The DNA template used for in vitro reconstitution of chromatin was the pBS HPRT 1.8-kb plasmid, which is a pBluescript-derived plasmid containing a 1.8-kb Eco RI-to- Bam HI fragment that includes the entire HPRT promoter. .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier.

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. The following hybridization probes were generated from purified cloned inserts: a 3.7 kb Eco RI fragment containing the 5.8S and 25S rRNA gene from plasmid pARR17 [ ] and a 1.7 kb Eco RI fragment from IGS clone, pAt4 [ , ].

    Article Title: Aberrant Methylation and Reduced Expression of LHX9 in Malignant Gliomas of Childhood 1
    Article Snippet: CpG-rich DNA fragments were isolated from the human CGI library and screened for the presence of Bst UI and Hpa II (New England Biolabs GmbH, Frankfurt, Germany) restriction sites. .. Sixteen thousand suitable fragments were polymerase chain reaction (PCR)-amplified using plasmid primer and spotted onto UltraGAPS microarrays (Corning, Acton, MA).

    Software:

    Article Title: Natural variation in DNA methylation in ribosomal RNA genes of Arabidopsis thaliana
    Article Snippet: Genomic DNA was digested with Hpa II or Hpa II+Eco RI according to the manufacturer's (New England Biolabs, USA) instructions. .. CR and IGS DNA methylation levels were determined from phosphorimager (Bio-Rad, USA) files using Quantity One™ (Bio-Rad, USA) software.

    Recombinant:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: The synthesized promoter was cloned into the pGL4.10 (Promega, Madison, WI, USA), a promoterless luciferase expression vector, to produce the pGL4.10-CHD5-2000 to −1 recombinant vector. .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions.

    In Vitro:

    Article Title: Silencing of CHD5 Gene by Promoter Methylation in Leukemia
    Article Snippet: .. A 321 bp region located at –560 to –240 was PCR amplified from pGL4.10-CHD5-2000 to −1 and the fragments were methylated in vitro with Sss I, Hpa II and Hha I methylases (New England Biolabs), or no enzyme (mock), according to the manufacturer’s instructions. .. Sss I methylates all 5′-CpG-3′ sites, Hpa II methylates only the CpG within the sequence 5′-CCGG-3′ , and Hha I methylates only the CpG within the sequence 5′-GCGC-3′ .

    Article Title: Nucleosomes Are Translationally Positioned on the Active Allele and Rotationally Positioned on the Inactive Allele of the HPRT Promoter
    Article Snippet: .. DNA methylation at CpG dinucleotides in vitro was performed using Hpa II, Hha I, and Sss I methylases (New England Biolabs) essentially as described by the supplier. .. The methylated DNA was then extracted with phenol-chloroform, precipitated, and resuspended at 100 ng/μl in TE.

    Sampling:

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Directing sequencing of MYH6 was conducted on at least 2 individuals from each sampling locality (n = 64; Additional file ; GenBank FJ638345 – FJ638410 ).

    Alkaline Lysis:

    Article Title: Localization of polyketide synthase encoding genes to the toxic dinoflagellate Karenia brevis
    Article Snippet: The plasmid alkaline lysis minipreparation procedure was performed as described previously ( ). .. Five microliters of each PCR was used for each of the four digestions using the enzymes Hind III, Sau 3AI, Hpa II and Rsa I (NEB).

    Staining:

    Article Title: Evidence for Widespread Genomic Methylation in the Migratory Locust, Locusta migratoria (Orthoptera: Acrididae)
    Article Snippet: Restriction digests with Hpa II and Msp I (New England Biolabs) were carried out for 4 hr at 37°C with 2 µg of genomic DNA and 10 U of enzyme in a final volume of 50 µl. .. Digested DNA was separated on 0.8% agarose/TAE gel and visualised using ethidium bromide staining.

    Article Title: Historic hybridization and persistence of a novel mito-nuclear combination in red-backed voles (genus Myodes)
    Article Snippet: Restriction enzyme digestion was completed using 5 μl of the PCR product, 7.5 units Hpa II, and 5 μl Buffer1 (New England Biolabs). .. Digested products were run on 2% agarose stained with ethidium bromide.

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    New England Biolabs hpa ii
    Methylation analysis of the ZPBP gene in whole blood samples of Berkshire sows by PMP assay. gDNAs were cut with the methylation-sensitive restriction enzymes <t>Hpa</t> II/ <t>Msp</t> I. M: size marker; U: undigested DNA; SLG: smaller litter size group; LLG: larger litter size group.
    Hpa Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hpa ii/product/New England Biolabs
    Average 95 stars, based on 87 article reviews
    Price from $9.99 to $1999.99
    hpa ii - by Bioz Stars, 2020-02
    95/100 stars
      Buy from Supplier

    99
    New England Biolabs methylation sensitive restriction enzyme hpa ii
    Representative examples of the electrophoretic pattern. ( A ) Male control (digestion control; polymerase chain reaction [PCR] products from undigested <t>DNA</t> appear as a single band, while PCR products from digested DNA does not have bands, this shows DNA is completely digested with <t>Hpa</t> II). ( B ) Excluded sample (could not distinguish two bands of undigested PCR products because two alleles from the maternally and paternally inherited CAG repeats were the same). ( C ) Not skewed (degree of X chromosome inactivation [XCI] skewing: 55.3%). ( D ) Skewed (degree of XCI skewing: 88.2%). Lane: + and − indicate undigestioned and digestion by Hpa II, respectively.
    Methylation Sensitive Restriction Enzyme Hpa Ii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/methylation sensitive restriction enzyme hpa ii/product/New England Biolabs
    Average 99 stars, based on 14 article reviews
    Price from $9.99 to $1999.99
    methylation sensitive restriction enzyme hpa ii - by Bioz Stars, 2020-02
    99/100 stars
      Buy from Supplier

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    Methylation analysis of the ZPBP gene in whole blood samples of Berkshire sows by PMP assay. gDNAs were cut with the methylation-sensitive restriction enzymes Hpa II/ Msp I. M: size marker; U: undigested DNA; SLG: smaller litter size group; LLG: larger litter size group.

    Journal: Archives Animal Breeding

    Article Title: Hypomethylation in the promoter region of ZPBP as a potential litter size indicator in Berkshire pigs

    doi: 10.5194/aab-62-69-2019

    Figure Lengend Snippet: Methylation analysis of the ZPBP gene in whole blood samples of Berkshire sows by PMP assay. gDNAs were cut with the methylation-sensitive restriction enzymes Hpa II/ Msp I. M: size marker; U: undigested DNA; SLG: smaller litter size group; LLG: larger litter size group.

    Article Snippet: To verify the result of GWBS, whole blood samples were collected from Berkshire sows to isolate gDNA using a Wizard Genomic DNA Purification Kit (Promega, Madison, WI, USA) according to the manufacturer's instructions and digested with Hpa II (NEB, Hitchin, UK) and Msp I (NEB), a pair of methylation-sensitive isoschizomers that have the same recognition site (CC|GG).

    Techniques: Methylation, Marker

    P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P

    Journal: Disease Models & Mechanisms

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    doi: 10.1242/dmm.022350

    Figure Lengend Snippet: P3 peptide treatment suppressed nucleolar stress in cells expressing expanded CAG RNA. (A) Dose-dependent effect of synthetic TAT-P3WT on the inhibition of cell death in EGFP CAG78 RNA-expressing HEK293 cells. A lactate dehydrogenase (LDH) cytotoxicity assay was performed. The IC 50 value represents the concentration of TAT-P3WT that reduced LDH enzyme activity by 50% when compared with the no-peptide treatment control group. Data are expressed as mean±s.e.m. for at least three independent experiments. (B,C) Synthetic TAT-P3WT peptide (12 μM) treatment restored pre-45s rRNA (B) and 18S rRNA (C) levels in EGFP CAG78 RNA-expressing HEK293 cells. Cells were treated with 12 μM of corresponding P3 peptides. Real-time PCR was performed to determine the level of pre-45s rRNA. ‘P3WT’ represents synthetic P3 peptide without the TAT fusion. This serves as a control to demonstrate that TAT-mediated intracellular delivery of P3 is crucial for its action. Experiments were repeated at least three times and data are expressed as mean±s.d. (D) Synthetic TAT-P3WT treatment resumed the interaction between NCL and UCE in EGFP CAG78 RNA-expressing HEK293 cells. Following chromatin immunoprecipitation, real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. Experiments were repeated at least three times and data are expressed as mean±s.d. (E) TAT-P3WT peptide treatment resumed the DNA methylation status of UCE in EGFP CAG78 RNA-expressing HEK293 cells. ‘–’ indicates cells that were not treated with peptides. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. Digested DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. Only representative gel photos are shown. (F) Synthetic TAT-P3WT peptide treatment inhibited p53 protein expression in EGFP CAG78 RNA-expressing HEK293 cells. Western blotting was performed to determine the p53 expression level. Tubulin was used as a loading control. The experiment was repeated three times with consistent results obtained. Only representative blots are shown. (G) Synthetic TAT-P3WT peptide treatment suppressed cell death in HEK293 cells expressing EGFP CAG78 RNA. Caspase 9 activity was determined. P3WT represents Peptide 3 wild type and P3MT5 represents P3 mutant 5. Experiments were repeated at least three times and data are expressed as mean±s.d. * P

    Article Snippet: To perform the Hpa II methylation assay, genomic DNA was extracted from cells, followed by digestion with 2 units of Hpa II or Msp I (New England Biolabs) for 4 h at 37°C.

    Techniques: Expressing, Inhibition, LDH Cytotoxicity Assay, Concentration Assay, Activity Assay, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, DNA Methylation Assay, Methylation, Polymerase Chain Reaction, Amplification, Western Blot, Mutagenesis

    Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P

    Journal: Disease Models & Mechanisms

    Article Title: Assessing a peptidylic inhibitor-based therapeutic approach that simultaneously suppresses polyglutamine RNA- and protein-mediated toxicities in patient cells and Drosophila

    doi: 10.1242/dmm.022350

    Figure Lengend Snippet: Expression of P3 suppressed nucleolar stress in cells expressed with expanded-CAG RNA. (A) Amino acid sequence of nucleolin (NCL) peptides used in this study. (B) The P3 and P5 peptides disrupted the interaction between expanded-CAG RNA and NCL. After in vitro binding of CAG 78 RNA and GST-NCL protein in the presence of NCL peptides, reverse-transcription PCR was performed to detect the binding of CAG 78 RNA to GST-NCL. (C) Amino acid sequences of mutant (MT) P3 peptides. The mutated residues are underlined. (D) Expression of P3WT resumed the expression level of pre-45s rRNA in EGFP CAG78 RNA-expressing HEK293 cells. Real-time PCR was performed to determine the expression level of pre-45s rRNA in cells co-transfected with EGFP CAG and P3 constructs. (E) Expression of P3WT resumed the physical interaction between NCL and upstream control element (UCE) in EGFP CAG78 RNA-expressing HEK293 cells. Chromatin immunoprecipitation was performed. Real-time PCR was performed to determine the amount of UCE in the immunoprecipitant. (F) Expression of P3WT resumed the DNA methylation status of UCE. ‘–’ represents cells that were transfected with pcDNA3.1 empty vector. Genomic DNA was treated with either Hpa II or Msp I. Hpa II is a methylation-sensitive restriction enzyme, whereas Msp I is a methylation-insensitive restriction enzyme. The enzyme-treated DNA was used in PCR. Amplicon UCE was amplified. Msp I-treated samples were used as loading control. (G) Expression of P3WT suppressed caspase 9 activity in HEK293 cells expressing EGFP CAG78 RNA. Experiments were repeated at least three times and data are expressed as mean±s.d. *** P

    Article Snippet: To perform the Hpa II methylation assay, genomic DNA was extracted from cells, followed by digestion with 2 units of Hpa II or Msp I (New England Biolabs) for 4 h at 37°C.

    Techniques: Expressing, Sequencing, In Vitro, Binding Assay, Polymerase Chain Reaction, Mutagenesis, Real-time Polymerase Chain Reaction, Transfection, Construct, Chromatin Immunoprecipitation, DNA Methylation Assay, Plasmid Preparation, Methylation, Amplification, Activity Assay

    Representative examples of the electrophoretic pattern. ( A ) Male control (digestion control; polymerase chain reaction [PCR] products from undigested DNA appear as a single band, while PCR products from digested DNA does not have bands, this shows DNA is completely digested with Hpa II). ( B ) Excluded sample (could not distinguish two bands of undigested PCR products because two alleles from the maternally and paternally inherited CAG repeats were the same). ( C ) Not skewed (degree of X chromosome inactivation [XCI] skewing: 55.3%). ( D ) Skewed (degree of XCI skewing: 88.2%). Lane: + and − indicate undigestioned and digestion by Hpa II, respectively.

    Journal: Thyroid

    Article Title: The Relationship Between Skewed X Chromosome Inactivation and the Prognosis of Graves' and Hashimoto's Diseases

    doi: 10.1089/thy.2014.0318

    Figure Lengend Snippet: Representative examples of the electrophoretic pattern. ( A ) Male control (digestion control; polymerase chain reaction [PCR] products from undigested DNA appear as a single band, while PCR products from digested DNA does not have bands, this shows DNA is completely digested with Hpa II). ( B ) Excluded sample (could not distinguish two bands of undigested PCR products because two alleles from the maternally and paternally inherited CAG repeats were the same). ( C ) Not skewed (degree of X chromosome inactivation [XCI] skewing: 55.3%). ( D ) Skewed (degree of XCI skewing: 88.2%). Lane: + and − indicate undigestioned and digestion by Hpa II, respectively.

    Article Snippet: Isolated DNA was digested with the methylation sensitive restriction enzyme Hpa II (New England BioLabs, Inc., Beverly, NA) to obtain polymerase chain reaction (PCR) templates.

    Techniques: Polymerase Chain Reaction

    Detailed MSAP-Seq assay overview (A) Genomic DNA is cleaved using rare cutter ( Eco RI) and methylation sensitive restriction enzyme ( Hpa II); only unmethylated recognition sites are digested by Hpa II. Then adapters specific to sticky ends are ligated and obtained fragments are amplified in PCR using selective primers; only fragments generated from unmethylated regions are amplified as they contain ends complementary to adapters. Products are then purified and fragmented by sonication to create shorter tags. (B) Purified fragments are used for standard library preparation involving following steps: end repair, adenylation, barcoded adapters ligation and purification, PCR amplification and purification. Then libraries quality and quantity is estimated. (C) Prepared libraries are pooled and processed thru cluster generation and high-throughput sequencing. (D) Sequencing data are analyzed using dedicated automatic pipeline—MSEQER. Firstly, reads are filtered for presence of Hpa II adapter and adapters are clipped. Then only reads containing CGG tags on the ends are mapped to the reference genome and functionally annotated. Obtained counts at each of the CCGG sites are normalized and differential methylation analysis among sets of samples is performed.

    Journal: Frontiers in Plant Science

    Article Title: Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

    doi: 10.3389/fpls.2017.02056

    Figure Lengend Snippet: Detailed MSAP-Seq assay overview (A) Genomic DNA is cleaved using rare cutter ( Eco RI) and methylation sensitive restriction enzyme ( Hpa II); only unmethylated recognition sites are digested by Hpa II. Then adapters specific to sticky ends are ligated and obtained fragments are amplified in PCR using selective primers; only fragments generated from unmethylated regions are amplified as they contain ends complementary to adapters. Products are then purified and fragmented by sonication to create shorter tags. (B) Purified fragments are used for standard library preparation involving following steps: end repair, adenylation, barcoded adapters ligation and purification, PCR amplification and purification. Then libraries quality and quantity is estimated. (C) Prepared libraries are pooled and processed thru cluster generation and high-throughput sequencing. (D) Sequencing data are analyzed using dedicated automatic pipeline—MSEQER. Firstly, reads are filtered for presence of Hpa II adapter and adapters are clipped. Then only reads containing CGG tags on the ends are mapped to the reference genome and functionally annotated. Obtained counts at each of the CCGG sites are normalized and differential methylation analysis among sets of samples is performed.

    Article Snippet: Identification of differentially methylated sites (DMS) using MSAP-Seq Five hundred nanograms of genomic DNA was cut with 2.5 U of the frequent-cutting methylation sensitive restriction enzyme Hpa II (New England Biolabs, Ipswich, USA) and 2.5 U of the rare-cutting Eco RI (New England Biolabs, Ipswich, USA) in a 20 μL reaction with 1x NEB1 buffer (New England Biolabs, Ipswich, USA) at 37°C for 6 h. Enzymes were inactivated at 80°C for 20 min. Next, 12 μL of ligation mixture containing 60 pmol of Hpa II-related adapter, 6 pmol of Eco RI-related adapter (Table ), 1x T4 ligase buffer (Thermo Scientific, Waltham, USA) and 1.2 U of T4 DNA ligase (Thermo Scientific, Waltham, USA) were added.

    Techniques: Methylation, Amplification, Polymerase Chain Reaction, Generated, Purification, Sonication, Ligation, Next-Generation Sequencing, Sequencing