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    Structured Review

    Thermo Fisher dpni
    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes <t>BsuRI</t> (A) and BamHI, <t>DpnI,</t> and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.
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    1) Product Images from "The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius"

    Article Title: The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00137

    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.
    Figure Legend Snippet: Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Techniques Used: Methylation, Whole Genome Amplification, Sequencing, Negative Control, Agarose Gel Electrophoresis, Staining

    2) Product Images from "Dnmt1/Transcription Factor Interactions"

    Article Title: Dnmt1/Transcription Factor Interactions

    Journal: Genes & Cancer

    doi: 10.1177/1947601910373794

    Description of the cooperation between Dnmt1/Sp1 and Dnmt1-PCNA-UHRF1 to maintain the DNA methylation. ( A ) The kinetic of ChIP experiments was realized at an indicated time after cell (U87) synchronization (72 hours of serum deprivation) and α-amanitin treatment. ChIP was performed with the EZ ChIP kit (Millipore, Molsheim, France) and with indicated antibodies. The amount of immunoprecipitated promoter was quantified by semiquantitative PCR and normalized to input. ( B ) Identification by methylation-specific PCR (MSP) of the methylation status of CG dinucleotides localized into the −526/−408 region of the SLIT2 gene in U87 cells. MSPa was designed to investigate the methylation status of CG dinucleotides #1, #2, and #3; MSPb was designed to investigate the methylation status of CG dinucleotide #4; and MSPc was designed to investigate the methylation status of CG dinucleotides #5 and #6. The green box indicates the presence of Sp1 binding site according to the use of the Patch program. U = unmethylated; M = methylated. Black boxes represent primers. ( C ) Impact of siRNA-induced decrease expression of Sp1 on the methylation status of specific CG dinucloetides of the SLIT2 genes. Methylation status of specific CG dicnuleotides was determined by methylation sensitive restriction assay (MSRA). DNA methylation blocks the cleavage of BstUI and DpnI when the CGCG and GATCg sequences are methylated, respectively. Thus, when DNA is initially unmethylated, no or weak DNA is amplified by PCR. Black triangles represent primers.
    Figure Legend Snippet: Description of the cooperation between Dnmt1/Sp1 and Dnmt1-PCNA-UHRF1 to maintain the DNA methylation. ( A ) The kinetic of ChIP experiments was realized at an indicated time after cell (U87) synchronization (72 hours of serum deprivation) and α-amanitin treatment. ChIP was performed with the EZ ChIP kit (Millipore, Molsheim, France) and with indicated antibodies. The amount of immunoprecipitated promoter was quantified by semiquantitative PCR and normalized to input. ( B ) Identification by methylation-specific PCR (MSP) of the methylation status of CG dinucleotides localized into the −526/−408 region of the SLIT2 gene in U87 cells. MSPa was designed to investigate the methylation status of CG dinucleotides #1, #2, and #3; MSPb was designed to investigate the methylation status of CG dinucleotide #4; and MSPc was designed to investigate the methylation status of CG dinucleotides #5 and #6. The green box indicates the presence of Sp1 binding site according to the use of the Patch program. U = unmethylated; M = methylated. Black boxes represent primers. ( C ) Impact of siRNA-induced decrease expression of Sp1 on the methylation status of specific CG dinucloetides of the SLIT2 genes. Methylation status of specific CG dicnuleotides was determined by methylation sensitive restriction assay (MSRA). DNA methylation blocks the cleavage of BstUI and DpnI when the CGCG and GATCg sequences are methylated, respectively. Thus, when DNA is initially unmethylated, no or weak DNA is amplified by PCR. Black triangles represent primers.

    Techniques Used: DNA Methylation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Methylation, Binding Assay, Expressing, Restriction Assay, Amplification

    3) Product Images from "In Vivo and In Vitro Intragenomic Rearrangement of TT Viruses ▿"

    Article Title: In Vivo and In Vitro Intragenomic Rearrangement of TT Viruses ▿

    Journal:

    doi: 10.1128/JVI.00781-07

    In vitro replication and intragenomic rearrangement of tth8 in the L428 cell line. (A) Southern blot analyses of total cellular DNA harvested at different time points after transfection. DNA was digested with DpnI to fragmentize input viral DNA (#). Hybridization
    Figure Legend Snippet: In vitro replication and intragenomic rearrangement of tth8 in the L428 cell line. (A) Southern blot analyses of total cellular DNA harvested at different time points after transfection. DNA was digested with DpnI to fragmentize input viral DNA (#). Hybridization

    Techniques Used: In Vitro, Southern Blot, Transfection, DNA Hybridization

    4) Product Images from "Identification of the Merkel Cell Polyomavirus Large Tumor Antigen Ubiquitin Conjugation Residue"

    Article Title: Identification of the Merkel Cell Polyomavirus Large Tumor Antigen Ubiquitin Conjugation Residue

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms22137169

    LT K585 is required for MCPyV replication. ( A ) LT K585R mutation maintains origin-binding ability. Early gene (ER) transcription activity was measured by luciferase activity. The reporters (Rep+ and Rep-, 0.5 μg) [ 4 ] were respectively transfected with either LT.wt or LT K585R mutant (0.5 μg). LT K585R similarly downregulated MCPyV ER transcription compared to LT.wt. Mean values, error bars representing the standard error of the mean (SEM), and p -values were calculated using GraphPad Prism software (**** p ≤ 0.0001, ns = not significant). Data were analyzed using three biological replicates, n = 3. ( B ) K585 is required for MCPyV viral replication. MCPyV origin replication was determined from samples tested in Figure 5 A (Rep+). Episomal DNAs were isolated and digested with DpnI. Replicated origin DNA was measured by qPCR. Mean values, error bars representing the standard error of the mean (SEM), and p -value were calculated using GraphPad Prism software (**** p ≤ 0.0001). Data were analyzed using three biological replicates per experiment, n = 3.
    Figure Legend Snippet: LT K585 is required for MCPyV replication. ( A ) LT K585R mutation maintains origin-binding ability. Early gene (ER) transcription activity was measured by luciferase activity. The reporters (Rep+ and Rep-, 0.5 μg) [ 4 ] were respectively transfected with either LT.wt or LT K585R mutant (0.5 μg). LT K585R similarly downregulated MCPyV ER transcription compared to LT.wt. Mean values, error bars representing the standard error of the mean (SEM), and p -values were calculated using GraphPad Prism software (**** p ≤ 0.0001, ns = not significant). Data were analyzed using three biological replicates, n = 3. ( B ) K585 is required for MCPyV viral replication. MCPyV origin replication was determined from samples tested in Figure 5 A (Rep+). Episomal DNAs were isolated and digested with DpnI. Replicated origin DNA was measured by qPCR. Mean values, error bars representing the standard error of the mean (SEM), and p -value were calculated using GraphPad Prism software (**** p ≤ 0.0001). Data were analyzed using three biological replicates per experiment, n = 3.

    Techniques Used: Mutagenesis, Binding Assay, Activity Assay, Luciferase, Transfection, Software, Isolation, Real-time Polymerase Chain Reaction

    5) Product Images from "The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells"

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116151

    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
    Figure Legend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Techniques Used: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.
    Figure Legend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Techniques Used: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

    6) Product Images from "Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency"

    Article Title: Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135770

    HPV Replication assays. A Transient replication assay of HPV-18 URR plasmid E2BS #1 and #2 linker sequence mutants (0-Linker, 1-Linker (wt), 2-Linker, 3-Linker). 25 ng of the E1 and E2 expression vectors were cotransfected, and total DNA extracts were obtained 24 h and 48 h post-transfection. DNA was linearized using Hind III enzyme, and input DNA was eliminated by digestion with DpnI. Normalized qPCR data are presented in the left panel, and the Southern blot analysis is shown in the right panel. A 2-sample t-test was performed and statistically significant (P
    Figure Legend Snippet: HPV Replication assays. A Transient replication assay of HPV-18 URR plasmid E2BS #1 and #2 linker sequence mutants (0-Linker, 1-Linker (wt), 2-Linker, 3-Linker). 25 ng of the E1 and E2 expression vectors were cotransfected, and total DNA extracts were obtained 24 h and 48 h post-transfection. DNA was linearized using Hind III enzyme, and input DNA was eliminated by digestion with DpnI. Normalized qPCR data are presented in the left panel, and the Southern blot analysis is shown in the right panel. A 2-sample t-test was performed and statistically significant (P

    Techniques Used: Plasmid Preparation, Sequencing, Expressing, Transfection, Real-time Polymerase Chain Reaction, Southern Blot

    7) Product Images from "Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome"

    Article Title: Replication, Gene Expression and Particle Production by a Consensus Merkel Cell Polyomavirus (MCPyV) Genome

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029112

    R17a replication in PFSK-1 cells. 2 µg low molecular weight DNA from PFSK-1 cells transfected with MCVSyn or R17a viral DNA was EcoRI and DpnI digested and separated on an agarose gel, followed by EtBr staining (left panel). The DNA was transferred via southern blotting and probed with a radioactively labelled LT-Ag PCR fragment (right panel). The blot was exposed for 24 h.
    Figure Legend Snippet: R17a replication in PFSK-1 cells. 2 µg low molecular weight DNA from PFSK-1 cells transfected with MCVSyn or R17a viral DNA was EcoRI and DpnI digested and separated on an agarose gel, followed by EtBr staining (left panel). The DNA was transferred via southern blotting and probed with a radioactively labelled LT-Ag PCR fragment (right panel). The blot was exposed for 24 h.

    Techniques Used: Molecular Weight, Transfection, Agarose Gel Electrophoresis, Staining, Southern Blot, Polymerase Chain Reaction

    SV40 replication and gene expression in CV-1 cells transfected with SV40 DNA. (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.
    Figure Legend Snippet: SV40 replication and gene expression in CV-1 cells transfected with SV40 DNA. (A) 100 ng of intramolecular religated SV40 viral DNA was transfected in CV-1 cells and cells were lysed 12 h, 24 h, 36 h, 2d and 7d post transfection. Protein lysates were subsequently analyzed for SV40 LT-Ag (Pab419 antibody) and VP1 expression (α-VP1 polyclonal rabbit serum) by SDS-page and Western Blotting. Staining of actin was used to ensure that equal protein amounts were loaded per lane. (B) Low molecular weight DNA was isolated from SV40 DNA transfected CV-1 cells at the indicated time points by HIRT extraction, 1 µg DNA was DpnI and EcoRI digested; DNA was separated on an agarose gel and stained with EtBr (left panel), followed by southern blotting and detection of viral DNA using a 32 PdCTP-labeled SV40 LT-Ag PCR fragment as a probe. The blot was exposed for 30 min. Numbers below the lanes correspond to the quantification of newly replicated DNA using a Fuji phosphoimager FLA7000 and MultiGauge software.

    Techniques Used: Expressing, Transfection, SDS Page, Western Blot, Staining, Molecular Weight, Isolation, Agarose Gel Electrophoresis, Southern Blot, Labeling, Polymerase Chain Reaction, Software

    MCVSyn replication assays in H1299, PFSK-1 and 293 cells. 5 µg low molecular weight DNA isolated from cell cultures at the indicated time points post-transfection with MCVSyn DNA was digested with DpnI and EcoRI, separated on an agarose gel and stained with EtBr (left panels), then transferred via southern blot and probed with a radioactively labelled LT-Ag PCR fragment [13] . The blot was exposed for 24 h and scanned using a Fuji phosphoimager FLA7000; MultiGauge software was used for quantification.
    Figure Legend Snippet: MCVSyn replication assays in H1299, PFSK-1 and 293 cells. 5 µg low molecular weight DNA isolated from cell cultures at the indicated time points post-transfection with MCVSyn DNA was digested with DpnI and EcoRI, separated on an agarose gel and stained with EtBr (left panels), then transferred via southern blot and probed with a radioactively labelled LT-Ag PCR fragment [13] . The blot was exposed for 24 h and scanned using a Fuji phosphoimager FLA7000; MultiGauge software was used for quantification.

    Techniques Used: Molecular Weight, Isolation, Transfection, Agarose Gel Electrophoresis, Staining, Southern Blot, Polymerase Chain Reaction, Software

    8) Product Images from "Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency"

    Article Title: Human Papillomavirus Type 18 cis-Elements Crucial for Segregation and Latency

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0135770

    HPV Replication assays. A Transient replication assay of HPV-18 URR plasmid E2BS #1 and #2 linker sequence mutants (0-Linker, 1-Linker (wt), 2-Linker, 3-Linker). 25 ng of the E1 and E2 expression vectors were cotransfected, and total DNA extracts were obtained 24 h and 48 h post-transfection. DNA was linearized using Hind III enzyme, and input DNA was eliminated by digestion with DpnI. Normalized qPCR data are presented in the left panel, and the Southern blot analysis is shown in the right panel. A 2-sample t-test was performed and statistically significant (P
    Figure Legend Snippet: HPV Replication assays. A Transient replication assay of HPV-18 URR plasmid E2BS #1 and #2 linker sequence mutants (0-Linker, 1-Linker (wt), 2-Linker, 3-Linker). 25 ng of the E1 and E2 expression vectors were cotransfected, and total DNA extracts were obtained 24 h and 48 h post-transfection. DNA was linearized using Hind III enzyme, and input DNA was eliminated by digestion with DpnI. Normalized qPCR data are presented in the left panel, and the Southern blot analysis is shown in the right panel. A 2-sample t-test was performed and statistically significant (P

    Techniques Used: Plasmid Preparation, Sequencing, Expressing, Transfection, Real-time Polymerase Chain Reaction, Southern Blot

    9) Product Images from "A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence"

    Article Title: A Comprehensive Analysis of Replicating Merkel Cell Polyomavirus Genomes Delineates the Viral Transcription Program and Suggests a Role for mcv-miR-M1 in Episomal Persistence

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1004974

    A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication. (A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI -resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI -sensitive input DNA are visible near the bottom of the blot.
    Figure Legend Snippet: A mcv-miR-M1 knockout mutant exhibits increased LT-Ag expression and enhanced viral DNA replication. (A) Predicted secondary structures of the wt (left) and mutated (right) mcv-miR-M1 pre-miRNA sequences. Regions encoding mature miRNAs are shown in bold, mutated nucleotides are highlighted in red. (B) Small RNA Northern Blot analysis of mcv-miR-M1 expression 24 and 48 hours after transfection of MCVSyn (left panel) or MCVSyn-hpko (right panel) genomes. Mock transfected cells or cells transfected with a plasmid containing the mcv-miR-M1 pre-miRNA cloned behind a CMV promoter served as negative and positive controls, respectively. Positions of pre- and mature miRNAs are marked by arrows. (C) Western Blot analysis of LT antigen expression in PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko at 4d post transfection. The position of LT-Ag is marked by an arrow. (D) Analysis of de novo replicated viral DNA by Southern Blot analysis of HIRT extracts prepared from PFSK-1 cells transfected with MCVSyn or MCVSyn-hpko genomes 4d post transfection. The position of DpnI -resistant replicated DNA is marked with an arrow. Faster migrating bands representing digestion products of the DpnI -sensitive input DNA are visible near the bottom of the blot.

    Techniques Used: Knock-Out, Mutagenesis, Expressing, Northern Blot, Transfection, Plasmid Preparation, Clone Assay, Western Blot, Southern Blot

    10) Product Images from "The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius"

    Article Title: The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.00137

    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.
    Figure Legend Snippet: Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Techniques Used: Methylation, Whole Genome Amplification, Sequencing, Negative Control, Agarose Gel Electrophoresis, Staining

    11) Product Images from "Repurposing a microfluidic formulation device for automated DNA construction"

    Article Title: Repurposing a microfluidic formulation device for automated DNA construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0242157

    DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.
    Figure Legend Snippet: DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.

    Techniques Used: Purification, Polymerase Chain Reaction, Generated, Chromatin Immunoprecipitation, Transformation Assay, Sequencing

    12) Product Images from "The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome"

    Article Title: The transcription map of HPV11 in U2OS cells adequately reflects the initial and stable replication phases of the viral genome

    Journal: Virology Journal

    doi: 10.1186/s12985-015-0292-6

    Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.
    Figure Legend Snippet: Transient, stable and amplificational replication of the HPV11wt and HPV11E8- genomes in U2OS cells. The mock-transfected cells were used as a negative control (A, lane 7, B, lane 23 and C, lane 10). The linearized HPV11 genome of 100 copies (A, lane 8, B, lane 24 and C, lane 11) and DpnI fragments (C, lane 12) was used as size markers, also indicated with arrows. (A) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-3) or E8- (lanes 4-6) genome. Extrachromosomal DNA was extracted via Hirt lysis at 3, 4 and 5 days post-transfection, digested with HindIII and with DpnI. The replication signal was detected via the Southern blot method with a radiolabelled HPV11 genome probe. (B) U2OS cells were transfected with 500 ng of the HPV11wt (lanes 1-11) or HPV11E8- (lanes 12-22) genome, together with 2 μg of the linearized pBabe-Neo construct. The transfected cells were selected with the antibiotic G418, and at 10 days post-transfection, the cells were split and cultivated under either subconfluent (lanes 2-6 and 13-17) or confluent conditions (lanes 7-11 and 18-22). Total DNA was extracted at the time points indicated at top of the figure, and 3 μg of each sample was analyzed as indicated in A . (C) Effect of E8˄E1 and E8˄E2 proteins on viral genome replication. U2OS cells were transfected with 500 ng of the HPV11E8- genome with increasing amounts of either the E8˄E1 or E8˄E2 expression plasmid. Total DNA was extracted at 4 days post-transfection, and 3 μg of each sample was analyzed as indicated in A . (D) The quantitation of HPV11wt and E8- genome DNA replication signals at different time points at subconfluent and confluent culture conditions. The signals were normalized to 10 th day time point. Shown is one of two independent experiments.

    Techniques Used: Transfection, Negative Control, Lysis, Southern Blot, Construct, Expressing, Plasmid Preparation, Quantitation Assay

    HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B) . (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1 A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.
    Figure Legend Snippet: HPV11 E1 expression from E1 plasmids and transient replication of the URR-plasmid in the presence of the E1 and E2 proteins. (A, B) Detection of the HA-epitope tagged HPV11 E1 protein from transiently transfected U2OS cells. The cells were transfected with 1-5000 ng of different HPV11 E1 expression plasmids (indicated at the top of the figure). Western blot analysis was performed at the 24 h time point to detect the HPV11 E1 protein (A) and the cellular marker α-tubulin (B) . (C) The HPV11 E1 and E2 proteins initiated DNA replication from the episomal HPV11 URR plasmid in transiently transfected U2OS cells. U2OS cells were co-transfected with 100 ng of the HPV11 URR plasmid, 100 ng of E2 and 1-5000 ng of different E1 expression plasmids. Extrachromosomal DNA was extracted at 24 and 48 h post-transfection via Hirt lysis, and ½ of each sample was analyzed as indicated in Figure 1 A. The replication signal was detected with a radiolabelled HPV11 URR specific probe. Mock-transfected U2OS cells were used as a negative control (lane 27), and 200 pg of the linearized HPV11 URR plasmid (lane 28) was used as a size marker. The linear HPV11 URR and DpnI fragments are indicated with arrows.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Western Blot, Marker, Lysis, Negative Control

    13) Product Images from "The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing"

    Article Title: The molecular biology and HPV drug responsiveness of cynomolgus macaque papillomaviruses support their use in the development of a relevant in vivo model for antiviral drug testing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0211235

    The physical state of the MfPV genomes during transient DNA replication in U2OS cells. U2OS cells were transfected with 4 μg E8^E2C mutant minicircle DNA of MfPV1 (lanes 1–3), MfPV5 (lanes 4–6), and MfPV8 (lanes 7–9). LMW DNA extracted via Hirt lysis 3, 4 and 5 days posttransfection (shown on the top), the samples were treated with DpnI, and analyzed by Southern blotting. Abbreviations: oc, open circular molecules; ccc, covalently closed circular molecules; lin, linear molecules; the numbers 1 and 2 refer to monomers and dimers, respectively.
    Figure Legend Snippet: The physical state of the MfPV genomes during transient DNA replication in U2OS cells. U2OS cells were transfected with 4 μg E8^E2C mutant minicircle DNA of MfPV1 (lanes 1–3), MfPV5 (lanes 4–6), and MfPV8 (lanes 7–9). LMW DNA extracted via Hirt lysis 3, 4 and 5 days posttransfection (shown on the top), the samples were treated with DpnI, and analyzed by Southern blotting. Abbreviations: oc, open circular molecules; ccc, covalently closed circular molecules; lin, linear molecules; the numbers 1 and 2 refer to monomers and dimers, respectively.

    Techniques Used: Transfection, Mutagenesis, Lysis, Southern Blot, Countercurrent Chromatography

    The transient replication of the MfPV1, 5 and 8 genomes in U2OS cells. U2OS cells were transfected with 5 μg of MfPV1 (lanes 1–3) and 4 μg of MfPV5 (lanes 7–9) and 8 (12–14) minicircle genomes. Low molecular weight (LMW) DNA was extracted at the indicated time points (days) and analyzed through 1D gel electrophoresis, followed by Southern blotting. Prior to analysis, the samples were treated with DpnI and specific linearizing endonuclease (SdaI for MfPV1, BamHI for MfPV5, and BglI for MfPV8) (Thermo Scientific). Lanes 4, 10 and 15 are size markers for linear MfPV1, 5 and 8 DNA, respectively. Lanes 5, 6 and 11 are size markers for the DpnI fragments of MfPV1, 5 and 8 genomes, respectively. DpnI, the fragments of DpnI-digested unreplicated genomes. Arrow marks the linear MfPV genomes.
    Figure Legend Snippet: The transient replication of the MfPV1, 5 and 8 genomes in U2OS cells. U2OS cells were transfected with 5 μg of MfPV1 (lanes 1–3) and 4 μg of MfPV5 (lanes 7–9) and 8 (12–14) minicircle genomes. Low molecular weight (LMW) DNA was extracted at the indicated time points (days) and analyzed through 1D gel electrophoresis, followed by Southern blotting. Prior to analysis, the samples were treated with DpnI and specific linearizing endonuclease (SdaI for MfPV1, BamHI for MfPV5, and BglI for MfPV8) (Thermo Scientific). Lanes 4, 10 and 15 are size markers for linear MfPV1, 5 and 8 DNA, respectively. Lanes 5, 6 and 11 are size markers for the DpnI fragments of MfPV1, 5 and 8 genomes, respectively. DpnI, the fragments of DpnI-digested unreplicated genomes. Arrow marks the linear MfPV genomes.

    Techniques Used: Transfection, Molecular Weight, Nucleic Acid Electrophoresis, Southern Blot

    Transient replication analysis of the MfPV wild-type and mutant genomes in the U2OS cell line. U2OS cells were transfected with 4 μg of wild-type (wt) or mutant minicircle genomes of MfPV5 (A), MfPV8 (B), and MfPV1 (C) defective in E1 (E1-), E2 (E2-) and/or E8^E2C (E8-) expression. For the complementation assay, the cells were cotransfected with E1- and E2-defective genomes (4 μg each). Episomal DNA was extracted via Hirt lysis at the indicated time points (days) and subjected to Southern blotting. Prior to analysis, the samples were treated with DpnI and specific endonucleases (Thermo Scientific). (A) In the case of MfPV5, the E8-E1- and E8-E2- double mutant genomes were used. MfPV5 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme BamHI, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with BamHI and SacI (only cuts the E2 mutant genome) to facilitate distinction between cotransfected plasmids. (B) MfPV8 wt (lines 1–3), E1- (lines 4–6) and E2- (lines 7–9) samples were digested with the linearizing enzyme BglI, while E1- plus E2- samples (lines 10–12) were double digested with BglI and BglII (only cuts the E2 mutant genome). (C) In the case of MfPV1, the E8-E1- and E8-E2- double mutant genomes were also used. MfPV1 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme Eam1105I, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with Eam1105I and HindIII (only cuts the E1 mutant genome). DpnI-treated, linear and double digested marker plasmids for MfPV5, MfPV8 or MfPV1 are shown in lanes 16 to 18. In the case of complementation assay, linear viral genome is indicated with arrows, double digested genome fragments with arrowheads. Abbreviations: E1-, E1 mutant genomes; E2-, E2 mutant genomes.
    Figure Legend Snippet: Transient replication analysis of the MfPV wild-type and mutant genomes in the U2OS cell line. U2OS cells were transfected with 4 μg of wild-type (wt) or mutant minicircle genomes of MfPV5 (A), MfPV8 (B), and MfPV1 (C) defective in E1 (E1-), E2 (E2-) and/or E8^E2C (E8-) expression. For the complementation assay, the cells were cotransfected with E1- and E2-defective genomes (4 μg each). Episomal DNA was extracted via Hirt lysis at the indicated time points (days) and subjected to Southern blotting. Prior to analysis, the samples were treated with DpnI and specific endonucleases (Thermo Scientific). (A) In the case of MfPV5, the E8-E1- and E8-E2- double mutant genomes were used. MfPV5 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme BamHI, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with BamHI and SacI (only cuts the E2 mutant genome) to facilitate distinction between cotransfected plasmids. (B) MfPV8 wt (lines 1–3), E1- (lines 4–6) and E2- (lines 7–9) samples were digested with the linearizing enzyme BglI, while E1- plus E2- samples (lines 10–12) were double digested with BglI and BglII (only cuts the E2 mutant genome). (C) In the case of MfPV1, the E8-E1- and E8-E2- double mutant genomes were also used. MfPV1 wt (lines 1–3), E8-E1- (lines 4–6) and E8-E2- (lines 7–9) samples were digested with the linearizing enzyme Eam1105I, while E8-E1- plus E8-E2- samples (lines 10–12) were double digested with Eam1105I and HindIII (only cuts the E1 mutant genome). DpnI-treated, linear and double digested marker plasmids for MfPV5, MfPV8 or MfPV1 are shown in lanes 16 to 18. In the case of complementation assay, linear viral genome is indicated with arrows, double digested genome fragments with arrowheads. Abbreviations: E1-, E1 mutant genomes; E2-, E2 mutant genomes.

    Techniques Used: Mutagenesis, Transfection, Expressing, Lysis, Southern Blot, Marker

    The inhibition of initial amplification of the MfPV8 genomes by the high-risk HPV-specific compounds. U2OS cells were transfected with 4 μg of the MfPV8 minicircle genomes and grown with the compounds for 5 days at the indicated concentrations. Genomic DNA was extracted, MfPV8 DNA was linearized and treated with DpnI, and analyzed by Southern blotting. The DNA replication signals of the MfPV8 were quantified (ImageQuant TL 8.1, GE Healthcare), expressed relative to the vehicle control (DMSO), and the inhibition curves and IC50 values for each compound were calculated by curve fitting ([inhibitor] vs. concentration, variable slope, 4 parameters). Error bars represent the standard deviation from 2–3 independent experiments.
    Figure Legend Snippet: The inhibition of initial amplification of the MfPV8 genomes by the high-risk HPV-specific compounds. U2OS cells were transfected with 4 μg of the MfPV8 minicircle genomes and grown with the compounds for 5 days at the indicated concentrations. Genomic DNA was extracted, MfPV8 DNA was linearized and treated with DpnI, and analyzed by Southern blotting. The DNA replication signals of the MfPV8 were quantified (ImageQuant TL 8.1, GE Healthcare), expressed relative to the vehicle control (DMSO), and the inhibition curves and IC50 values for each compound were calculated by curve fitting ([inhibitor] vs. concentration, variable slope, 4 parameters). Error bars represent the standard deviation from 2–3 independent experiments.

    Techniques Used: Inhibition, Amplification, Transfection, Southern Blot, Concentration Assay, Standard Deviation

    14) Product Images from "OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments"

    Article Title: OEPR Cloning: an Efficient and Seamless Cloning Strategy for Large- and Multi-Fragments

    Journal: Scientific Reports

    doi: 10.1038/srep44648

    Schematic diagram and flowchart of OEPR cloning for insertion of multiple fragments. The cloning method requires three procedures: ( A ) amplification of insert fragments a, b and c with primers F1 and R1, F2 and R2 and F3 and R3, respectively; ( B ) exponential amplification using fragments (a, b, and c) generated in the first PCR and reverse primer R; and ( C ) digestion by DpnI to eliminate the parent DNA and direct transformation into chemically competent Top10F’ cells.
    Figure Legend Snippet: Schematic diagram and flowchart of OEPR cloning for insertion of multiple fragments. The cloning method requires three procedures: ( A ) amplification of insert fragments a, b and c with primers F1 and R1, F2 and R2 and F3 and R3, respectively; ( B ) exponential amplification using fragments (a, b, and c) generated in the first PCR and reverse primer R; and ( C ) digestion by DpnI to eliminate the parent DNA and direct transformation into chemically competent Top10F’ cells.

    Techniques Used: Clone Assay, Amplification, Generated, Polymerase Chain Reaction, Transformation Assay

    Schematic diagram and flowchart of OEPR cloning for large fragment insertion. The cloning method requires only three steps: ( A ) amplification of insert fragments using primer F1 and R1; ( B ) exponential amplification using fragments generated in the first PCR and reverse primer R; and ( C ) digestion by DpnI to eliminate the parent DNA and direct transformation into chemically competent Top10F’ cells.
    Figure Legend Snippet: Schematic diagram and flowchart of OEPR cloning for large fragment insertion. The cloning method requires only three steps: ( A ) amplification of insert fragments using primer F1 and R1; ( B ) exponential amplification using fragments generated in the first PCR and reverse primer R; and ( C ) digestion by DpnI to eliminate the parent DNA and direct transformation into chemically competent Top10F’ cells.

    Techniques Used: Clone Assay, Amplification, Generated, Polymerase Chain Reaction, Transformation Assay

    15) Product Images from "Repurposing a microfluidic formulation device for automated DNA construction"

    Article Title: Repurposing a microfluidic formulation device for automated DNA construction

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0242157

    DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.
    Figure Legend Snippet: DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.

    Techniques Used: Purification, Polymerase Chain Reaction, Generated, Chromatin Immunoprecipitation, Transformation Assay, Sequencing

    16) Product Images from "The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells"

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0116151

    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
    Figure Legend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Techniques Used: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.
    Figure Legend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Techniques Used: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

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    Thermo Fisher fast digest dpni
    DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and <t>DpnI-digested</t> <t>PCR</t> products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.
    Fast Digest Dpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fast digest dpni/product/Thermo Fisher
    Average 96 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fast digest dpni - by Bioz Stars, 2022-10
    96/100 stars
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    97
    Thermo Fisher digestion with dpni
    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected <t>with</t> 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized <t>with</t> <t>BglI</t> and treated with <t>DpnI</t> to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.
    Digestion With Dpni, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 97/100, based on 83 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/digestion with dpni/product/Thermo Fisher
    Average 97 stars, based on 83 article reviews
    Price from $9.99 to $1999.99
    digestion with dpni - by Bioz Stars, 2022-10
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    DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.

    Journal: PLoS ONE

    Article Title: Repurposing a microfluidic formulation device for automated DNA construction

    doi: 10.1371/journal.pone.0242157

    Figure Lengend Snippet: DNA design and construction workflow on and off a microfluidic device. User requests are sent from the TeselaGen DESIGN module to j5 to generate a scar-less combinatorial Golden Gate DNA assembly protocol. The automation module allocates resources, and initiates construction. Gel-purified, BsaI and DpnI-digested PCR products are generated off-chip according to the protocol, and then supplied to and then assembled together on the microfluidic chip. Assembled DNA (plasmids) are transformed into a host system of interest ( E . coli ) off-chip. The assembled DNA can then be sequence validated, and resulting reports generated.

    Article Snippet: DpnI digest and purificationFollowing PCR amplification, residual (methylated) DNA template in each PCR reaction was DpnI digested at 37°C for 1 h. Each 110 μL digest reaction consisted of 95 μL PCR product, 11 μL 10x Fast Digest buffer, 1.5 μL Fast Digest DpnI (Thermo Fisher Scientific; Waltham, MA), and 2.5 μL deionized water.

    Techniques: Purification, Polymerase Chain Reaction, Generated, Chromatin Immunoprecipitation, Transformation Assay, Sequencing

    Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Mutational analysis of the functions of the putative E2C1 and E2C2 proteins expressed from promoter P3385. A: Southern blot analysis of the transient replication of different HPV18 genome mutants. U2OS cells were transfected with 2 µg of HPV18 wt , E8-, E2C2-, 2-E2C-, E8-E2C2-, E8-2-E2C-, E2C-1 or E8-E2C1- minicircles. Genomic DNA was extracted 3 and 5 days after the transfection, linearized with BglI and treated with DpnI to distinguish between transfected and replicated DNA. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. Size markers for linearized HPV18 (lanes 11 and 17) and for the DpnI-digested fragments of the HPV18 (lanes 12 and 18) are included B: U2OS cells were transfected with 2 µg of the indicated HPV18 genome mutants, and genomic DNA was extracted 3 and 5 days after the transfection. Samples were digested with BglI and DpnI, and the replication of different HPV18 genome mutants was measured by a qPCR-based analysis of the viral relative copy number (C N ). The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. The average and standard deviation (SD) of at least three independent experiments are shown. C: U2OS cells were transfected with the expression plasmids of HPV18 full-length E2, E8E2, E2C1 and E2C2. IP-Western Blot analyses was performed to evaluate the expression levels and MWs of different HPV18 E2 variants. Arrows indicate the positions of the full-length E2 (lane 1), E8 ∧ E2 (lane 2), E2C1 (lane 3) and E2C2 (lane 4). Mock transfection is shown in lane 5. D and E: U2OS cells were transfected with 2 µg of HPV18 wt minicircle plasmid alone or together with different concentrations (10, 50 and 250 ng) of either the expression vector or the E2C-1 or E2C-2 proteins. The E8ˇE2 expression vector (250 ng) was added as a control. Genomic DNA was extracted 3 and 4 days after the transfection, linearized with BglI and treated with DpnI. A qPCR-based analysis of the viral relative copy number (C N ) was performed. The value obtained from the HPV18 wt 3-day time point was set to 1, and the C N values of other samples are expressed relative to this point. Panel D shows the effect of overexpression of E2C-1 on HPV18 wt replication, whereas panel E shows the effect of E2C-2.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Hybridization, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing, Western Blot, Plasmid Preparation, Over Expression

    Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Journal: PLoS ONE

    Article Title: The Transcription Map of Human Papillomavirus Type 18 during Genome Replication in U2OS Cells

    doi: 10.1371/journal.pone.0116151

    Figure Lengend Snippet: Southern blot analysis of HPV18 genome replication in U2OS cells that were transfected with 500 ng of the HPV18 genome miniplasmid. Extrachromosomal DNA samples were digested with BglI to linearize the HPV18 miniplasmid and with DpnI to fragment the bacterially produced input non-replicated plasmid. The samples were analyzed by Southern blotting after hybridization with an HPV18-specific radiolabeled probe. The DNA extraction timepoints (22, 46 and 71 hours) are indicated at the top. Extrachromosomal DNA extracted from mock-transfected U2OS cells was used as a negative control (lane 4). Size markers for the linearized HPV18 genome (lane 5, indicated by arrow) and for the DpnI+BglI digested fragments of the HPV18 genome miniplasmid DNA (lane 6) are included.

    Article Snippet: The samples were linearized by digestion with BglI (Thermo Scientific), and the bacterially produced input DNA was fragmented by digestion with DpnI (Thermo Scientific).

    Techniques: Southern Blot, Transfection, Produced, Plasmid Preparation, Hybridization, DNA Extraction, Negative Control

    Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Journal: Frontiers in Microbiology

    Article Title: The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

    doi: 10.3389/fmicb.2018.00137

    Figure Lengend Snippet: Identification of methylated recognition sequences in S. acidocaldarius genomic DNA by digestion assays. Restriction enzymes BsuRI (A) and BamHI, DpnI, and MboI (B) were used to highlight the presence/absence of methylated 5′-GGCC-3′ and 5′-GATC-3′ palindromes, respectively, in genomic DNA of S. acidocaldarius . Two types of substrates were digested: genomic DNA (gDNA) and a whole genome amplification of the genomic DNA (WGA). WGA is identical to gDNA in terms of nucleic sequence but it does not contain epigenetic marks (see material and methods). This negative control is only used in (A) . The addition of different restriction enzymes is symbolized by a positive sign “+” while reaction mix without restriction enzyme is represented by a negative sign “–”. Molecular size markers (GeneRuler DNA Ladder, Thermo Scientific) were loaded in both gels (M). Digestion patterns were obtained in 0.8% agarose gel stained with ethidium bromide and visualized under UV light.

    Article Snippet: Digestion assays Genomic DNA isolated from asynchronous S. acidocaldarius cells was cleaved independently with a specific REase in order to decipher the presence or the absence of m4C or m6A: BsuRI, BamHI, MboI, and DpnI (Thermo Scientific).

    Techniques: Methylation, Whole Genome Amplification, Sequencing, Negative Control, Agarose Gel Electrophoresis, Staining

    Description of the cooperation between Dnmt1/Sp1 and Dnmt1-PCNA-UHRF1 to maintain the DNA methylation. ( A ) The kinetic of ChIP experiments was realized at an indicated time after cell (U87) synchronization (72 hours of serum deprivation) and α-amanitin treatment. ChIP was performed with the EZ ChIP kit (Millipore, Molsheim, France) and with indicated antibodies. The amount of immunoprecipitated promoter was quantified by semiquantitative PCR and normalized to input. ( B ) Identification by methylation-specific PCR (MSP) of the methylation status of CG dinucleotides localized into the −526/−408 region of the SLIT2 gene in U87 cells. MSPa was designed to investigate the methylation status of CG dinucleotides #1, #2, and #3; MSPb was designed to investigate the methylation status of CG dinucleotide #4; and MSPc was designed to investigate the methylation status of CG dinucleotides #5 and #6. The green box indicates the presence of Sp1 binding site according to the use of the Patch program. U = unmethylated; M = methylated. Black boxes represent primers. ( C ) Impact of siRNA-induced decrease expression of Sp1 on the methylation status of specific CG dinucloetides of the SLIT2 genes. Methylation status of specific CG dicnuleotides was determined by methylation sensitive restriction assay (MSRA). DNA methylation blocks the cleavage of BstUI and DpnI when the CGCG and GATCg sequences are methylated, respectively. Thus, when DNA is initially unmethylated, no or weak DNA is amplified by PCR. Black triangles represent primers.

    Journal: Genes & Cancer

    Article Title: Dnmt1/Transcription Factor Interactions

    doi: 10.1177/1947601910373794

    Figure Lengend Snippet: Description of the cooperation between Dnmt1/Sp1 and Dnmt1-PCNA-UHRF1 to maintain the DNA methylation. ( A ) The kinetic of ChIP experiments was realized at an indicated time after cell (U87) synchronization (72 hours of serum deprivation) and α-amanitin treatment. ChIP was performed with the EZ ChIP kit (Millipore, Molsheim, France) and with indicated antibodies. The amount of immunoprecipitated promoter was quantified by semiquantitative PCR and normalized to input. ( B ) Identification by methylation-specific PCR (MSP) of the methylation status of CG dinucleotides localized into the −526/−408 region of the SLIT2 gene in U87 cells. MSPa was designed to investigate the methylation status of CG dinucleotides #1, #2, and #3; MSPb was designed to investigate the methylation status of CG dinucleotide #4; and MSPc was designed to investigate the methylation status of CG dinucleotides #5 and #6. The green box indicates the presence of Sp1 binding site according to the use of the Patch program. U = unmethylated; M = methylated. Black boxes represent primers. ( C ) Impact of siRNA-induced decrease expression of Sp1 on the methylation status of specific CG dinucloetides of the SLIT2 genes. Methylation status of specific CG dicnuleotides was determined by methylation sensitive restriction assay (MSRA). DNA methylation blocks the cleavage of BstUI and DpnI when the CGCG and GATCg sequences are methylated, respectively. Thus, when DNA is initially unmethylated, no or weak DNA is amplified by PCR. Black triangles represent primers.

    Article Snippet: Methylation sensitive restriction assay (MSRA) was performed by digesting 1 µg of genomic DNA with BstUI (Ozyme, Saint-Quentin-en-Yvelines, France) or DpnI (Fermentas, St. Rémy Les Chevreuse, France) for 16 hours.

    Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Polymerase Chain Reaction, Methylation, Binding Assay, Expressing, Restriction Assay, Amplification