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ChIP analysis of DMR1 occupancy by <t>Ctcf,</t> Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific <t>DNA</t> sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P
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1) Product Images from "ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites"

Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites

Journal: Biochemical Journal

doi: 10.1042/BJ20111417

ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P
Figure Legend Snippet: ChIP analysis of DMR1 occupancy by Ctcf, Parp1, PARs and Dnmt1 ( A ) Schematic map of the locus. The DMR1 region is expanded to show the approximate position of putative Ctcf-binding sites and of PCR primers used to detect the presence of specific DNA sequences in ChIP complexes. Circles represent CpG dinuclotides. H, HpaII sites. ( B ) ChIP analysis of DMR1 region carried out with anti-Ctcf, anti-Parp1, anti-Dnmt1 and anti-PAR Abs. Beads alone (No Ab) and anti-IgG Abs were used as negative controls. Real-time PCR data are expressed as percentage of the signal detected for the non-immunoprecipitated input (4% of the chromatin subjected to immunoprecipitation) taken as 100%. The Actb promoter served as a control. ( C ) Standard ChIP assay for Ctcf was followed by RE-ChIP to assess the co-occupancy of Ctcf, with Parp1, PARs and Dnmt1 at the Ctcf DNA target sites within the DMR1. Real-time PCR was performed for the DMR1 b and c fragments. The efficiency of RE-ChIP at each of the sites was calculated as a percentage of the chromatin input that co-purified with Ctcf in the first ChIP (10% of the Ctcf ChIP fraction). Results are means±S.E.M. calculated from three experiments. * P

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

Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.
Figure Legend Snippet: Ctcf, Parp1, and Dnmt1 bind to non-methylated DNA molecules within the DMR1 Ctcf, Parp1, and Dnmt1 ChIPs were performed for the DMR1 b and c fragments. End-point PCR was performed after digestion of ChIP fractions DNA and inputs with either HpaII (H, methylation sensitive) or MspI (M, methylation insensitive) following heat inactivation of the restriction enzymes. The input (4% of the chromatin subjected to immunoprecipitation) was diluted 1/100 or 1/200 before restriction. The uncut (U) fractions consisted in HpaII digestions preventively blocked by heat inactivation. W, PCR performed in the absence of added template.

Techniques Used: Methylation, Polymerase Chain Reaction, Chromatin Immunoprecipitation, Immunoprecipitation

2) Product Images from "Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas"

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas

Journal: Human Mutation

doi: 10.1002/humu.23137

A : Pathogenic germline and somatic mutations in BRCA1 and BRCA2 detected using smMIP‐based targeted sequencing of FFPE tumor material. Lollipops above the bar: germline mutations detected in 47 FFPE ovarian carcinomas derived from 38 patients. Lollipops below the bar: somatic pathogenic mutations observed in seven FFPE ovarian carcinomas derived from five patients. B : Genomic location of 43 germline SNPs in BRCA1 and BRCA2 that were selected to determine the sensitivity of smMIP‐based next‐generation sequencing. Depicted base substitutions (lollipops) represent 15 and 28 benign germline variants in the ORF of BRCA1 and BRCA2 , respectively. These variants were known to be present in the germline of a subset of the included ovarian carcinoma patients prior to smMIP‐based sequencing of BRCA1 and BRCA2 in the corresponding FFPE ovarian carcinomas. Numbers depict the total number of the corresponding germline variant observed in these ovarian carcinoma patients. All germline SNPs could successfully be detected using smMIP‐based targeted sequencing on DNA derived from the corresponding FFPE ovarian carcinoma sample.
Figure Legend Snippet: A : Pathogenic germline and somatic mutations in BRCA1 and BRCA2 detected using smMIP‐based targeted sequencing of FFPE tumor material. Lollipops above the bar: germline mutations detected in 47 FFPE ovarian carcinomas derived from 38 patients. Lollipops below the bar: somatic pathogenic mutations observed in seven FFPE ovarian carcinomas derived from five patients. B : Genomic location of 43 germline SNPs in BRCA1 and BRCA2 that were selected to determine the sensitivity of smMIP‐based next‐generation sequencing. Depicted base substitutions (lollipops) represent 15 and 28 benign germline variants in the ORF of BRCA1 and BRCA2 , respectively. These variants were known to be present in the germline of a subset of the included ovarian carcinoma patients prior to smMIP‐based sequencing of BRCA1 and BRCA2 in the corresponding FFPE ovarian carcinomas. Numbers depict the total number of the corresponding germline variant observed in these ovarian carcinoma patients. All germline SNPs could successfully be detected using smMIP‐based targeted sequencing on DNA derived from the corresponding FFPE ovarian carcinoma sample.

Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Derivative Assay, Next-Generation Sequencing, Variant Assay

Related Articles

Ligation:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: If the extension or ligation arm targeted a common SNP (MAF > 1%), two different smMIPs were designed to recognize and target both alleles. .. Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O).

Methylation:

Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites
Article Snippet: The primers and annealing temperatures are indicated in the Supplementary Materials and methods section. .. For ChIP-CHOP analysis of the DMR1 b and c fragments, the Ctcf-, Parp1- and Dnmt-pulled down DNA (20 μl), and input DNA (20 μl after 1:100 and 1:200 dilutions) from ChIP assays were digested for 1 h at 37°C with 10 units of either the methylation-sensitive HpaII restriction enzyme or its methylation-insensitive isoschizomer MspI (New England Biolabs). .. Digestion was followed by heat inactivation.

Polymerase Chain Reaction:

Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites
Article Snippet: For ChIP-CHOP analysis of the DMR1 b and c fragments, the Ctcf-, Parp1- and Dnmt-pulled down DNA (20 μl), and input DNA (20 μl after 1:100 and 1:200 dilutions) from ChIP assays were digested for 1 h at 37°C with 10 units of either the methylation-sensitive HpaII restriction enzyme or its methylation-insensitive isoschizomer MspI (New England Biolabs). .. As an uncut control, ChIP DNA fractions were prepared for HpaII digestion and immediately heat inactivated to prevent restriction.

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O). .. Incubation was followed by exonuclease treatment: 0.5μl exonuclease I (New England Biolabs), 0.5μl exonuclease III (New England Biolabs), 0.2μl 10x ampligase buffer (Illumina), and 0.8μl H2 O was added to the (cooled) capture samples (consecutively incubated at 37°C and 95°C for 45 and 2 min, respectively).

Size-exclusion Chromatography:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O). .. Incubation was followed by exonuclease treatment: 0.5μl exonuclease I (New England Biolabs), 0.5μl exonuclease III (New England Biolabs), 0.2μl 10x ampligase buffer (Illumina), and 0.8μl H2 O was added to the (cooled) capture samples (consecutively incubated at 37°C and 95°C for 45 and 2 min, respectively).

Sequencing:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Paragraph title: Targeted Sequencing of BRCA1 and BRCA2 by smMIPs ... Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O).

Formalin-fixed Paraffin-Embedded:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Targeted sequencing of BRCA1 and BRCA2 using DNA derived from FFPE OCs was performed as previously described [Weren et al., ], using a slightly modified smMIP capture protocol [O'Roak et al., ; Hiatt et al., ]. .. Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O).

Modification:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Targeted sequencing of BRCA1 and BRCA2 using DNA derived from FFPE OCs was performed as previously described [Weren et al., ], using a slightly modified smMIP capture protocol [O'Roak et al., ; Hiatt et al., ]. .. Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O).

Chromatin Immunoprecipitation:

Article Title: ADP-ribose polymers localized on Ctcf-Parp1-Dnmt1 complex prevent methylation of Ctcf target sites
Article Snippet: The primers and annealing temperatures are indicated in the Supplementary Materials and methods section. .. For ChIP-CHOP analysis of the DMR1 b and c fragments, the Ctcf-, Parp1- and Dnmt-pulled down DNA (20 μl), and input DNA (20 μl after 1:100 and 1:200 dilutions) from ChIP assays were digested for 1 h at 37°C with 10 units of either the methylation-sensitive HpaII restriction enzyme or its methylation-insensitive isoschizomer MspI (New England Biolabs). .. Digestion was followed by heat inactivation.

Derivative Assay:

Article Title: Novel BRCA1 and BRCA2 Tumor Test as Basis for Treatment Decisions and Referral for Genetic Counselling of Patients with Ovarian Carcinomas
Article Snippet: Targeted sequencing of BRCA1 and BRCA2 using DNA derived from FFPE OCs was performed as previously described [Weren et al., ], using a slightly modified smMIP capture protocol [O'Roak et al., ; Hiatt et al., ]. .. Briefly, smMIP capture was performed on 10μl of input DNA (20–500ng) supplied with 15μl capture mixture (0.01μl ampligase DNA ligase [100U/μl; Illumina, Madison, WI], 2.5μl 10x ampligase buffer [Illumina], 0.27μl smMIP pool dilution [6.6x105 μM], 0.32μl Hemo Klentaq [10U/μl; New England Biolabs], 0.03μl dNTPs [0.25mM], and 11.9μl H2 O).