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  • 86
    ATUM unimmunoprecipitated dna
    Unimmunoprecipitated Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 86/100, based on 14 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ATUM k562 dna
    K562 Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 85/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATUM input dna
    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
    Input Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    r dna  (ATUM)
    92
    ATUM r dna
    Design of logic gates constructed with MTs. For the YES gate, the l <t>-DNA1</t> signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r <t>-DNA</t> to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.
    R Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ATUM cy3 labeled dna
    Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of <t>Cy3-labeled</t> total genomic <t>DNA</t> versus Cy5-labeled, E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.
    Cy3 Labeled Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 88/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM salmon sperm dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Salmon Sperm Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 321 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM high quality dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    High Quality Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM adaptor ligated dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Adaptor Ligated Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM sperm dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Sperm Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 68 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    ATUM mbd2 enriched dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Mbd2 Enriched Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM bisulfite converted dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Bisulfite Converted Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 80 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM dna 2 0 grna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Dna 2 0 Grna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    ATUM genomic dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Genomic Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 96/100, based on 2002 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ip dna  (ATUM)
    90
    ATUM ip dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    Ip Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 90/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM h3k4me3 chip dna
    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the <t>DNA–protein</t> complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. <t>Preimmune</t> IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.
    H3k4me3 Chip Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM high dose hbv dna
    HBc mutant I97L exhibited an immature secretion phenotype by in vivo hydrodynamic delivery. BALB/c mice were hydrodynamically injected with 30 μg of plasmid DNAs of a WT <t>HBV</t> ( adr ) or mutant I97L. At 3 dpi, the mice were sacrificed, and both liver and serum samples were collected for assays of HBV replication and virion secretion. (A) Extracellular HBV DNAs were prepared directly from equal volumes of mouse sera of WT and mutant I97L injections before Southern blot analysis. An immature secretion phenotype of HBc mutant I97L in tissue culture was recapitulated in this in vivo experimental setting. The red asterisk highlights the lessened abundance of fully mature full-length RC <t>DNA</t> associated with mutant I97L. (B) Purified HBV particles in mouse sera were pooled from fractions 9 to 12 by gradient centrifugation (see Materials and Methods) before Southern blot analysis. Unlike the WT, mutant I97L displayed an excessive number of immature genomes. Dane, Dane particles refer to enveloped virions. NakedC, nonenveloped naked core particles were not detected in the higher-density fractions (see the text). The results here represent one of two independent repeat experiments. (C) No naked core particles can be detected in serum samples of wild-type HBV DNA-injected BALB/c mice. Serum samples were subjected to cesium chloride gradient centrifugation, and HBsAg-positive fractions were detected by HBsAg ELISA (see Materials and Methods). No positive signal was detected in any fractions by HBeAg ELISA. (D) Intracellular core-associated HBV DNA was extracted from the liver tissue and subjected to Southern blotting. The full-length RC form was almost undetectable in mutant I97L. Each lane here represents different DNA samples from each block of approximately 100 mg of liver mass dissected from each injected mouse. This same protocol was used in most of the experiments in other figures. The dotted vertical line indicates splicing from the same gel. The results here represent one of three independent repeat experiments. The amounts of total DNA were quantified by measuring the intensities of full-length RC and full-length SS DNAs using densitometry and ImageJ software. The averaged total DNAs are calculated from two mice injected with WT HBV DNA and normalized to the averaged value from three mice injected with mutant I97L. (E) Detection of only slightly reduced amounts of HBV core protein in the liver lysates of mutant I97L by Western blotting. Each lane represents one liver sample from one injected mouse. (F) Plasmid SEAP encoding a secretable alkaline phosphatase was coinjected with an HBV tandem dimer in panel A. The SEAP activities in the sera indicated similar transfection efficiencies between WT and I97L in hydrodynamic delivery.
    High Dose Hbv Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATUM crispr grna designing tool
    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled <t>CRISPR</t> guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and <t>gRNA</t> separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
    Crispr Grna Designing Tool, supplied by ATUM, used in various techniques. Bioz Stars score: 91/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    ATUM atum grna design tool
    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled <t>CRISPR</t> guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and <t>gRNA</t> separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9
    Atum Grna Design Tool, supplied by ATUM, used in various techniques. Bioz Stars score: 93/100, based on 25 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATUM dnazyme subunits
    Schematic representation of the XOR + NOR logic gate that consists of the DNA template (DNA1), the <t>DNAzyme</t> subunits (DNA2–DNA7), the substrate (hairpin DNA8, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.
    Dnazyme Subunits, supplied by ATUM, used in various techniques. Bioz Stars score: 91/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATUM minion low input library preparation kit
    Function and Taxonomy of Ice Wedge soil. Ice wedge soil metagenomes sequenced using the <t>MinION</t> rapid and <t>low</t> <t>input</t> <t>kit</t> (A) Taxonomy at the domain level (B) Clusters of Orthologous Groups (COG) categories (level 2).
    Minion Low Input Library Preparation Kit, supplied by ATUM, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ATUM crispr gdna design tool dna2 0
    Function and Taxonomy of Ice Wedge soil. Ice wedge soil metagenomes sequenced using the <t>MinION</t> rapid and <t>low</t> <t>input</t> <t>kit</t> (A) Taxonomy at the domain level (B) Clusters of Orthologous Groups (COG) categories (level 2).
    Crispr Gdna Design Tool Dna2 0, supplied by ATUM, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    ATUM dnmt pulled
    Function and Taxonomy of Ice Wedge soil. Ice wedge soil metagenomes sequenced using the <t>MinION</t> rapid and <t>low</t> <t>input</t> <t>kit</t> (A) Taxonomy at the domain level (B) Clusters of Orthologous Groups (COG) categories (level 2).
    Dnmt Pulled, supplied by ATUM, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    l dna1  (ATUM)
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    ATUM l dna1
    Design of logic gates constructed with MTs. For the YES gate, the l <t>-DNA1</t> signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.
    L Dna1, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    cy5  (ATUM)
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    ATUM cy5
    Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of Cy3-labeled total genomic DNA versus <t>Cy5-labeled,</t> E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.
    Cy5, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 27 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    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

    Journal: Biochemical Journal

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

    doi: 10.1042/BJ20111417

    Figure Lengend 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

    Article Snippet: ChIP-CHOP assay 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).

    Techniques: 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.

    Journal: Biochemical Journal

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

    doi: 10.1042/BJ20111417

    Figure Lengend 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.

    Article Snippet: ChIP-CHOP assay 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).

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

    Design of logic gates constructed with MTs. For the YES gate, the l -DNA1 signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Design of logic gates constructed with MTs. For the YES gate, the l -DNA1 signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Construct, Concentration Assay, Conjugation Assay

    Orthogonal control of swarming of MTs. a Schematic representation and fluorescence microscopy image of MTs with different rigidity. The flexible MTs (red) were conjugated with r -DNA1 and r -DNA2, while the rigid MTs (green) were conjugated with r -DNA5 and r -DNA6. b Upon inputting l -DNA1, the flexible MTs associated into circular shaped swarms through hybridization of r -DNA1 and r -DNA2 with l -DNA1 and appeared in red. c Green swarms with translational motion associated with rigid MTs were formed through hybridization of r -DNA5 and r -DNA6 with l -DNA5. d Swarms with translational and circular motions were simultaneously formed in response to the introduction of both input DNA signals. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin was 0.3 µM and that of each l -DNA was 0.6 µM. Scale bars: 20 µm

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Orthogonal control of swarming of MTs. a Schematic representation and fluorescence microscopy image of MTs with different rigidity. The flexible MTs (red) were conjugated with r -DNA1 and r -DNA2, while the rigid MTs (green) were conjugated with r -DNA5 and r -DNA6. b Upon inputting l -DNA1, the flexible MTs associated into circular shaped swarms through hybridization of r -DNA1 and r -DNA2 with l -DNA1 and appeared in red. c Green swarms with translational motion associated with rigid MTs were formed through hybridization of r -DNA5 and r -DNA6 with l -DNA5. d Swarms with translational and circular motions were simultaneously formed in response to the introduction of both input DNA signals. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin was 0.3 µM and that of each l -DNA was 0.6 µM. Scale bars: 20 µm

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Fluorescence, Microscopy, Hybridization, Concentration Assay, Conjugation Assay

    Regulation of swarming mode of MTs by tuning their physical properties. a , b Fluorescence microscopy images of rigid and flexible MTs, respectively; schematic illustrations showing different swarming modes of the MTs. c Time-lapse fluorescence microscopy images showing the formation of a swarm group with a circular motion from flexible red and green MTs with lengths of 22 ± 13 µm and 16 ± 9 µm (average ± s.d.), respectively. d Time-lapse fluorescence microscopy images showing the formation of swarms with a circular motion in the presence of l -DNA1 (0.6 µM). e Dissociation of a swarm group with a circular motion triggered by the d -DNA input signal (0.6 µM). The swarm group was made of ~300 single MTs. f Change of the association ratio over time upon addition of l -DNA1 and d -DNA. The concentration of the red and green MTs was 0.6 µM each, and the conjugation ratio of r -DNA1 or r -DNA2 to MTs was ~100%. The concentration of kinesin was 0.3 µM. Scale bar: 20 µm ( a – e ). Error bar: s.e.m

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Regulation of swarming mode of MTs by tuning their physical properties. a , b Fluorescence microscopy images of rigid and flexible MTs, respectively; schematic illustrations showing different swarming modes of the MTs. c Time-lapse fluorescence microscopy images showing the formation of a swarm group with a circular motion from flexible red and green MTs with lengths of 22 ± 13 µm and 16 ± 9 µm (average ± s.d.), respectively. d Time-lapse fluorescence microscopy images showing the formation of swarms with a circular motion in the presence of l -DNA1 (0.6 µM). e Dissociation of a swarm group with a circular motion triggered by the d -DNA input signal (0.6 µM). The swarm group was made of ~300 single MTs. f Change of the association ratio over time upon addition of l -DNA1 and d -DNA. The concentration of the red and green MTs was 0.6 µM each, and the conjugation ratio of r -DNA1 or r -DNA2 to MTs was ~100%. The concentration of kinesin was 0.3 µM. Scale bar: 20 µm ( a – e ). Error bar: s.e.m

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Fluorescence, Microscopy, Concentration Assay, Conjugation Assay

    Preparation of MTs and control of their swarming. a Conjugation of r -DNA to azide-functionalized MTs by a click reaction. b TAMRA (red) and FAM (green) labeled DNA-conjugated MTs with sequences of T 16 ( r -DNA1) and (TTG) 5 ( r -DNA2), respectively. Scale bar: 5 µm. c Schematic of red and green MTs gliding on kinesins. d Time-lapse images showing motility of rigid MTs with lengths of 5 ± 2 and 5 ± 3 µm (average ± s.d.) conjugated to T 16 and (TTG) 5 , respectively. Scale bar: 10 µm. e Schematic of the association of red and green MTs by l -DNA1 and their dissociation by d -DNA via extraction of l -DNA1 through a DNA strand exchange reaction. f Association and dissociation of MTs by DNA base pair interaction. g Time-lapse images showing swarming of MTs. h Dissociation of swarms of MTs. Scale bar: 20 µm ( g and h ). i Change of association ratio over time after introduction of l -DNA1 (0.6 µM) and d -DNA (0.6 µM). The concentration of the red and green MTs was 0.6 µM each, and the kinesin concentration was 0.3 µM. Error bar: standard error (s.e.m.)

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Preparation of MTs and control of their swarming. a Conjugation of r -DNA to azide-functionalized MTs by a click reaction. b TAMRA (red) and FAM (green) labeled DNA-conjugated MTs with sequences of T 16 ( r -DNA1) and (TTG) 5 ( r -DNA2), respectively. Scale bar: 5 µm. c Schematic of red and green MTs gliding on kinesins. d Time-lapse images showing motility of rigid MTs with lengths of 5 ± 2 and 5 ± 3 µm (average ± s.d.) conjugated to T 16 and (TTG) 5 , respectively. Scale bar: 10 µm. e Schematic of the association of red and green MTs by l -DNA1 and their dissociation by d -DNA via extraction of l -DNA1 through a DNA strand exchange reaction. f Association and dissociation of MTs by DNA base pair interaction. g Time-lapse images showing swarming of MTs. h Dissociation of swarms of MTs. Scale bar: 20 µm ( g and h ). i Change of association ratio over time after introduction of l -DNA1 (0.6 µM) and d -DNA (0.6 µM). The concentration of the red and green MTs was 0.6 µM each, and the kinesin concentration was 0.3 µM. Error bar: standard error (s.e.m.)

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Conjugation Assay, Labeling, Concentration Assay

    Light-switched, repeated swarming of MTs. a Reversible hydrogen bonding of photoresponsive DNA ( p -DNA) by light-induced cis–trans isomerization of azobenzene. b Schematic of selective association and dissociation of p -DNA-conjugated MTs under UV and visible light irradiation, respectively. c Visible light ( λ = 480 nm, I = 1.3 mW cm −2 ) induced isomerization of azobenzene from the cis to the trans form, which resulted in translational swarming of the p -DNA-conjugated rigid MTs with length of 4 ± 2 µm (average ± s.d.). The swarms were then exposed to UV light ( λ = 365 nm, I = 0.4 mW cm −2 ) for 6 min that isomerized the azobenzene from the trans to the cis form. The swarms dissociated into single MTs within 12 min of the onset of photoirradiation. This cycle was repeated three times. Visible light irradiation to flexible MTs with length of 12 ± 1 µm (average ± s.d.) generated swarms with circular motion. d Changes in the association ratio upon repeated irradiation by visible and UV light. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of p -DNA1 or p -DNA2 to MTs was ~100%. The concentration of kinesin was 0.8 µM. Scale bars: 20 µm. Error bar: s.e.m.

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Light-switched, repeated swarming of MTs. a Reversible hydrogen bonding of photoresponsive DNA ( p -DNA) by light-induced cis–trans isomerization of azobenzene. b Schematic of selective association and dissociation of p -DNA-conjugated MTs under UV and visible light irradiation, respectively. c Visible light ( λ = 480 nm, I = 1.3 mW cm −2 ) induced isomerization of azobenzene from the cis to the trans form, which resulted in translational swarming of the p -DNA-conjugated rigid MTs with length of 4 ± 2 µm (average ± s.d.). The swarms were then exposed to UV light ( λ = 365 nm, I = 0.4 mW cm −2 ) for 6 min that isomerized the azobenzene from the trans to the cis form. The swarms dissociated into single MTs within 12 min of the onset of photoirradiation. This cycle was repeated three times. Visible light irradiation to flexible MTs with length of 12 ± 1 µm (average ± s.d.) generated swarms with circular motion. d Changes in the association ratio upon repeated irradiation by visible and UV light. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of p -DNA1 or p -DNA2 to MTs was ~100%. The concentration of kinesin was 0.8 µM. Scale bars: 20 µm. Error bar: s.e.m.

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Irradiation, Generated, Concentration Assay, Conjugation Assay

    Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of Cy3-labeled total genomic DNA versus Cy5-labeled, E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.

    Journal: Genes & Development

    Article Title: E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints

    doi: 10.1101/gad.949802

    Figure Lengend Snippet: Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of Cy3-labeled total genomic DNA versus Cy5-labeled, E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.

    Article Snippet: Each hybridization reaction included the Cy5- and Cy3-labeled DNA, 20 μg of human Cot-1 DNA enriched for repetitive DNA sequences such as the Alu and Kpn family members (Invitrogen), 40 μg of yeast tRNA (Sigma), 2.2× SSC, 0.1% SDS, 0.12% BSA (Sigma), 28% formamide (Sigma), and 5.7% dextran sulfate (Sigma), in a total of 50 μL.

    Techniques: Labeling, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Immunoprecipitation

    Strategy for mammalian factor location analysis using the 1.5K DNA microarray. ( A ) are indicated in red. Primary human cells (WI-38) were cross-linked and sonicated, and chromatin was immunoprecipitated with anti-E2F antibodies. The resulting ChIP-enriched DNA was purified, amplified by LM-PCR together with total genomic DNA, labeled with the Cy5 and Cy3 fluorophores using random priming, and hybridized to the 1.5K microarray in the presence of Cot-1 DNA to reduce nonspecific hybridization. ( B ).

    Journal: Genes & Development

    Article Title: E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints

    doi: 10.1101/gad.949802

    Figure Lengend Snippet: Strategy for mammalian factor location analysis using the 1.5K DNA microarray. ( A ) are indicated in red. Primary human cells (WI-38) were cross-linked and sonicated, and chromatin was immunoprecipitated with anti-E2F antibodies. The resulting ChIP-enriched DNA was purified, amplified by LM-PCR together with total genomic DNA, labeled with the Cy5 and Cy3 fluorophores using random priming, and hybridized to the 1.5K microarray in the presence of Cot-1 DNA to reduce nonspecific hybridization. ( B ).

    Article Snippet: Each hybridization reaction included the Cy5- and Cy3-labeled DNA, 20 μg of human Cot-1 DNA enriched for repetitive DNA sequences such as the Alu and Kpn family members (Invitrogen), 40 μg of yeast tRNA (Sigma), 2.2× SSC, 0.1% SDS, 0.12% BSA (Sigma), 28% formamide (Sigma), and 5.7% dextran sulfate (Sigma), in a total of 50 μL.

    Techniques: Microarray, Sonication, Immunoprecipitation, Chromatin Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Labeling, Hybridization

    Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the DNA–protein complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. Preimmune IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.

    Journal: Biochemical Journal

    Article Title: Regulation of the human involucrin gene promoter by co-activator proteins

    doi: 10.1042/BJ20031653

    Figure Lengend Snippet: Fra-1, FosB and JunB proteins bind to the proximal and distal AP-1 sites of the involucrin promoter and immunoprecipitate with CBP ( A ) Nuclear extract from SCC12 cells was incubated with radiolabelled oligonucleotide probes corresponding to the −125 (AP1-1) or −2122 (AP1-5) AP-1 sites of the involucrin promoter. Anti-AP-1 antibodies were used to identify specific components of the DNA–protein complexes. Unlabelled competitor or mutant oligonucleotides were included in some reactions to demonstrate the binding specificity. No extract was used as the negative control. The positions of the supershifted (SS) complexes and the free probe are shown. ( B ) CBP was immunoprecipitated from stable clones (IP CBP) as described in the Materials and methods section. Preimmune IgG was used as the negative control antibody (IP IgG). Immunoprecipitated proteins were subjected to Western-blot analysis with anti-Fra-1, FosB and JunB antibodies. Blots were incubated with anti-CBP antibody to determine the relative amounts of immunoprecipitated protein in each lane. These experiments were repeated three times with similar results. Representative gels and blots are shown.

    Article Snippet: Cells were washed with PBS and lysed in immunoprecipitation buffer containing protease inhibitors for 30 min at 4 °C, sheared, and centrifuged at 10000 g for 10 min. Supernatants were cleared with 2 μg of sheared salmon sperm DNA, 20 μl of preimmune serum and 20 μl of Protein A/G–Sepharose beads for 2 h at 4 °C.

    Techniques: Incubation, Mutagenesis, Binding Assay, Negative Control, Immunoprecipitation, Clone Assay, Western Blot

    HBc mutant I97L exhibited an immature secretion phenotype by in vivo hydrodynamic delivery. BALB/c mice were hydrodynamically injected with 30 μg of plasmid DNAs of a WT HBV ( adr ) or mutant I97L. At 3 dpi, the mice were sacrificed, and both liver and serum samples were collected for assays of HBV replication and virion secretion. (A) Extracellular HBV DNAs were prepared directly from equal volumes of mouse sera of WT and mutant I97L injections before Southern blot analysis. An immature secretion phenotype of HBc mutant I97L in tissue culture was recapitulated in this in vivo experimental setting. The red asterisk highlights the lessened abundance of fully mature full-length RC DNA associated with mutant I97L. (B) Purified HBV particles in mouse sera were pooled from fractions 9 to 12 by gradient centrifugation (see Materials and Methods) before Southern blot analysis. Unlike the WT, mutant I97L displayed an excessive number of immature genomes. Dane, Dane particles refer to enveloped virions. NakedC, nonenveloped naked core particles were not detected in the higher-density fractions (see the text). The results here represent one of two independent repeat experiments. (C) No naked core particles can be detected in serum samples of wild-type HBV DNA-injected BALB/c mice. Serum samples were subjected to cesium chloride gradient centrifugation, and HBsAg-positive fractions were detected by HBsAg ELISA (see Materials and Methods). No positive signal was detected in any fractions by HBeAg ELISA. (D) Intracellular core-associated HBV DNA was extracted from the liver tissue and subjected to Southern blotting. The full-length RC form was almost undetectable in mutant I97L. Each lane here represents different DNA samples from each block of approximately 100 mg of liver mass dissected from each injected mouse. This same protocol was used in most of the experiments in other figures. The dotted vertical line indicates splicing from the same gel. The results here represent one of three independent repeat experiments. The amounts of total DNA were quantified by measuring the intensities of full-length RC and full-length SS DNAs using densitometry and ImageJ software. The averaged total DNAs are calculated from two mice injected with WT HBV DNA and normalized to the averaged value from three mice injected with mutant I97L. (E) Detection of only slightly reduced amounts of HBV core protein in the liver lysates of mutant I97L by Western blotting. Each lane represents one liver sample from one injected mouse. (F) Plasmid SEAP encoding a secretable alkaline phosphatase was coinjected with an HBV tandem dimer in panel A. The SEAP activities in the sera indicated similar transfection efficiencies between WT and I97L in hydrodynamic delivery.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: HBc mutant I97L exhibited an immature secretion phenotype by in vivo hydrodynamic delivery. BALB/c mice were hydrodynamically injected with 30 μg of plasmid DNAs of a WT HBV ( adr ) or mutant I97L. At 3 dpi, the mice were sacrificed, and both liver and serum samples were collected for assays of HBV replication and virion secretion. (A) Extracellular HBV DNAs were prepared directly from equal volumes of mouse sera of WT and mutant I97L injections before Southern blot analysis. An immature secretion phenotype of HBc mutant I97L in tissue culture was recapitulated in this in vivo experimental setting. The red asterisk highlights the lessened abundance of fully mature full-length RC DNA associated with mutant I97L. (B) Purified HBV particles in mouse sera were pooled from fractions 9 to 12 by gradient centrifugation (see Materials and Methods) before Southern blot analysis. Unlike the WT, mutant I97L displayed an excessive number of immature genomes. Dane, Dane particles refer to enveloped virions. NakedC, nonenveloped naked core particles were not detected in the higher-density fractions (see the text). The results here represent one of two independent repeat experiments. (C) No naked core particles can be detected in serum samples of wild-type HBV DNA-injected BALB/c mice. Serum samples were subjected to cesium chloride gradient centrifugation, and HBsAg-positive fractions were detected by HBsAg ELISA (see Materials and Methods). No positive signal was detected in any fractions by HBeAg ELISA. (D) Intracellular core-associated HBV DNA was extracted from the liver tissue and subjected to Southern blotting. The full-length RC form was almost undetectable in mutant I97L. Each lane here represents different DNA samples from each block of approximately 100 mg of liver mass dissected from each injected mouse. This same protocol was used in most of the experiments in other figures. The dotted vertical line indicates splicing from the same gel. The results here represent one of three independent repeat experiments. The amounts of total DNA were quantified by measuring the intensities of full-length RC and full-length SS DNAs using densitometry and ImageJ software. The averaged total DNAs are calculated from two mice injected with WT HBV DNA and normalized to the averaged value from three mice injected with mutant I97L. (E) Detection of only slightly reduced amounts of HBV core protein in the liver lysates of mutant I97L by Western blotting. Each lane represents one liver sample from one injected mouse. (F) Plasmid SEAP encoding a secretable alkaline phosphatase was coinjected with an HBV tandem dimer in panel A. The SEAP activities in the sera indicated similar transfection efficiencies between WT and I97L in hydrodynamic delivery.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: Mutagenesis, In Vivo, Mouse Assay, Injection, Plasmid Preparation, Southern Blot, Purification, Gradient Centrifugation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Software, Western Blot, Transfection

    Reduction in the persistence of intracellular HBV DNA level of mutant I97L in the mouse liver by hydrodynamic delivery. (A) Liver samples were collected at 3 days or 1 week after the hydrodynamic injection of 14 μg of HBV plasmid DNAs of WT or mutant I97L. The intrahepatic levels of viral DNA and RNA were determined using Southern and Northern blot analyses. The averaged amounts of total viral DNA were measured and calculated as described in Fig. 2D . Some differences in the intensities of viral RNAs are not representative and are likely due to experimental variations from individual littermate. The viral DNA level of mutant I97L appeared to be much lower than those of WT HBV at day 3 or week 1 postinjection. The intrahepatic levels of ribosomal 28S and 18S RNAs were included as the sample loading control for Northern blotting. The intrahepatic levels of capsid particles were determined by native agarose gel electrophoresis and subsequent immunoblotting with anti-HBc antibodies. The results here represent one of four independent repeat experiments. (B) The serum HBeAg titers in mice receiving WT (●) or I97L (■) DNAs were determined at the indicated time points with an enzyme immunoassay (see Materials and Methods). S/Co, signal/cutoff. Positivity for HBeAg was defined as an S/Co value of ≥ 1. (C) More rapid decay of viral DNA of mutant I97L in hepatocytes was observed by hydrodynamic delivery. As described above, liver samples were collected for Southern and Northern blot analysis at 3 days or 2 weeks after the hydrodynamic injections of 10 μg of WT or 30 μg of mutant I97L HBV DNA. Although the viral DNA levels were comparable between 10 μg of WT and 30 μg of I97L at 3 dpi (left panel), no viral DNA signal of I97L was detectable at 2 weeks postinjection (right panel). (D) Detection of extracellular levels of HBeAg was as described above.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: Reduction in the persistence of intracellular HBV DNA level of mutant I97L in the mouse liver by hydrodynamic delivery. (A) Liver samples were collected at 3 days or 1 week after the hydrodynamic injection of 14 μg of HBV plasmid DNAs of WT or mutant I97L. The intrahepatic levels of viral DNA and RNA were determined using Southern and Northern blot analyses. The averaged amounts of total viral DNA were measured and calculated as described in Fig. 2D . Some differences in the intensities of viral RNAs are not representative and are likely due to experimental variations from individual littermate. The viral DNA level of mutant I97L appeared to be much lower than those of WT HBV at day 3 or week 1 postinjection. The intrahepatic levels of ribosomal 28S and 18S RNAs were included as the sample loading control for Northern blotting. The intrahepatic levels of capsid particles were determined by native agarose gel electrophoresis and subsequent immunoblotting with anti-HBc antibodies. The results here represent one of four independent repeat experiments. (B) The serum HBeAg titers in mice receiving WT (●) or I97L (■) DNAs were determined at the indicated time points with an enzyme immunoassay (see Materials and Methods). S/Co, signal/cutoff. Positivity for HBeAg was defined as an S/Co value of ≥ 1. (C) More rapid decay of viral DNA of mutant I97L in hepatocytes was observed by hydrodynamic delivery. As described above, liver samples were collected for Southern and Northern blot analysis at 3 days or 2 weeks after the hydrodynamic injections of 10 μg of WT or 30 μg of mutant I97L HBV DNA. Although the viral DNA levels were comparable between 10 μg of WT and 30 μg of I97L at 3 dpi (left panel), no viral DNA signal of I97L was detectable at 2 weeks postinjection (right panel). (D) Detection of extracellular levels of HBeAg was as described above.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: Mutagenesis, Injection, Plasmid Preparation, Northern Blot, Agarose Gel Electrophoresis, Mouse Assay, Enzyme-linked Immunosorbent Assay

    Mutant I97L can synthesize full-length RC form by in vitro endogenous DNA polymerase reaction (EPR). (A) Two different hypotheses for the cause-effect relationship between genome maturation and virion secretion (see Discussion for details). The left panel postulates that an intracellular defect in genome maturation is the primary cause of the extracellular immature secretion of mutant I97L. The right panel postulates that an overly efficient and less selective envelopment is the primary cause of the intracellular deficiency in the fully mature RC DNA in mutant I97L. (B) As described in the Fig. 5 legend, BALB/c mice were hydrodynamically injected with four different HBV constructs (WT, I97L, P130T, and I97L/P130T). At 3 dpi, liver lysate and pooled sera were used to purify HBV capsids for EPR as described previously (see Materials and Methods). In vitro -elongated viral DNAs were extracted after EPR. As an internal control, intracellular capsids were measured by native agarose gel electrophoresis, followed by Western blotting using anti-core antibody. Red asterisks highlight the fully mature full-length, double-stranded DNA of the RC form. **, the minor difference in the mobility of SS DNA between WT and I97L (lanes 1 and 2) is not reproducible, probably due to experimental variation in salt concentrations between these two particular samples. The results represent one of three independent repeat experiments.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: Mutant I97L can synthesize full-length RC form by in vitro endogenous DNA polymerase reaction (EPR). (A) Two different hypotheses for the cause-effect relationship between genome maturation and virion secretion (see Discussion for details). The left panel postulates that an intracellular defect in genome maturation is the primary cause of the extracellular immature secretion of mutant I97L. The right panel postulates that an overly efficient and less selective envelopment is the primary cause of the intracellular deficiency in the fully mature RC DNA in mutant I97L. (B) As described in the Fig. 5 legend, BALB/c mice were hydrodynamically injected with four different HBV constructs (WT, I97L, P130T, and I97L/P130T). At 3 dpi, liver lysate and pooled sera were used to purify HBV capsids for EPR as described previously (see Materials and Methods). In vitro -elongated viral DNAs were extracted after EPR. As an internal control, intracellular capsids were measured by native agarose gel electrophoresis, followed by Western blotting using anti-core antibody. Red asterisks highlight the fully mature full-length, double-stranded DNA of the RC form. **, the minor difference in the mobility of SS DNA between WT and I97L (lanes 1 and 2) is not reproducible, probably due to experimental variation in salt concentrations between these two particular samples. The results represent one of three independent repeat experiments.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: Mutagenesis, In Vitro, Electron Paramagnetic Resonance, Mouse Assay, Injection, Construct, Agarose Gel Electrophoresis, Western Blot

    Mutant I97L replicates less efficiently than wild-type HBV in three different kinds of immunodeficient mice. (A) IFN-α receptor knockout (IFNAR −/− ) mice were hydrodynamically injected with 30 μg of DNA of WT or mutant I97L HBV. Mouse liver was collected on 3 dpi, and HBV DNA was extracted for Southern blot analysis. Each lane represents one viral DNA sample extracted from 100 mg of the liver mass of one injected mouse. The overall viral DNA synthesis of mutant I97L is significantly reduced relative to WT HBV. The results here represent one of two independent repeat experiments. (B) STAT1 −/− mice were hydrodynamically injected with 30 μg of HBV plasmid DNA. The viral DNA levels of mutant I97L, particularly the RC form, were greatly reduced in the liver at 3 dpi by Southern blotting. Each lane represents one viral DNA sample extracted from 100 mg of the liver mass of one injected mouse. The averaged amounts of total viral DNA were measured and calculated as described in Fig. 2D . (C, left panel) At different time points postinjection into NOD/SCID mice, intracellular core-associated HBV DNA in the liver (upper panel) and extracellular HBV DNA in the sera (lower panel) were compared between WT HBV and mutant I97L by Southern blotting. In both panels, each lane represents pooled HBV DNA samples from three to four mice in the same experimental group. (Upper right panel) A faster decline rate of mutant I97L SS DNA was observed in a time course experiment. (Lower right panel) Similar levels of serum HBeAg were observed by ELISA in the same time course experiment. The results here represent one of two repeat experiments. (D) No naked core particles can be detected in serum samples of HBV DNA-injected NOD/SCID mice. Serum samples were subjected to cesium chloride gradient centrifugation, and HBsAg-positive fractions were detected by HBsAg ELISA (see Materials and Methods). No positive signal was detected in all fractions by HBeAg ELISA. (E) Anti-core antibody can be detected in the serum samples of hydrodynamically injected BALB/c mice but not in immunodeficient NOD/SCID mice. Anti-core antibody was measured by the ELISA (see Materials and Methods). 4/4, four of four mice were serum anti-HBc positive; 0/4, none of four mice was serum anti-HBc positive.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: Mutant I97L replicates less efficiently than wild-type HBV in three different kinds of immunodeficient mice. (A) IFN-α receptor knockout (IFNAR −/− ) mice were hydrodynamically injected with 30 μg of DNA of WT or mutant I97L HBV. Mouse liver was collected on 3 dpi, and HBV DNA was extracted for Southern blot analysis. Each lane represents one viral DNA sample extracted from 100 mg of the liver mass of one injected mouse. The overall viral DNA synthesis of mutant I97L is significantly reduced relative to WT HBV. The results here represent one of two independent repeat experiments. (B) STAT1 −/− mice were hydrodynamically injected with 30 μg of HBV plasmid DNA. The viral DNA levels of mutant I97L, particularly the RC form, were greatly reduced in the liver at 3 dpi by Southern blotting. Each lane represents one viral DNA sample extracted from 100 mg of the liver mass of one injected mouse. The averaged amounts of total viral DNA were measured and calculated as described in Fig. 2D . (C, left panel) At different time points postinjection into NOD/SCID mice, intracellular core-associated HBV DNA in the liver (upper panel) and extracellular HBV DNA in the sera (lower panel) were compared between WT HBV and mutant I97L by Southern blotting. In both panels, each lane represents pooled HBV DNA samples from three to four mice in the same experimental group. (Upper right panel) A faster decline rate of mutant I97L SS DNA was observed in a time course experiment. (Lower right panel) Similar levels of serum HBeAg were observed by ELISA in the same time course experiment. The results here represent one of two repeat experiments. (D) No naked core particles can be detected in serum samples of HBV DNA-injected NOD/SCID mice. Serum samples were subjected to cesium chloride gradient centrifugation, and HBsAg-positive fractions were detected by HBsAg ELISA (see Materials and Methods). No positive signal was detected in all fractions by HBeAg ELISA. (E) Anti-core antibody can be detected in the serum samples of hydrodynamically injected BALB/c mice but not in immunodeficient NOD/SCID mice. Anti-core antibody was measured by the ELISA (see Materials and Methods). 4/4, four of four mice were serum anti-HBc positive; 0/4, none of four mice was serum anti-HBc positive.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: Mutagenesis, Mouse Assay, Knock-Out, Injection, Southern Blot, DNA Synthesis, Plasmid Preparation, Enzyme-linked Immunosorbent Assay, Gradient Centrifugation

    HBV core variants I97L and P130T exhibited abnormal behaviors in viral DNA synthesis and virion secretion. (A) Two frequent natural mutations of HBV core (HBc) antigen occur at amino acids 97 and 130 in chronic hepatitis B patients. The image is a schematic representation of the fold of the HBV capsid protein monomer based on the published crystal structure (PDB code 1QGT ) by use of the PyMOL program. Red, wild-type amino acid; green, HBc mutants. Cell culture phenotypes are as explained below for panels B and C. (B) Illustrations of mature and immature HBV genomes. pgRNA, pregenomic RNA; SS DNA, single-stranded (–) DNA reversed transcribed from pgRNA (+); RC DNA, partially double-stranded relaxed circle DNA. DL, low-abundance double-stranded linear DNA, which can be separated from RC DNA after a longer period of electrophoresis. A dotted line of RC molecules represents the single-strand gap region with a variable size in an HBV population. (C) Different Southern blot patterns of virion-associated HBV DNA genomes from the wild type and HBc mutants. (D) Diagram of the trilateral relationships among persistence, genome maturity, and virion secretion.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: HBV core variants I97L and P130T exhibited abnormal behaviors in viral DNA synthesis and virion secretion. (A) Two frequent natural mutations of HBV core (HBc) antigen occur at amino acids 97 and 130 in chronic hepatitis B patients. The image is a schematic representation of the fold of the HBV capsid protein monomer based on the published crystal structure (PDB code 1QGT ) by use of the PyMOL program. Red, wild-type amino acid; green, HBc mutants. Cell culture phenotypes are as explained below for panels B and C. (B) Illustrations of mature and immature HBV genomes. pgRNA, pregenomic RNA; SS DNA, single-stranded (–) DNA reversed transcribed from pgRNA (+); RC DNA, partially double-stranded relaxed circle DNA. DL, low-abundance double-stranded linear DNA, which can be separated from RC DNA after a longer period of electrophoresis. A dotted line of RC molecules represents the single-strand gap region with a variable size in an HBV population. (C) Different Southern blot patterns of virion-associated HBV DNA genomes from the wild type and HBc mutants. (D) Diagram of the trilateral relationships among persistence, genome maturity, and virion secretion.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: DNA Synthesis, Cell Culture, Electrophoresis, Southern Blot

    Correlation between HBV genome maturation and in vivo persistence. Both intracellular and extracellular viral DNAs were compared among four different groups of BALB/c mice hydrodynamically injected with 30 μg of plasmid DNAs of WT, mutant I97L, mutant P130T, and double mutant I97L/P130T, respectively. (A) Extracellular virion-associated HBV DNAs were prepared from pooled mouse sera of the same experimental group at 3 and 7 dpi, followed by Southern blotting. As highlighted by a red asterisk, fully mature full-length RC DNAs (lanes 2 and 4) in mutant I97L virions were significantly reduced. Mutation P130T appeared to partially upshift the immature SS DNA of mutant I97L to the higher-MW RC form at 3 dpi (lane 2 versus lane 4). The RC/SS ratio reflects the degree of genome maturity. Because of the smearing signals of the nascent RC replicative intermediates, quantitation of the RC DNA here was done by measuring all of the signals above the full-length SS DNA position. In the middle panel, mutation I97L resulted in higher levels of extracellular virions at 3 dpi by native agarose gel and Western blot analysis with anti-core antibody. In the bottom panel, poor persistence of I97L viral DNA was not rescued by P130T at 1 week postinjection (lane 6 versus lane 8). (B) Intracellular core-associated HBV DNA was extracted from liver tissues at 3 days, 1 week, and 2 weeks postinjection before Southern blot analysis. The characteristic hypermaturation phenotype of single mutant P130T was most pronounced at 1 week postinjection (red asterisk in the middle panel). Intracellular viral DNA of mutant P130T was also more persistent than WT and I97L mutants at 1 and 2 weeks postinjection. Mutation I97L appeared to be dominant over mutation P130T in vivo , since both the intracellular deficiency in RC DNA and poor persistence of mutant I97L were not significantly rescued by the mutation P130T. The results here represent one of three independent repeat experiments.

    Journal: Journal of Virology

    Article Title: Persistence of Hepatitis B Virus DNA and the Tempos between Virion Secretion and Genome Maturation in a Mouse Model

    doi: 10.1128/JVI.01001-19

    Figure Lengend Snippet: Correlation between HBV genome maturation and in vivo persistence. Both intracellular and extracellular viral DNAs were compared among four different groups of BALB/c mice hydrodynamically injected with 30 μg of plasmid DNAs of WT, mutant I97L, mutant P130T, and double mutant I97L/P130T, respectively. (A) Extracellular virion-associated HBV DNAs were prepared from pooled mouse sera of the same experimental group at 3 and 7 dpi, followed by Southern blotting. As highlighted by a red asterisk, fully mature full-length RC DNAs (lanes 2 and 4) in mutant I97L virions were significantly reduced. Mutation P130T appeared to partially upshift the immature SS DNA of mutant I97L to the higher-MW RC form at 3 dpi (lane 2 versus lane 4). The RC/SS ratio reflects the degree of genome maturity. Because of the smearing signals of the nascent RC replicative intermediates, quantitation of the RC DNA here was done by measuring all of the signals above the full-length SS DNA position. In the middle panel, mutation I97L resulted in higher levels of extracellular virions at 3 dpi by native agarose gel and Western blot analysis with anti-core antibody. In the bottom panel, poor persistence of I97L viral DNA was not rescued by P130T at 1 week postinjection (lane 6 versus lane 8). (B) Intracellular core-associated HBV DNA was extracted from liver tissues at 3 days, 1 week, and 2 weeks postinjection before Southern blot analysis. The characteristic hypermaturation phenotype of single mutant P130T was most pronounced at 1 week postinjection (red asterisk in the middle panel). Intracellular viral DNA of mutant P130T was also more persistent than WT and I97L mutants at 1 and 2 weeks postinjection. Mutation I97L appeared to be dominant over mutation P130T in vivo , since both the intracellular deficiency in RC DNA and poor persistence of mutant I97L were not significantly rescued by the mutation P130T. The results here represent one of three independent repeat experiments.

    Article Snippet: However, hydrodynamic injection with high-dose HBV DNA (≥20 μg DNA/mouse) could induce alpha/beta interferon (IFN-α/β), which in turn suppressed HBV replication ( ).

    Techniques: In Vivo, Mouse Assay, Injection, Plasmid Preparation, Mutagenesis, Southern Blot, Quantitation Assay, Agarose Gel Electrophoresis, Western Blot

    Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Overview of In-Fusion enzyme-based cloning of a single sgRNA. a Schematics of the final assembled CRISPR guide RNA cassette, along with the location of primers is shown in the top panel. b Using a set of two universal primers (p1F and g2R) and two sgRNA-specific primers (g1F and p1R), two fragments, A and B, are PCR-amplified using pEN-Chimera-ccdB plasmid as a template that contains AtU6-26(P) promoter and gRNA separated by the ccdB gene (Fig. 1 ). This PCR incorporates the 20-nt protospacer sgRNA sequence to the 3′ end of AtU6-26 promoter in fragments A, and a 15-bp overlap of the 3′ end of the protospacer sgRNA to the 5′ end of fragment B. c These two fragments are then fused using the In-Fusion ® HD cloning kit with the Cas9-containig pDe-Cas9 fragment, which is amplified with primers 3-AvrII and 5-MluI or obtained by digestion with Avr II/ Mlu I restriction enzymes. Alternatively, fragments A and B can also be fused with pUC57GW amplified with primers 3-AvrII and 5-MluI, which contains the attL1 and attL2 sites for subsequent Gateway ® LR cloning in a plant expression destination vector that contains R1 and R2 sites such as pDe-Cas9

    Article Snippet: For the Cas9-D10A nickase, two 20 nucleotides-long guide RNAs (gRNA) for each gene (YFP, NbPDS and mCherry) were selected using an online CRISPR gRNA designing tool (DNA 2.0 Incorporation; https://www.dna20.com/eCommerce/Cas9/input ).

    Techniques: Clone Assay, CRISPR, Polymerase Chain Reaction, Amplification, Plasmid Preparation, Sequencing, Expressing

    Mutation detection using Agrobacterium -mediated transient expression in N. benthamiana. a Schematic illustrations showing the locations of gRNAs, PAM positions, and primers in the genes , NbPDS , PIP2 - 1 - mCherry and YFP targeted for genome editing. b Resolvase assay for detecting mutation in NbPDS and mCherry targeted by the gRNA constructs pDe-Cas9-D10A-gNbPDS and pDe-Cas9-D10A-gmCherry. High fidelity PCR was performed with primers NbPDS-F and NbPDS-R for the NbPDS target, and mCherry-F and mCherry-R for the mCherry target (panel A and Table 1 ) using template DNA isolated from leaf spots on N. benthamiana infiltrated with Agrobacterium tumefaciens carrying the indicated CRISPR–Cas9 constructs; N. benthamiana was stably transformed with the PIP2 - 1 - mCherry gene, which served as a transgene target for the gmCherry gRNA. Amplicons were subjected to Resolvase assays (Guide-it™ Mutation Detection Kit, Cat#631443, Clontech) and reactions were run on a 1.5% agarose gels. Digested fragments, which result from gRNA-induced mutations, are indicated by *, and their sizes are ~ 570 and ~ 150 bp for NbPDS, and ~ 277 and ~ 150 bp for mCherry. Undigested fragments are 727 bp for NbPDS and 427 bp for mCherry. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs. c Surveyor (CEL II)—nuclease assay for detecting mutation in a transiently transformed YFP gene carried on pGWB415-35S::HA-YFP plasmid. PCR was performed with forward primer (35SpromF) and reverse primer (EYFPStopXhoR) (panel A and Table 1 ) using template DNA from Wild Type N. benthamiana co-transformed with pGWB415-35S::HA-YFP along with either pDe-Cas9-D10A-gYFP or pDe-Cas9-D10A-gNbPDS (negative control). Surveyor (CEL II)—nuclease assay was performed with amplicons and reactions were run on 1.5% agarose gels (mismatch-specific Surveyor nuclease, Surveyor ® Mutation Detection Kit; Cat#706025, IDTdna.com). gRNA-induced mutations are revealed by the digested ~ 700 and ~ 400 bp fragments, marked by *; the undigested fragment is 1067 bp, which is shown in both lanes. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs

    Journal: Plant Methods

    Article Title: A highly efficient ligation-independent cloning system for CRISPR/Cas9 based genome editing in plants

    doi: 10.1186/s13007-017-0236-9

    Figure Lengend Snippet: Mutation detection using Agrobacterium -mediated transient expression in N. benthamiana. a Schematic illustrations showing the locations of gRNAs, PAM positions, and primers in the genes , NbPDS , PIP2 - 1 - mCherry and YFP targeted for genome editing. b Resolvase assay for detecting mutation in NbPDS and mCherry targeted by the gRNA constructs pDe-Cas9-D10A-gNbPDS and pDe-Cas9-D10A-gmCherry. High fidelity PCR was performed with primers NbPDS-F and NbPDS-R for the NbPDS target, and mCherry-F and mCherry-R for the mCherry target (panel A and Table 1 ) using template DNA isolated from leaf spots on N. benthamiana infiltrated with Agrobacterium tumefaciens carrying the indicated CRISPR–Cas9 constructs; N. benthamiana was stably transformed with the PIP2 - 1 - mCherry gene, which served as a transgene target for the gmCherry gRNA. Amplicons were subjected to Resolvase assays (Guide-it™ Mutation Detection Kit, Cat#631443, Clontech) and reactions were run on a 1.5% agarose gels. Digested fragments, which result from gRNA-induced mutations, are indicated by *, and their sizes are ~ 570 and ~ 150 bp for NbPDS, and ~ 277 and ~ 150 bp for mCherry. Undigested fragments are 727 bp for NbPDS and 427 bp for mCherry. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs. c Surveyor (CEL II)—nuclease assay for detecting mutation in a transiently transformed YFP gene carried on pGWB415-35S::HA-YFP plasmid. PCR was performed with forward primer (35SpromF) and reverse primer (EYFPStopXhoR) (panel A and Table 1 ) using template DNA from Wild Type N. benthamiana co-transformed with pGWB415-35S::HA-YFP along with either pDe-Cas9-D10A-gYFP or pDe-Cas9-D10A-gNbPDS (negative control). Surveyor (CEL II)—nuclease assay was performed with amplicons and reactions were run on 1.5% agarose gels (mismatch-specific Surveyor nuclease, Surveyor ® Mutation Detection Kit; Cat#706025, IDTdna.com). gRNA-induced mutations are revealed by the digested ~ 700 and ~ 400 bp fragments, marked by *; the undigested fragment is 1067 bp, which is shown in both lanes. Lower panel shows a representative N. benthamiana leaf infiltrated in three independent sites with the indicated CRISPR–Cas9 constructs

    Article Snippet: For the Cas9-D10A nickase, two 20 nucleotides-long guide RNAs (gRNA) for each gene (YFP, NbPDS and mCherry) were selected using an online CRISPR gRNA designing tool (DNA 2.0 Incorporation; https://www.dna20.com/eCommerce/Cas9/input ).

    Techniques: Mutagenesis, Expressing, Construct, Polymerase Chain Reaction, Isolation, CRISPR, Stable Transfection, Transformation Assay, Nuclease Assay, Plasmid Preparation, Negative Control

    Schematic representation of the XOR + NOR logic gate that consists of the DNA template (DNA1), the DNAzyme subunits (DNA2–DNA7), the substrate (hairpin DNA8, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Journal: Chemical Science

    Article Title: A label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c7sc04007e

    doi: 10.1039/c7sc04007e

    Figure Lengend Snippet: Schematic representation of the XOR + NOR logic gate that consists of the DNA template (DNA1), the DNAzyme subunits (DNA2–DNA7), the substrate (hairpin DNA8, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Article Snippet: In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs.

    Techniques: Sequencing

    Schematic representation of the AND logic gate that consists of the DNAzyme subunits (DNA1 and DNA2), the substrate (hairpin DNA3, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Journal: Chemical Science

    Article Title: A label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c7sc04007e

    doi: 10.1039/c7sc04007e

    Figure Lengend Snippet: Schematic representation of the AND logic gate that consists of the DNAzyme subunits (DNA1 and DNA2), the substrate (hairpin DNA3, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Article Snippet: In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs.

    Techniques: Sequencing

    Schematic representation of the XOR + AND logic gate that consists of the DNAzyme subunits (DNA1–DNA6), the substrate (hairpin DNA7, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Journal: Chemical Science

    Article Title: A label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c7sc04007e

    doi: 10.1039/c7sc04007e

    Figure Lengend Snippet: Schematic representation of the XOR + AND logic gate that consists of the DNAzyme subunits (DNA1–DNA6), the substrate (hairpin DNA7, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Article Snippet: In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs.

    Techniques: Sequencing

    Schematic representation of the XOR logic gate that consists of the DNAzyme subunits (DNA1–DNA4), the substrate (hairpin DNA5, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA. Throughout the paper the domains X and X′ in the DNA strands represent complementary base pair regions. Domains I and II are the catalytic core components of the Mg 2+ -dependent DNAzyme subunits.

    Journal: Chemical Science

    Article Title: A label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c7sc04007e

    doi: 10.1039/c7sc04007e

    Figure Lengend Snippet: Schematic representation of the XOR logic gate that consists of the DNAzyme subunits (DNA1–DNA4), the substrate (hairpin DNA5, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA. Throughout the paper the domains X and X′ in the DNA strands represent complementary base pair regions. Domains I and II are the catalytic core components of the Mg 2+ -dependent DNAzyme subunits.

    Article Snippet: In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs.

    Techniques: Sequencing

    Schematic representation of the OR logic gate that consists of the DNAzyme subunits (DNA1–DNA4), the substrate (hairpin DNA5, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Journal: Chemical Science

    Article Title: A label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer label-free and enzyme-free platform with a visible output for constructing versatile logic gates using caged G-quadruplex as the signal transducer †Electronic supplementary information (ESI) available: Experimental details and supplementary figures. See DOI: 10.1039/c7sc04007e

    doi: 10.1039/c7sc04007e

    Figure Lengend Snippet: Schematic representation of the OR logic gate that consists of the DNAzyme subunits (DNA1–DNA4), the substrate (hairpin DNA5, the caged G-quadruplex sequence in the stem structure of the hairpin is indicated in blue) and the input DNA.

    Article Snippet: In the presence of both inputs, the six DNAzyme subunits (DNA2–DNA7) cannot assemble into any active DNAzyme structure due to the preferred inter-input hybridization and the displacement reactions between the template and the inputs.

    Techniques: Sequencing

    Function and Taxonomy of Ice Wedge soil. Ice wedge soil metagenomes sequenced using the MinION rapid and low input kit (A) Taxonomy at the domain level (B) Clusters of Orthologous Groups (COG) categories (level 2).

    Journal: Frontiers in Microbiology

    Article Title: In Situ Field Sequencing and Life Detection in Remote (79°26′N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities

    doi: 10.3389/fmicb.2017.02594

    Figure Lengend Snippet: Function and Taxonomy of Ice Wedge soil. Ice wedge soil metagenomes sequenced using the MinION rapid and low input kit (A) Taxonomy at the domain level (B) Clusters of Orthologous Groups (COG) categories (level 2).

    Article Snippet: The MinION low input library preparation kit required much lower amounts of DNA (~20–100 ng), took ~160 min and required more instrumentation (a microcentrifuge, magnetic beads, additional enzymes, ligation of a hairpin adapter etc.)

    Techniques:

    Ice Wedge soil bacterial community composition detected by the MinION. Bacterial reads from ice wedge soil metagenomes sequenced using the MinION rapid and low input kit compared with Bacterial composition inferred from amplicon sequencing of 16S rRNA gene.

    Journal: Frontiers in Microbiology

    Article Title: In Situ Field Sequencing and Life Detection in Remote (79°26′N) Canadian High Arctic Permafrost Ice Wedge Microbial Communities

    doi: 10.3389/fmicb.2017.02594

    Figure Lengend Snippet: Ice Wedge soil bacterial community composition detected by the MinION. Bacterial reads from ice wedge soil metagenomes sequenced using the MinION rapid and low input kit compared with Bacterial composition inferred from amplicon sequencing of 16S rRNA gene.

    Article Snippet: The MinION low input library preparation kit required much lower amounts of DNA (~20–100 ng), took ~160 min and required more instrumentation (a microcentrifuge, magnetic beads, additional enzymes, ligation of a hairpin adapter etc.)

    Techniques: Amplification, Sequencing

    Design of logic gates constructed with MTs. For the YES gate, the l -DNA1 signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Design of logic gates constructed with MTs. For the YES gate, the l -DNA1 signal was inputted into the system and swarming was obtained as the output signal (1 to 1). For the AND gate, l -DNA2 and l -DNA3 had both to be present to obtain swarming. For the OR gate, the presence of either l -DNA1 or l -DNA4 was sufficient to obtain swarming. The concentration of red and green MTs was 0.6 µM, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin and each l -DNA was 0.3 µM and 0.6 µM, respectively. Scale bar: 20 µm. Error bars: s.e.m.

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Construct, Concentration Assay, Conjugation Assay

    Orthogonal control of swarming of MTs. a Schematic representation and fluorescence microscopy image of MTs with different rigidity. The flexible MTs (red) were conjugated with r -DNA1 and r -DNA2, while the rigid MTs (green) were conjugated with r -DNA5 and r -DNA6. b Upon inputting l -DNA1, the flexible MTs associated into circular shaped swarms through hybridization of r -DNA1 and r -DNA2 with l -DNA1 and appeared in red. c Green swarms with translational motion associated with rigid MTs were formed through hybridization of r -DNA5 and r -DNA6 with l -DNA5. d Swarms with translational and circular motions were simultaneously formed in response to the introduction of both input DNA signals. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin was 0.3 µM and that of each l -DNA was 0.6 µM. Scale bars: 20 µm

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Orthogonal control of swarming of MTs. a Schematic representation and fluorescence microscopy image of MTs with different rigidity. The flexible MTs (red) were conjugated with r -DNA1 and r -DNA2, while the rigid MTs (green) were conjugated with r -DNA5 and r -DNA6. b Upon inputting l -DNA1, the flexible MTs associated into circular shaped swarms through hybridization of r -DNA1 and r -DNA2 with l -DNA1 and appeared in red. c Green swarms with translational motion associated with rigid MTs were formed through hybridization of r -DNA5 and r -DNA6 with l -DNA5. d Swarms with translational and circular motions were simultaneously formed in response to the introduction of both input DNA signals. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of each r -DNA to MTs was ~100%. The concentration of kinesin was 0.3 µM and that of each l -DNA was 0.6 µM. Scale bars: 20 µm

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Fluorescence, Microscopy, Hybridization, Concentration Assay, Conjugation Assay

    Regulation of swarming mode of MTs by tuning their physical properties. a , b Fluorescence microscopy images of rigid and flexible MTs, respectively; schematic illustrations showing different swarming modes of the MTs. c Time-lapse fluorescence microscopy images showing the formation of a swarm group with a circular motion from flexible red and green MTs with lengths of 22 ± 13 µm and 16 ± 9 µm (average ± s.d.), respectively. d Time-lapse fluorescence microscopy images showing the formation of swarms with a circular motion in the presence of l -DNA1 (0.6 µM). e Dissociation of a swarm group with a circular motion triggered by the d -DNA input signal (0.6 µM). The swarm group was made of ~300 single MTs. f Change of the association ratio over time upon addition of l -DNA1 and d -DNA. The concentration of the red and green MTs was 0.6 µM each, and the conjugation ratio of r -DNA1 or r -DNA2 to MTs was ~100%. The concentration of kinesin was 0.3 µM. Scale bar: 20 µm ( a – e ). Error bar: s.e.m

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Regulation of swarming mode of MTs by tuning their physical properties. a , b Fluorescence microscopy images of rigid and flexible MTs, respectively; schematic illustrations showing different swarming modes of the MTs. c Time-lapse fluorescence microscopy images showing the formation of a swarm group with a circular motion from flexible red and green MTs with lengths of 22 ± 13 µm and 16 ± 9 µm (average ± s.d.), respectively. d Time-lapse fluorescence microscopy images showing the formation of swarms with a circular motion in the presence of l -DNA1 (0.6 µM). e Dissociation of a swarm group with a circular motion triggered by the d -DNA input signal (0.6 µM). The swarm group was made of ~300 single MTs. f Change of the association ratio over time upon addition of l -DNA1 and d -DNA. The concentration of the red and green MTs was 0.6 µM each, and the conjugation ratio of r -DNA1 or r -DNA2 to MTs was ~100%. The concentration of kinesin was 0.3 µM. Scale bar: 20 µm ( a – e ). Error bar: s.e.m

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Fluorescence, Microscopy, Concentration Assay, Conjugation Assay

    Preparation of MTs and control of their swarming. a Conjugation of r -DNA to azide-functionalized MTs by a click reaction. b TAMRA (red) and FAM (green) labeled DNA-conjugated MTs with sequences of T 16 ( r -DNA1) and (TTG) 5 ( r -DNA2), respectively. Scale bar: 5 µm. c Schematic of red and green MTs gliding on kinesins. d Time-lapse images showing motility of rigid MTs with lengths of 5 ± 2 and 5 ± 3 µm (average ± s.d.) conjugated to T 16 and (TTG) 5 , respectively. Scale bar: 10 µm. e Schematic of the association of red and green MTs by l -DNA1 and their dissociation by d -DNA via extraction of l -DNA1 through a DNA strand exchange reaction. f Association and dissociation of MTs by DNA base pair interaction. g Time-lapse images showing swarming of MTs. h Dissociation of swarms of MTs. Scale bar: 20 µm ( g and h ). i Change of association ratio over time after introduction of l -DNA1 (0.6 µM) and d -DNA (0.6 µM). The concentration of the red and green MTs was 0.6 µM each, and the kinesin concentration was 0.3 µM. Error bar: standard error (s.e.m.)

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Preparation of MTs and control of their swarming. a Conjugation of r -DNA to azide-functionalized MTs by a click reaction. b TAMRA (red) and FAM (green) labeled DNA-conjugated MTs with sequences of T 16 ( r -DNA1) and (TTG) 5 ( r -DNA2), respectively. Scale bar: 5 µm. c Schematic of red and green MTs gliding on kinesins. d Time-lapse images showing motility of rigid MTs with lengths of 5 ± 2 and 5 ± 3 µm (average ± s.d.) conjugated to T 16 and (TTG) 5 , respectively. Scale bar: 10 µm. e Schematic of the association of red and green MTs by l -DNA1 and their dissociation by d -DNA via extraction of l -DNA1 through a DNA strand exchange reaction. f Association and dissociation of MTs by DNA base pair interaction. g Time-lapse images showing swarming of MTs. h Dissociation of swarms of MTs. Scale bar: 20 µm ( g and h ). i Change of association ratio over time after introduction of l -DNA1 (0.6 µM) and d -DNA (0.6 µM). The concentration of the red and green MTs was 0.6 µM each, and the kinesin concentration was 0.3 µM. Error bar: standard error (s.e.m.)

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Conjugation Assay, Labeling, Concentration Assay

    Light-switched, repeated swarming of MTs. a Reversible hydrogen bonding of photoresponsive DNA ( p -DNA) by light-induced cis–trans isomerization of azobenzene. b Schematic of selective association and dissociation of p -DNA-conjugated MTs under UV and visible light irradiation, respectively. c Visible light ( λ = 480 nm, I = 1.3 mW cm −2 ) induced isomerization of azobenzene from the cis to the trans form, which resulted in translational swarming of the p -DNA-conjugated rigid MTs with length of 4 ± 2 µm (average ± s.d.). The swarms were then exposed to UV light ( λ = 365 nm, I = 0.4 mW cm −2 ) for 6 min that isomerized the azobenzene from the trans to the cis form. The swarms dissociated into single MTs within 12 min of the onset of photoirradiation. This cycle was repeated three times. Visible light irradiation to flexible MTs with length of 12 ± 1 µm (average ± s.d.) generated swarms with circular motion. d Changes in the association ratio upon repeated irradiation by visible and UV light. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of p -DNA1 or p -DNA2 to MTs was ~100%. The concentration of kinesin was 0.8 µM. Scale bars: 20 µm. Error bar: s.e.m.

    Journal: Nature Communications

    Article Title: DNA-assisted swarm control in a biomolecular motor system

    doi: 10.1038/s41467-017-02778-5

    Figure Lengend Snippet: Light-switched, repeated swarming of MTs. a Reversible hydrogen bonding of photoresponsive DNA ( p -DNA) by light-induced cis–trans isomerization of azobenzene. b Schematic of selective association and dissociation of p -DNA-conjugated MTs under UV and visible light irradiation, respectively. c Visible light ( λ = 480 nm, I = 1.3 mW cm −2 ) induced isomerization of azobenzene from the cis to the trans form, which resulted in translational swarming of the p -DNA-conjugated rigid MTs with length of 4 ± 2 µm (average ± s.d.). The swarms were then exposed to UV light ( λ = 365 nm, I = 0.4 mW cm −2 ) for 6 min that isomerized the azobenzene from the trans to the cis form. The swarms dissociated into single MTs within 12 min of the onset of photoirradiation. This cycle was repeated three times. Visible light irradiation to flexible MTs with length of 12 ± 1 µm (average ± s.d.) generated swarms with circular motion. d Changes in the association ratio upon repeated irradiation by visible and UV light. The concentration of red and green MTs was 0.6 µM each, and the conjugation ratio of p -DNA1 or p -DNA2 to MTs was ~100%. The concentration of kinesin was 0.8 µM. Scale bars: 20 µm. Error bar: s.e.m.

    Article Snippet: The OR logic gate was implemented by conjugating pairs of r -DNA to the MTs ( r -DNA1 and r -DNA3 labeled with TAMRA; r -DNA2 and r -DNA4 labeled with FAM), and using l -DNA1 (complementary to r- DNA1 and r -DNA2) and l -DNA4 (complementary to r -DNA3 and r -DNA4) as the two input signals.

    Techniques: Irradiation, Generated, Concentration Assay, Conjugation Assay

    Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of Cy3-labeled total genomic DNA versus Cy5-labeled, E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.

    Journal: Genes & Development

    Article Title: E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints

    doi: 10.1101/gad.949802

    Figure Lengend Snippet: Location analysis of E2F4 on human promoters and identification of previously known E2F target genes. ( A ) Scatter plot analysis of Cy3-labeled total genomic DNA versus Cy5-labeled, E2F4 ChIP-enriched DNA. A P -value cutoff of 0.01 is shown. ( B ) Confirmation of promoter occupancy by E2Fs in quiescent WI-38 cells using a standard ChIPs protocol. E2Fs were enriched at several promoters involved in cell cycle control and DNA replication. In contrast, we did not detect enrichment of a negative control β-interferon fragment with anti-E2F4 antibodies. Nor did we observe significant promoter enrichment with an irrelevant antibody control (mock) lanes. Input lanes correspond to PCR reactions with 0.5% of total chromatin in immunoprecipitation reactions.

    Article Snippet: Each hybridization reaction included the Cy5- and Cy3-labeled DNA, 20 μg of human Cot-1 DNA enriched for repetitive DNA sequences such as the Alu and Kpn family members (Invitrogen), 40 μg of yeast tRNA (Sigma), 2.2× SSC, 0.1% SDS, 0.12% BSA (Sigma), 28% formamide (Sigma), and 5.7% dextran sulfate (Sigma), in a total of 50 μL.

    Techniques: Labeling, Chromatin Immunoprecipitation, Negative Control, Polymerase Chain Reaction, Immunoprecipitation

    Strategy for mammalian factor location analysis using the 1.5K DNA microarray. ( A ) are indicated in red. Primary human cells (WI-38) were cross-linked and sonicated, and chromatin was immunoprecipitated with anti-E2F antibodies. The resulting ChIP-enriched DNA was purified, amplified by LM-PCR together with total genomic DNA, labeled with the Cy5 and Cy3 fluorophores using random priming, and hybridized to the 1.5K microarray in the presence of Cot-1 DNA to reduce nonspecific hybridization. ( B ).

    Journal: Genes & Development

    Article Title: E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints

    doi: 10.1101/gad.949802

    Figure Lengend Snippet: Strategy for mammalian factor location analysis using the 1.5K DNA microarray. ( A ) are indicated in red. Primary human cells (WI-38) were cross-linked and sonicated, and chromatin was immunoprecipitated with anti-E2F antibodies. The resulting ChIP-enriched DNA was purified, amplified by LM-PCR together with total genomic DNA, labeled with the Cy5 and Cy3 fluorophores using random priming, and hybridized to the 1.5K microarray in the presence of Cot-1 DNA to reduce nonspecific hybridization. ( B ).

    Article Snippet: Each hybridization reaction included the Cy5- and Cy3-labeled DNA, 20 μg of human Cot-1 DNA enriched for repetitive DNA sequences such as the Alu and Kpn family members (Invitrogen), 40 μg of yeast tRNA (Sigma), 2.2× SSC, 0.1% SDS, 0.12% BSA (Sigma), 28% formamide (Sigma), and 5.7% dextran sulfate (Sigma), in a total of 50 μL.

    Techniques: Microarray, Sonication, Immunoprecipitation, Chromatin Immunoprecipitation, Purification, Amplification, Polymerase Chain Reaction, Labeling, Hybridization