r0176  (New England Biolabs)

Journal: PLoS Pathogens

Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

doi: 10.1371/journal.ppat.1009230

Figure Lengend Snippet: Effect of linker mutations on core DNA. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and were harvested seven days post-transfection. HBV NC-associated DNA (core DNA) was extracted from cytoplasmic lysate without ( A ) or with TURBO DNase digestion ( B ), and detected by Southern blot analysis. Input plasmid DNA (but not viral replicative DNA) was removed with Dpn I before Southern blot analysis in A . The viral DNA signals (RC and immature DS DNA) digested by the nuclease were marked by the dotted boxes ( B ). RC, RC DNA; SS, SS DNA. C. Quantitative results from multiple experiments shown in A. SS DNA synthesis efficiency was determined by normalizing the levels of SS DNA to the pgRNA signals in Fig 4 . RC DNA synthesis efficiency was determined by normalizing the levels of RC DNA to the SS DNA signals. The efficiencies from WT was set to 1.0. *, P

Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

Techniques: Transfection, Expressing, Construct, Southern Blot, Plasmid Preparation, DNA Synthesis

Journal: PLoS Pathogens

Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

doi: 10.1371/journal.ppat.1009230

Figure Lengend Snippet: Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein. The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L - derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P

Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

Techniques: Countercurrent Chromatography, Mutagenesis, Transfection, Southern Blot

Journal: PLoS Pathogens

Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

doi: 10.1371/journal.ppat.1009230

Figure Lengend Snippet: Effects of linker mutations on CCC DNA formation. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids ( A ), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA ( B ), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P

Article Snippet: Where indicated, Dpn I (NEB) digestion, instead of TURBO DNase, was used to remove the transfected plasmid after DNA purification [ ].

Techniques: Countercurrent Chromatography, Transfection, Expressing, Construct, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis

Journal: PLoS ONE

Article Title: Non-Hemagglutinating Flaviviruses: Molecular Mechanisms for the Emergence of New Strains via Adaptation to European Ticks

doi: 10.1371/journal.pone.0007295

Figure Lengend Snippet: Mutagenesis of the infectious clone of TBEV. The plasmid pGGVs 660–1982 H [29] that contains the partial PrM-E gene fragment between nucleotides 660–1882 of the Vs virus genome was used as a template in PCR to synthesize megaprimers E-1171 A/G (genome positions and lengths are specified). Primers used to produce megaprimers, with targeted mutations (circles) are represented by thick arrows. Subsequently the megaprimer without the other pair of primers was used to amplify plasmid pGGVs 660–1982 H (solid circular line). The produced linear newly-synthesized complementary ssDNA molecules (nicked circular dotted line) with acquired mutations were annealed during the last step of PCR, randomly producing twice-nic ked circular DNA. Parent Dam+ methylated DNA of the pGGVs 660–1982 H was removed by DpnI endonuclease digestion to facilitate clone selection.

Article Snippet: To facilitate screening, the dam-methylated bacterial (template) DNA was digested with 40 U of DpnI (New England Biolabs) at 37°C for 1 h. Following this, PCR products were electroporated into AbleK bacterial cells (Stratagene) and selected clones were completely sequenced.

Techniques: Mutagenesis, Plasmid Preparation, Polymerase Chain Reaction, Produced, Synthesized, Methylation, Selection

Journal: Molecular Systems Biology

Article Title: DamID in C. elegans reveals longevity-associated targets of DAF-16/FoxO

doi: 10.1038/msb.2010.54

Figure Lengend Snippet: The DAM identification (DamID) procedure in C. elegans . ( A ) How DamID works. A fusion protein consisting of DNA adenine methyltransferase (DAM) and the protein of interest methylates GATC sites near binding sites. Genomic DNA is digested with Dpn I, which cuts only methylated GATC sites. Adaptors are added, and DNA is digested with Dpn II (which cuts at unmethylated GATC sites) to assure selective amplification of methylated DNA. A parallel DAM-only experiment is also performed to control for non-specific methylation. Samples are then labeled and hybridized to arrays. ( B ) Schematic of plasmid constructs used for preparation of transgenic strains. ( C , D ) Transgene expression. Nuclear localization of GFP was detected in UL1782 animals (expressing DAM∷DAF-16∷GFP) (marked with arrows in C) in body wall muscle and anterior bulb of pharynx (circled) following heat shock. UL1787 animals (expressing DAM∷GFP) (D) do not show nuclear localization. ( E ) DAF-16∷DAM methylation profile for ist-1 , one of several evolutionarily conserved FoxO targets identified. ( F ) Average distribution of methylation (DAF-16∷DAM versus DAM) from peak center for 1135 peaks identified.

Article Snippet: Briefly, genomic DNA was isolated using a DNAeasy kit (Qiagen) and then digested overnight with Dpn I (New England Biolabs (NEB)) to cut at GAm TC sites.

Techniques: Binding Assay, Methylation, Amplification, Labeling, Plasmid Preparation, Construct, Transgenic Assay, Expressing

Journal: Nature Communications

Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

doi: 10.1038/ncomms11301

Figure Lengend Snippet: Detection of 6mA by DA-6mA-seq. ( a ) Flowchart of DA-6mA-seq. DpnI cleaves fully methylated G(6mA)TC sites, whereas the cleavage of DpnII is hindered by hemi- or fully-methylated 6mA. After treatment with restriction enzyme, DNA segments are further sheared by sonication to ∼300 bp followed by standard Illumina DNA library construction procedures. ( b ) DA-6mA-seq identifies consistent 6mA sites as reported previously 8 . ( c ) The genomic distribution of 6mA sites in promoter, genic and intergenic regions. Promoter is defined as −1,000 to +1,000 bp region around transcription start sites (TSS). ( d ) The periodic distribution pattern of base-resolution 6mA sites identified by DA-6mA-seq around TSS.

Article Snippet: DA-6mA-seq Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S).

Techniques: Methylation, Sonication

Journal: Nature Communications

Article Title: Characterization of eukaryotic DNA N6-methyladenine by a highly sensitive restriction enzyme-assisted sequencing

doi: 10.1038/ncomms11301

Figure Lengend Snippet: DpnI cleavage assay on fully- or hemi-methylated GATC and CATC/GATG DNA probes. The PAGE gel shows the formation of digested products. ( a ) DNA probes containing fully methylated GATC (F-GATC, 10 pmol) or hemi-methylated GATC (H-GATC, 10 pmol) were treated with DpnI (10 units) for 30 min or overnight at 37 °C. ( b ) DNA probes containing fully methylated CATC/GATG (F-CATC, 10 pmol) or hemi-methylated CATC/GATG (H-CATC, 10 pmol) were treated with DpnI for 30 min or overnight. All sequences used are listed in Supplementary Table 1 .

Article Snippet: DA-6mA-seq Approximately 10 ng purified genomic DNA was digested in 0.5h or 12 h by using 20 U DpnI (NEB, Cat. no. R0176S).

Techniques: Cleavage Assay, Methylation, Polyacrylamide Gel Electrophoresis