dna polymerase  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 86

    Structured Review

    New England Biolabs dna polymerase
    Optimization of NRCA reaction conditions for the detection of SARS-COV-2 nucleic acid. (A-C) The ratio of fluorescent intensity between amplification products of target and negative control adopting Bst. <t>DNA</t> polymerase/Nb. BsrDI, <t>Klenow</t> fragment exo-/Nb.BbvCI, phi29 DNA polymerase/Nb.BbvCI in different buffer systems. (D) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of the padlock probe. (E) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of nicking enzyme (Nb.BbvCI). (F) The ratio of fluorescent intensity between amplification products of target and negative control at different concentration of primer 1 and primer 2.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2022-07
    86/100 stars

    Images

    1) Product Images from "Paper-based netlike rolling circle amplification (NRCA) for ultrasensitive and visual detection of SARS-CoV-2"

    Article Title: Paper-based netlike rolling circle amplification (NRCA) for ultrasensitive and visual detection of SARS-CoV-2

    Journal: Sensors and Actuators. B, Chemical

    doi: 10.1016/j.snb.2022.131460

    Optimization of NRCA reaction conditions for the detection of SARS-COV-2 nucleic acid. (A-C) The ratio of fluorescent intensity between amplification products of target and negative control adopting Bst. DNA polymerase/Nb. BsrDI, Klenow fragment exo-/Nb.BbvCI, phi29 DNA polymerase/Nb.BbvCI in different buffer systems. (D) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of the padlock probe. (E) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of nicking enzyme (Nb.BbvCI). (F) The ratio of fluorescent intensity between amplification products of target and negative control at different concentration of primer 1 and primer 2.
    Figure Legend Snippet: Optimization of NRCA reaction conditions for the detection of SARS-COV-2 nucleic acid. (A-C) The ratio of fluorescent intensity between amplification products of target and negative control adopting Bst. DNA polymerase/Nb. BsrDI, Klenow fragment exo-/Nb.BbvCI, phi29 DNA polymerase/Nb.BbvCI in different buffer systems. (D) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of the padlock probe. (E) The ratio of fluorescent intensity between amplification products of target and negative control at different concentrations of nicking enzyme (Nb.BbvCI). (F) The ratio of fluorescent intensity between amplification products of target and negative control at different concentration of primer 1 and primer 2.

    Techniques Used: Amplification, Negative Control, Concentration Assay

    2) Product Images from "Directed evolution of protein enzymes using nonhomologous random recombination"

    Article Title: Directed evolution of protein enzymes using nonhomologous random recombination

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0402202101

    Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry
    Figure Legend Snippet: Protein NRR. One or more parental genes are digested with DNase I. Fragments are blunt-ended with T4 DNA polymerase, size-selected, and ligated under conditions that favor intermolecular ligation. Two hairpin sequences are added in a defined stoichiometry

    Techniques Used: Ligation

    3) Product Images from "Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe"

    Article Title: Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe

    Journal: bioRxiv

    doi: 10.1101/2020.10.05.327460

    Alternative approaches to testing hybridization between osmylated probes and targets. (a) Enzymatic elongation of osmylated primers using ssM13mp18 DNA as the template and DNA polymerase; time points obtained at 5, 10 and 20min. No primer and M13 rev (−48) used as negative controls. With the exception of BJ1, all the other osmylated primers exhibit enzymatic elongation comparable to the positive control M13 fwd (6097). Absence of elongation with BJ1 is attributed to the presence of a T(OsBp) at the 3’end. (b), (c) and (d) Overlapping HPLC profiles from the analyses of different samples, with samples at about 5μM in about 90% ONT buffer. The same HPLC method B was used for all the samples (see Experimental Section). Intact ss oligos and ds oligos appear as sharp peaks, whereas osmylated oligos and hybrids with one osmylated strand appear as broad peaks; hybrids elute later compared to ss nucleic acids. (b) Sample composition: intact BJ2 (blue trace), intact complement of primerM13 for (−41) (red trace). The HPLC profile of their equimolar mixture is consistent with hybridization (green trace). (c) Sample composition: miRNA21 (blue trace), probe 21EXT carrying 8 dT(OsBp) moieties (red trace), and equimolar mixture of the two (green trace). HPLC profile of the mixture consistent with NO hybridization, attributed to the high number of single OsBp tags, 6 within a sequence of 22nt, likely to distort the helical structure of the probe, and prevent ds formation. (d) Sample composition practically the same as under (b), with the exception that in these samples BJ2 carries 6 dT(OsBp) moieties (first peak with broad shape and absorbance at 312nm). HPLC profile of the mixture is consistent with hybridization, attributed to the fact that most of the OsBp moieties are adjacent, so that the rest of the sequence can still hybridize with the target.
    Figure Legend Snippet: Alternative approaches to testing hybridization between osmylated probes and targets. (a) Enzymatic elongation of osmylated primers using ssM13mp18 DNA as the template and DNA polymerase; time points obtained at 5, 10 and 20min. No primer and M13 rev (−48) used as negative controls. With the exception of BJ1, all the other osmylated primers exhibit enzymatic elongation comparable to the positive control M13 fwd (6097). Absence of elongation with BJ1 is attributed to the presence of a T(OsBp) at the 3’end. (b), (c) and (d) Overlapping HPLC profiles from the analyses of different samples, with samples at about 5μM in about 90% ONT buffer. The same HPLC method B was used for all the samples (see Experimental Section). Intact ss oligos and ds oligos appear as sharp peaks, whereas osmylated oligos and hybrids with one osmylated strand appear as broad peaks; hybrids elute later compared to ss nucleic acids. (b) Sample composition: intact BJ2 (blue trace), intact complement of primerM13 for (−41) (red trace). The HPLC profile of their equimolar mixture is consistent with hybridization (green trace). (c) Sample composition: miRNA21 (blue trace), probe 21EXT carrying 8 dT(OsBp) moieties (red trace), and equimolar mixture of the two (green trace). HPLC profile of the mixture consistent with NO hybridization, attributed to the high number of single OsBp tags, 6 within a sequence of 22nt, likely to distort the helical structure of the probe, and prevent ds formation. (d) Sample composition practically the same as under (b), with the exception that in these samples BJ2 carries 6 dT(OsBp) moieties (first peak with broad shape and absorbance at 312nm). HPLC profile of the mixture is consistent with hybridization, attributed to the fact that most of the OsBp moieties are adjacent, so that the rest of the sequence can still hybridize with the target.

    Techniques Used: Hybridization, Positive Control, High Performance Liquid Chromatography, Sequencing

    4) Product Images from "Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii"

    Article Title: Molecular Factors of Hypochlorite Tolerance in the Hypersaline Archaeon Haloferax volcanii

    Journal: Genes

    doi: 10.3390/genes9110562

    Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.
    Figure Legend Snippet: Schematic diagram of the inverted-nested two-step PCR (INT-PCR, left) and the semi-random two-step PCR (ST-PCR, right) strategies to identify the transposon insertion sites in Haloferax volcanii . The transposable (Tn) element includes the following: two Mu repeats (MuR), a chloramphenicol acetyltransferase ( cat ) gene, a P cat promoter, a tryptophan synthase ( trpA ) gene, and a ferredoxin promoter (P fdx ). NdeI and HindIII are examples of the restriction enzyme (RE) sites used to cleave the genomic DNA prior to blunt-end ligation to form the circular DNA template used in the INT-PCR method. Primer pairs used for the two PCR steps (PCR1 and PCR2) and the DNA sequencing are color coded (red, blue, and purple) and numbered according to Table S1 . See Methods for details.

    Techniques Used: Polymerase Chain Reaction, Ligation, DNA Sequencing

    5) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.
    Figure Legend Snippet: Role of conserved residue Y172 in the interaction with the DNAP. ( A ) Template-independent initiation products of increasing concentrations of wild type and Y172F and Y172A TP mutants. Reactions were carried out with the indicated concentrations of TP and triggered with 1 mM MnCl 2 . ( B ) Comparative analysis of wild type and Y172A and Y172F mutant TPs interaction with the DNA polymerase. Shown are mean and standard error of three independent experiments. See Materials and Methods for details. ( C ) TP-primed replication of 29-mer single stranded oligonucleotide template containing the Bam35 genome origin sequence, using either wild type or Y172F TPs as primer. Time-course of full-length replication, relative to the initial events. Shown are mean and standard error of three independent experiments. The inset panel shows a representative SDS-PAGE of initiation (with dTTP) and replication (with all 4 dNTPs) products primed by the wild type or Y172F TPs after 30 min of reaction.

    Techniques Used: Mutagenesis, Sequencing, SDS Page

    Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.
    Figure Legend Snippet: Mapping Bam35 TP priming residue. ( A ) Multiple sequence alignment of Bam35 TP and related TPs. Sequences used were from putative TPs (proteins encoded by ORF4) of representative related Gram-positive tectiviruses Bam35 (NCBI ID NP_943750.1, 10), GIL16 (YP_224102.1, 47), AP50 (YP_002302516.1, 30), as well as other BLAST-retrieved orthologous sequences from NR protein database and tentatively annotated as bacterial proteins from Bacillus cereus (WP_001085581.1), Streptococcus pneumoniae (WP_050224775.1), Exiguobacerium antarticum (WP_026829749.1), Bacillus flexus (WP_025907183.1) and Brevibacillus sp. CF112 (WP_007784052.1). These bacterial proteins may correspond to TPs from uncharacterized tectivirus-like prophages or linear plasmids from Gram-positive hosts. Sequences were aligned with MUSCLE algorithm implemented in Geneious R8 software ( 48 ). The C-terminal fragment of all proteins that corresponds with the bromide cyanogen cleavage product is shadowed in blue and the tyrosine residues present in the Bam35 portion are highlighted in pink. Conserved Y172 and Y194 residues are marked with an asterisk above the sequences. ( B ) Determination of the nature of the Bam35 TP priming residue by alkali treatment. Control initiation reactions with Φ29 DNA polymerase and TP were performed in parallel. After the initiation reaction, samples were incubated for 6 min at 95°C in the absence or presence of 100 mM NaOH, and subsequently neutralized and analyzed by SDS-PAGE and autoradiography. ( C ) Mapping the Bam35 TP priming residue. The TP-AMP complexes were performed as described and afterward treated with 1.2 mM of cyanogen bromide (CNBr) and 200 mM HCl for 20 h at room temperature. Finally, the samples were neutralized and analyzed by SDS-18% polyacrylamide electrophoresis. ( D ) Identification of Y194 as the priming residue by TP-deoxyadenylation assays with 0.5 or 2 μl of cell-free extracts of bacterial cultures expressing the TP variants. Extracts prepared from bacteria harboring the empty plasmid (lanes 1, 2) and the wild type TP expression vector (lanes 3, 4) were also used as negative or positive controls, respectively. See Materials and Methods for details.

    Techniques Used: Sequencing, Software, Incubation, SDS Page, Autoradiography, Electrophoresis, Expressing, Plasmid Preparation

    Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.
    Figure Legend Snippet: Bam35 protein-primed genome replication. Alkaline agarose gel electrophoresis of TP-DNA replication products. Samples contained 11 nM B35DNAP and 133 nM TP, as indicated, and 100 ng of Bam35 TP-DNA. See Materials and Methods for details. ( A ) Reactions were triggered by addition of 10 mM MgCl 2 and incubated for 1, 2 and 4 h (lanes 3–5). ( B ) Reactions were triggered by 10 mM MgCl 2 and/or 1 mM MnCl 2 as indicated and incubated for 2 h. A λ-HindIII DNA ladder was loaded as a size marker, and the expected size of the Bam35 TP-DNA product is also indicated.

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Marker

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    6) Product Images from "A rapid method for assessing the RNA-binding potential of a protein"

    Article Title: A rapid method for assessing the RNA-binding potential of a protein

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks285

    Schematic of ssRNA Pentaprobe production. pcDNA3.1 vector containing a dsDNA Pentaprobe sequence under the control of a T7 promoter site is linearized and the in vitro transcribed to produce a ssRNA sequence encoding the Pentaprobe sequence. Highlighted is the ApaI restriction site (purple), T7 promoter site (pink), the encoded DNA sequence (blue) and the resulting ssRNA probe sequence (blue).
    Figure Legend Snippet: Schematic of ssRNA Pentaprobe production. pcDNA3.1 vector containing a dsDNA Pentaprobe sequence under the control of a T7 promoter site is linearized and the in vitro transcribed to produce a ssRNA sequence encoding the Pentaprobe sequence. Highlighted is the ApaI restriction site (purple), T7 promoter site (pink), the encoded DNA sequence (blue) and the resulting ssRNA probe sequence (blue).

    Techniques Used: Plasmid Preparation, Sequencing, In Vitro

    7) Product Images from "Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma"

    Article Title: Oxidative stress-induced chromosome breaks within the ABL gene: a model for chromosome rearrangement in nasopharyngeal carcinoma

    Journal: Human Genomics

    doi: 10.1186/s40246-018-0160-8

    A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]
    Figure Legend Snippet: A flowchart showing the manipulation steps in the preparation of genomic DNA for IPCR. The genomic DNA was subjected to RE digestions, Klenow fill-in and ligation prior to IPCR as reported before [ 80 ]

    Techniques Used: Ligation

    8) Product Images from "Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells"

    Article Title: Nonviral Direct Conversion of Primary Mouse Embryonic Fibroblasts to Neuronal Cells

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2012.25

    Immunofluorochemistry and synapsin reporter activity in NiNs generated with p(CBA-ABOL)/DNA polyplexes . All bars are 50 µm. Tau stain (first row): three doses of 1.0 µg pmax-BAM factors, ≥10 days in N3 medium, on TCPS. MAP2 stain (second row): three doses of 1.0 µg pmax-BAM factors (left panel), or 2.0 µg pUNO-AM/pmax-B factors (center and right panels), 16 days in N3, on poly-D-lysine/laminin-coated coverslips. Synaptophysin stain (third row): five (left and center panels) or three (right panel) doses of 1.0 µg pmax-BAM factors, 17 days in N3, on PDL/laminin-coated coverslips. Expression of RFP under control of the synapsin promoter (fourth row): three doses of 1.0 µg pmax-BAM factors (left and center panels), or 2.0 µg pUNO-AM/pmax-B factors (right panel), ≥10 days in N3, on PDL/laminin-coated coverslips. Synapsin-RFP images have not been false-colored red, in order to maximize visual contrast for thin cellular processes. Arrows indicate synapsin-RFP + cell bodies. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein; TCPS, tissue culture polystyrene.
    Figure Legend Snippet: Immunofluorochemistry and synapsin reporter activity in NiNs generated with p(CBA-ABOL)/DNA polyplexes . All bars are 50 µm. Tau stain (first row): three doses of 1.0 µg pmax-BAM factors, ≥10 days in N3 medium, on TCPS. MAP2 stain (second row): three doses of 1.0 µg pmax-BAM factors (left panel), or 2.0 µg pUNO-AM/pmax-B factors (center and right panels), 16 days in N3, on poly-D-lysine/laminin-coated coverslips. Synaptophysin stain (third row): five (left and center panels) or three (right panel) doses of 1.0 µg pmax-BAM factors, 17 days in N3, on PDL/laminin-coated coverslips. Expression of RFP under control of the synapsin promoter (fourth row): three doses of 1.0 µg pmax-BAM factors (left and center panels), or 2.0 µg pUNO-AM/pmax-B factors (right panel), ≥10 days in N3, on PDL/laminin-coated coverslips. Synapsin-RFP images have not been false-colored red, in order to maximize visual contrast for thin cellular processes. Arrows indicate synapsin-RFP + cell bodies. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein; TCPS, tissue culture polystyrene.

    Techniques Used: Activity Assay, Generated, Staining, Expressing

    Current clamp in the whole-cell configuration of synapsin-RFP + NiNs generated with p(CBA-ABOL)/DNA polyplexes . ( a , b ) Traces of cells that received three doses of 1.0 µg pmax-BAM factors, after 16–17 days of culture in N3 medium, on poly-D-lysine/laminin-coated coverslips. ( c , d ) Traces of cells that received three doses of 2.0 µg pUNO-AM/pmax-B factors, on day 12 of culture in N3, on PDL/laminin-coated coverslips. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein.
    Figure Legend Snippet: Current clamp in the whole-cell configuration of synapsin-RFP + NiNs generated with p(CBA-ABOL)/DNA polyplexes . ( a , b ) Traces of cells that received three doses of 1.0 µg pmax-BAM factors, after 16–17 days of culture in N3 medium, on poly-D-lysine/laminin-coated coverslips. ( c , d ) Traces of cells that received three doses of 2.0 µg pUNO-AM/pmax-B factors, on day 12 of culture in N3, on PDL/laminin-coated coverslips. NiN, non-virally induced neuronal cell; RFP, red fluorescent protein.

    Techniques Used: Generated

    Optimization of nonviral GFP reporter plasmid transfection in PMEFs . ( a ) Fluorescence micrographs of PMEFs transfected with increasing doses of DNA in p(CBA-ABOL)/pmax-GFP polyplexes (Bar = 500 µm), with 1.0 µg of pmax-GFP delivered with Lipofectamine 2000 (LF2k) included for comparison. Images were taken 24 hours after transfection. ( b ) AlamarBlue toxicity assay multiplexed with ( c ) flow cytometric measurement of GFP transfection efficiency and ( d ) % median fluorescence intensity increase of GFP + cells compared to nonfluorescent control transfections for one and two doses of p(CBA-ABOL)/pmax-GFP polyplexes, assayed 24 hours after transfection; 1.0 µg of pmax-GFP delivered with LF2k is again included for comparison. Error bars represent mean ± SEM of three separate experiments performed in triplicate. Two-way ANOVA were performed with P
    Figure Legend Snippet: Optimization of nonviral GFP reporter plasmid transfection in PMEFs . ( a ) Fluorescence micrographs of PMEFs transfected with increasing doses of DNA in p(CBA-ABOL)/pmax-GFP polyplexes (Bar = 500 µm), with 1.0 µg of pmax-GFP delivered with Lipofectamine 2000 (LF2k) included for comparison. Images were taken 24 hours after transfection. ( b ) AlamarBlue toxicity assay multiplexed with ( c ) flow cytometric measurement of GFP transfection efficiency and ( d ) % median fluorescence intensity increase of GFP + cells compared to nonfluorescent control transfections for one and two doses of p(CBA-ABOL)/pmax-GFP polyplexes, assayed 24 hours after transfection; 1.0 µg of pmax-GFP delivered with LF2k is again included for comparison. Error bars represent mean ± SEM of three separate experiments performed in triplicate. Two-way ANOVA were performed with P

    Techniques Used: Plasmid Preparation, Transfection, Fluorescence, Flow Cytometry

    9) Product Images from "Site-specific strand breaks in RNA produced by 125I radiodecay"

    Article Title: Site-specific strand breaks in RNA produced by 125I radiodecay

    Journal: Nucleic Acids Research

    doi:

    Binding of antisense oligonucleotides to target RNA and DNA analyzed by 12% native PAGE. Lanes 3 and 7, [ 32 P]DNA and [ 32 P]RNA targets, respectively; lanes 1, 2, 5 and 6, [ 32 P]DNA and [ 32 P]RNA targets incubated with [ 125 I]AO-I (lanes 2 and 6 with DMSO); lanes 4 and 8, [ 32 P]DNA and [ 32 P]RNA targets incubated with excess cold AO-I.
    Figure Legend Snippet: Binding of antisense oligonucleotides to target RNA and DNA analyzed by 12% native PAGE. Lanes 3 and 7, [ 32 P]DNA and [ 32 P]RNA targets, respectively; lanes 1, 2, 5 and 6, [ 32 P]DNA and [ 32 P]RNA targets incubated with [ 125 I]AO-I (lanes 2 and 6 with DMSO); lanes 4 and 8, [ 32 P]DNA and [ 32 P]RNA targets incubated with excess cold AO-I.

    Techniques Used: Binding Assay, Clear Native PAGE, Incubation

    Analysis of break distribution in target DNA and RNA. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]-AO-II; lane 3, [ 32 P]DNA with [ 125 I]AO-I; lane 4, Maxam–Gilbert G sequencing line of [ 32 P]DNA; lane 5, [ 32 P]RNA; lane 6, [ 32 P]RNA with 125 I-AO-II; lane 7, [ 32 P]RNA with [ 125 I]-AO-I; lane 8, RNase T1 G sequencing line of [ 32 P]RNA. ( B ) Percentages of breaks at individual bases of the target molecules calculated from the data of the gel in (A) for DNA (open squares) and RNA (filled circles).
    Figure Legend Snippet: Analysis of break distribution in target DNA and RNA. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]-AO-II; lane 3, [ 32 P]DNA with [ 125 I]AO-I; lane 4, Maxam–Gilbert G sequencing line of [ 32 P]DNA; lane 5, [ 32 P]RNA; lane 6, [ 32 P]RNA with 125 I-AO-II; lane 7, [ 32 P]RNA with [ 125 I]-AO-I; lane 8, RNase T1 G sequencing line of [ 32 P]RNA. ( B ) Percentages of breaks at individual bases of the target molecules calculated from the data of the gel in (A) for DNA (open squares) and RNA (filled circles).

    Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing

    Strand breaks in target DNA and RNA from two 125 I atoms incorporated in antisense oligonucleotides. ( A ) Scheme of the target and antisense oligonucleotides. ( B ) Autoradiogram of the 12% urea–PAGE showing RNA fragments after incubation of duplexes at –70°C (lanes 1–8) and at +5°C (lanes 9–12). Lanes 1, 3, 5, 7, 9 and 11, RNA targets without [ 125 I]oligonucleotides; lanes 2, 4, 6, 8, 10 and 12, RNA targets with [ 125 I]oligonucleotides, complementary AO-II (lanes 2, 4, 10 and 12) and non-complimentary AO-N (lanes 6 and 8). Samples in lanes 3, 4, 7, 8, 11 and 12 contained 10% DMSO. Lane 13, [ 32 P]DNA marker, 8–32 nt.
    Figure Legend Snippet: Strand breaks in target DNA and RNA from two 125 I atoms incorporated in antisense oligonucleotides. ( A ) Scheme of the target and antisense oligonucleotides. ( B ) Autoradiogram of the 12% urea–PAGE showing RNA fragments after incubation of duplexes at –70°C (lanes 1–8) and at +5°C (lanes 9–12). Lanes 1, 3, 5, 7, 9 and 11, RNA targets without [ 125 I]oligonucleotides; lanes 2, 4, 6, 8, 10 and 12, RNA targets with [ 125 I]oligonucleotides, complementary AO-II (lanes 2, 4, 10 and 12) and non-complimentary AO-N (lanes 6 and 8). Samples in lanes 3, 4, 7, 8, 11 and 12 contained 10% DMSO. Lane 13, [ 32 P]DNA marker, 8–32 nt.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Incubation, Marker

    Analysis of breaks in RNA and DNA with extended antisense oligonucleotide AO-L. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]AO-L; lane 3, [ 32 P]RNA; lane 4, [ 32 P]RNA with [ 125 I]AO-L; lane 5, Maxam–Gilbert G sequencing line of [ 32 P]DNA. ( B ) Percentages of breaks at individual bases of the target DNA (open squares) and RNA (filled circles) calculated from the data of the gel in (A). Error bars show standard deviations calculated from three independent measurements.
    Figure Legend Snippet: Analysis of breaks in RNA and DNA with extended antisense oligonucleotide AO-L. ( A ) 12% urea–PAGE. Lane 1, [ 32 P]DNA; lane 2, [ 32 P]DNA with [ 125 I]AO-L; lane 3, [ 32 P]RNA; lane 4, [ 32 P]RNA with [ 125 I]AO-L; lane 5, Maxam–Gilbert G sequencing line of [ 32 P]DNA. ( B ) Percentages of breaks at individual bases of the target DNA (open squares) and RNA (filled circles) calculated from the data of the gel in (A). Error bars show standard deviations calculated from three independent measurements.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Sequencing

    10) Product Images from "ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes"

    Article Title: ExoCET: exonuclease in vitro assembly combined with RecET recombination for highly efficient direct DNA cloning from complex genomes

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx1249

    Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).
    Figure Legend Snippet: Concerted action of in vitro assembly and full length RecE/RecT improves the efficiency of direct cloning. ( A ) A schematic diagram illustrating direct cloning of the 14-kb lux gene cluster from Photobacterium phosphoreum ANT-2200. The linear p15A-cm vector and target genomic segment have identical sequences at both ends. ( B ) Longer homology arms increase the cloning efficiency of ExoCET. The linear vector flanked by 25-, 40- or 80-bp homology arms was mixed with genomic DNA and treated with 0.02 U μl −1 T4pol at 25°C for 20 min before annealing and electroporation into arabinose induced Escherichia coli GB05-dir. Error bars, s.d.; n = 3. ( C ) Titration of T4pol amount for ExoCET. The linear vector with 80-bp homology arms and genomic DNA were treated as in (B) except the amount of T4pol was altered as indicated. ( D ) Incubation time of T4pol on cloning efficiency. As for (C) using 0.02 U μl −1 T4pol except the incubation time was altered as indicated. ( E ) Higher copy number of ETgA increases ExoCET cloning efficiency. As for (D) using 1 h and electroporation into arabinose induced E. coli GB05-dir (one copy of ETgA on the chromosome), GB2005 harboring pSC101-BAD-ETgA-tet (approximately five copies of ETgA on pSC101 plasmids) or GB05-dir harboring pSC101-BAD-ETgA-tet (approximately six copies of ETgA ) as indicated. ( F ) ExoCET increases direct cloning efficiency. As for (E) using E. coli GB05-dir harboring pSC101-BAD-ETgA-tet (ExoCET) or omission of T4pol from the in vitro assembly (ETgA) or omission of the arabinose induction of pSC101-BAD-ETgA-tet (T4pol). ( G ) As for (F) except the 53 kb plu2670 gene cluster was directly cloned. Accuracy denotes the success of direct cloning as evaluated by restriction digestions ( Supplementary Figure S4 ). Each experiment was performed in triplicate ( n = 3) and error bars show standard deviation (s.d).

    Techniques Used: In Vitro, Clone Assay, Plasmid Preparation, Electroporation, Titration, Incubation, Standard Deviation

    11) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.
    Figure Legend Snippet: Comparison of DNA polymerase activity in the presence of dUTP. The efficiency of dUTP utilization was compared among five DNA polymerases. Results are presented as percentages of incorporated radioactivity in the presence of [ 3 H]dUTP compared to [ 3 H]TTP. The relative efficiencies of dUTP utilization were 74.9% for Neq DNA polymerase, 71.3% for Taq DNA polymerase, 9.4% for Pfu DNA polymerase, 15.1% for Vent DNA polymerase, and 12.3% for KOD DNA polymerase. Columns are mean values obtained from three independent assays; bars indicate standard deviations.

    Techniques Used: Activity Assay, Radioactivity

    Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.
    Figure Legend Snippet: Comparison of PCR amplifications in the presence of deaminated bases. (a) PCR in the presence of dUTP or dITP. PCR was conducted using 250 μM dNTPs (lanes 1 to 5); 250 μM each of dATP, dCTP, dGTP, and dUTP (lanes 6 to 10); or 250 μM each of dATP, dCTP, dTTP, and dITP/dGTP (1:9 ratio) (lanes 11 to 15). Lane M, DNA molecular size markers; lanes 1, 6, and 11, Neq DNA polymerase; lanes 2, 7, and 12, Taq DNA polymerase; lanes 3, 8, and 13, Pfu DNA polymerase; lanes 4, 9, and 14, Vent DNA polymerase; lanes 5, 10, and 15, KOD DNA polymerase. (b) PCRs in the presence of different concentrations of dITP. PCR was conducted using dITP/dGTP mixtures in different ratios, in which the final concentration of the mixtures was maintained at 250 μM. The values on the gel indicate the percentages of dITP included in dITP/dGTP mixture. Lanes 1 to 5, Neq DNA polymerase; lanes 6 to 10, Taq DNA polymerase.

    Techniques Used: Polymerase Chain Reaction, Concentration Assay

    12) Product Images from "Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins"

    Article Title: Bam35 Tectivirus Intraviral Interaction Map Unveils New Function and Localization of Phage ORFan Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.00870-17

    Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.
    Figure Legend Snippet: Bam35 interactions found by different combinations of bait/prey fusion proteins. The numbers of reproducible protein-protein interactions (PPIs) detected with four, three, two, and one different combinations of vectors are indicated in red, green, blue, and black, respectively. The combination of vectors used, a graphic representation of the fusion protein obtained in each case, and the number of PPIs detected with each combination are indicated inside the boxes. AD, activation domain of the Gal4 transcription factor; DBD, DNA-binding domain of Gal4 transcription factor.

    Techniques Used: Activation Assay, Binding Assay

    Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.
    Figure Legend Snippet: Genetic map of phage Bam35. The boxes correspond to the ORFs predicted to exist in the Bam35 genome. The ORFs that overlap another ORF(s) are represented at the bottom. Namely, the last 5 amino acids of ORF4 overlap ORF5, the last 8 amino acids of ORF7 overlap ORF8, the last 62 amino acids of ORF10 and the first 51 amino acids of ORF12 overlap ORF11, the last 10 amino acids of ORF12 and the first 6 amino acids of ORF14 overlap ORF13, the last amino acid of ORF21 and first amino acid of ORF24 overlap ORF23, the last 17 amino acids of ORF26 overlap ORF27, ORF31 is constituted by nucleotides 148 to 527 (in +1 frame) of ORF30, and ORF32 is constituted by the 102 C-terminal amino acids of ORF30. The previously shown or suggested (*) function of the protein or the type of protein encoded by an ORF is indicated as follows: green, DNA-binding protein; blue, DNA replication; chartreuse, cycle regulator; salmon, assembly or DNA packaging and other special vertex components; cyan, major capsid and spike proteins; purple, membrane structural protein; and orange, lytic proteins. Oblique lines indicate a predicted transmembrane domain. The ruler at the bottom represents the number of base pairs in the Bam35 genome. ITR, inverted terminal repeat; HVR, highly variable region. See Table S1 in the supplemental material for further details.

    Techniques Used: Binding Assay

    13) Product Images from "MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression"

    Article Title: MrkH, a Novel c-di-GMP-Dependent Transcriptional Activator, Controls Klebsiella pneumoniae Biofilm Formation by Regulating Type 3 Fimbriae Expression

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1002204

    The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.
    Figure Legend Snippet: The mrkABCDF and mrkHIJ loci in K. pneumoniae AJ218. ( A ) Genetic organization of the mrkABCDF and mrkHIJ gene clusters from K. pneumoniae strains AJ218, NTUH-K2044 (GenBank Ref: AP006725), MGH 78578 (GenBank Ref: CP000647) and 342 (GenBank Ref: CP000964). ( B ) RT-PCR analysis of mrkHIJ transcription. PCR amplicon products of either RNA (−), reverse transcribed DNA (+) or genomic DNA (gDNA) were visualized on a 1% agarose gel. The mrkH-I product was generated with primer mrkI-R and amplified with primers mrkH-F and mrkI-R. The mrkI-J product was generated with primer mrkJ-R and amplified with primers mrkI-F and mrkJ-R.

    Techniques Used: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis, Generated

    Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.
    Figure Legend Snippet: Analysis of the binding of MrkH-8×His to the mrkA regulatory region by EMSA. The 32 P-labelled PCR fragment containing the mrkA regulatory region was generated using primer pairs 32 P-Px1mrkARev and mrk295F. The mrkA fragment was mixed with varying amounts of the purified MrkH-8×His protein (from 0 to 500 nM) in the absence or presence of c-di-GMP (200 µM). Following incubation at 30°C for 20 min, the samples were analyzed on native polyacrylamide gels. The right-hand panel shows control reactions with approximately 100-fold molar excess of the unlabeled (cold) mrkA promoter fragment (specific competitor DNA), used to demonstrate the specificity of the c-di-GMP-mediated MrkH binding to the mrkA promoter region. The unbound DNA (F) and protein-DNA complexes (C1, C2 and C3) are marked.

    Techniques Used: Binding Assay, Polymerase Chain Reaction, Generated, Purification, Incubation

    14) Product Images from "Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver"

    Article Title: Apobec1 complementation factor (A1CF) and RBM47 interact in tissue-specific regulation of C to U RNA editing in mouse intestine and liver

    Journal: RNA

    doi: 10.1261/rna.068395.118

    Generation and characterization of conditional Rbm47 knockout mice. ( A ) Endogenous mouse Rbm47 gene and targeting construct. Schematic illustration of genomic structure of Rbm47 allele containing nine exons (orange boxes). Schematic of Rbm47 tm1a composition. (Numbered oranges boxes) Rbm47 exons; (blue boxes) Lac Z and neo cassettes; (green triangles) FRT sites; (purple triangles) LoxP sites. FLP recombination eliminates the LacZ and neo cassettes. Exon 6 containing transcriptional start codon and sequences encoding RNA recognition motifs was targeted for Cre-dependent homologous recombination. ( B ) Representative DNA electrophoresis showing genotype of WT and targeted alleles using primers P1 and P2 surrounding the 3′arm LoxP site. Analysis of the WT allele results in a 490 bp PCR product (lane 1 ) whereas recombination at the floxed allele generates a 530 bp product (f/f) (lane 3 ). Cre activation resuts in the loss of exon 6, evidenced by the absence of PCR amplification (lane 5 ). Molecular weights (bp) are indicated to the left . ( C ) Western blot detection of RBM47 in liver of floxed and Rbm47 liver-specific knockout ( Rbm47 LKO ) mice with Actin used as loading control. ( D ) Western blot analysis of RBM47 in Rbm47 IKO mice showing no detectable protein in intestinal nuclear extract. ( E ) Hepatic apoB RNA editing profile in Rbm47 LKO mice, representative of three individual livers. Forty-two clones were sequenced, with each clone represented by a circle. Solid circles indicate editing at the specified position. We examined an apoB region encompassing nucleotides 6563–7210, with editing only at the canonical cytidine (6666) detected. ( F ) Intestinal apoB RNA editing profile in Rbm47 IKO mice. Representative distribution of RNA editing sites from the same region (6563–7210) revealing only 2/19 clones exhibiting RNA editing. ( G ) Western blot analysis of hepatic apoB100 and apoB48 isoforms (three to five mice per genotype) with actin as loading control. (Panel to the right ) Quantitation of apoB100 isoform as a fraction of total apoB showing significant increase of apoB100 in liver of Rbm47 LKO mice. ( H ) Western blot analysis of RBM47 and A1CF in Rbm47 LKO mice at baseline and following adenoviral APOBEC1 overexpression. ( I ) Editing and hyperediting profile of hepatic apoB RNA following adenoviral overexpression of APOBEC1 in two Rbm47 LKO mice. Representative distribution of edited sites from nucleotide 6563 to nucleotide 7210.
    Figure Legend Snippet: Generation and characterization of conditional Rbm47 knockout mice. ( A ) Endogenous mouse Rbm47 gene and targeting construct. Schematic illustration of genomic structure of Rbm47 allele containing nine exons (orange boxes). Schematic of Rbm47 tm1a composition. (Numbered oranges boxes) Rbm47 exons; (blue boxes) Lac Z and neo cassettes; (green triangles) FRT sites; (purple triangles) LoxP sites. FLP recombination eliminates the LacZ and neo cassettes. Exon 6 containing transcriptional start codon and sequences encoding RNA recognition motifs was targeted for Cre-dependent homologous recombination. ( B ) Representative DNA electrophoresis showing genotype of WT and targeted alleles using primers P1 and P2 surrounding the 3′arm LoxP site. Analysis of the WT allele results in a 490 bp PCR product (lane 1 ) whereas recombination at the floxed allele generates a 530 bp product (f/f) (lane 3 ). Cre activation resuts in the loss of exon 6, evidenced by the absence of PCR amplification (lane 5 ). Molecular weights (bp) are indicated to the left . ( C ) Western blot detection of RBM47 in liver of floxed and Rbm47 liver-specific knockout ( Rbm47 LKO ) mice with Actin used as loading control. ( D ) Western blot analysis of RBM47 in Rbm47 IKO mice showing no detectable protein in intestinal nuclear extract. ( E ) Hepatic apoB RNA editing profile in Rbm47 LKO mice, representative of three individual livers. Forty-two clones were sequenced, with each clone represented by a circle. Solid circles indicate editing at the specified position. We examined an apoB region encompassing nucleotides 6563–7210, with editing only at the canonical cytidine (6666) detected. ( F ) Intestinal apoB RNA editing profile in Rbm47 IKO mice. Representative distribution of RNA editing sites from the same region (6563–7210) revealing only 2/19 clones exhibiting RNA editing. ( G ) Western blot analysis of hepatic apoB100 and apoB48 isoforms (three to five mice per genotype) with actin as loading control. (Panel to the right ) Quantitation of apoB100 isoform as a fraction of total apoB showing significant increase of apoB100 in liver of Rbm47 LKO mice. ( H ) Western blot analysis of RBM47 and A1CF in Rbm47 LKO mice at baseline and following adenoviral APOBEC1 overexpression. ( I ) Editing and hyperediting profile of hepatic apoB RNA following adenoviral overexpression of APOBEC1 in two Rbm47 LKO mice. Representative distribution of edited sites from nucleotide 6563 to nucleotide 7210.

    Techniques Used: Knock-Out, Mouse Assay, Construct, Homologous Recombination, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Activation Assay, Amplification, Western Blot, Clone Assay, Quantitation Assay, Over Expression

    15) Product Images from "Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †"

    Article Title: Effects of Vinylphosphonate Internucleotide Linkages on the Cleavage Specificity of Exonuclease III and on the Activity of DNA Polymerase I †

    Journal: Biochemistry

    doi: 10.1021/bi026985+

    Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).
    Figure Legend Snippet: Primer extension reactions by DNA polymerase I (A) and Klenow (B). All reactions were carried out using DNA substrates P1 (unmodified) and P2 (modified) in the presence of dATP (lane 2), dATP and dGTP (lane 3), dATP, dGTP, and dCTP (lane 4), and dATP, dGTP, dCTP, and dTTP (lane 5). Lane 1 shows the radioactively labeled substrate, whereas the positions of the vinylphosphonate internucleotide linkages are marked with asterisks. Panel C shows similar reactions carried out with Klenow and DNA polymerase I but this time using DNA substrate P3 (one modification).

    Techniques Used: Modification, Labeling

    Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.
    Figure Legend Snippet: Synthetic oligonucleotides (A) and the DNA substrates (B) used in this study. The vinylphosphonate internucleotide linkages are denoted with asterisks, whereas the positions of the radioactive phosphate labels are denoted with bullets. Substrates N1–N4 and N7 were used for exonuclease III digestions and substrates N5 and N6 for mung bean nuclease digestions. Substrates P1–P3 were used for primer extension reactions catalyzed by Klenow and DNA polymerase I.

    Techniques Used:

    16) Product Images from "Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants"

    Article Title: Necessity of integrated genomic analysis to establish a designed knock-in mouse from CRISPR-Cas9-induced mutants

    Journal: bioRxiv

    doi: 10.1101/2022.06.08.495409

    Analysis of targeted gene recombination by using of Southern blotting. (A) Schematic representation of wild-type mouse Prlhr genomic locus (left) and genomic locus with designed recombination (right). The black bars show specific probes used in Southern blotting. The horizontal arrows denote the expected sizes of restriction DNA fragments. (B) Southern blot analysis of genomic DNA of the F0, F1, F2 generations and wild type (wt). Blue, green and red arrows show the parent-offspring relationships. Black and red arrowheads show the DNA fragment from wild-type and designed knock-in allele, respectively. Green and blue arrowheads show the DNA fragments from unintentional mutant alleles.
    Figure Legend Snippet: Analysis of targeted gene recombination by using of Southern blotting. (A) Schematic representation of wild-type mouse Prlhr genomic locus (left) and genomic locus with designed recombination (right). The black bars show specific probes used in Southern blotting. The horizontal arrows denote the expected sizes of restriction DNA fragments. (B) Southern blot analysis of genomic DNA of the F0, F1, F2 generations and wild type (wt). Blue, green and red arrows show the parent-offspring relationships. Black and red arrowheads show the DNA fragment from wild-type and designed knock-in allele, respectively. Green and blue arrowheads show the DNA fragments from unintentional mutant alleles.

    Techniques Used: Southern Blot, Knock-In, Mutagenesis

    Analysis of copy number of the Venus gene by using droplet digital PCR. (A) Reliable detection of the copy number of the Venus gene by using genomic DNA of oxytocin receptor-Venus knock-in heterozygous mice generated by embryonic stem cells. Representative droplet plots of oxytocin receptor Venus/+ and +/+ (Venus-positive droplets (Upper left), OXTR positive-droplet (lower left)). Calculated copy number of the Venus gene in oxytocin receptor Venus/+ and +/+ mice (right). (B) Calculated copy numbers of the Venus gene in F0, F1 and F2 generations of Prlhr knock-in mice. Black arrows show the parent-offspring relationships.
    Figure Legend Snippet: Analysis of copy number of the Venus gene by using droplet digital PCR. (A) Reliable detection of the copy number of the Venus gene by using genomic DNA of oxytocin receptor-Venus knock-in heterozygous mice generated by embryonic stem cells. Representative droplet plots of oxytocin receptor Venus/+ and +/+ (Venus-positive droplets (Upper left), OXTR positive-droplet (lower left)). Calculated copy number of the Venus gene in oxytocin receptor Venus/+ and +/+ mice (right). (B) Calculated copy numbers of the Venus gene in F0, F1 and F2 generations of Prlhr knock-in mice. Black arrows show the parent-offspring relationships.

    Techniques Used: Digital PCR, Knock-In, Mouse Assay, Generated

    CRISPR-Cas9-mediated knock-in strategy at the prolactin releasing-peptide receptor (Prlhr) locus and analysis of Venus integration by using conventional PCR. (A) Schematic representation of wild-type mouse Prlhr genomic locus, sgRNA targeting site, targeting vector containing the Venus - polyadenylation signal and primer sets (internal primer pair, Internal-1and Internal-2: primer external to the targeting vector and internal primer pairs, 5’ext-1, 3’ext-1, 5’ext-2: primer pair external to the targeting vector, Full ext-1). Red and black arrows show the primer external to the targeting vector and internal primer, respectively. (B) Target sequence of Prlhr sgRNA on the C57BL6/N genome. (C) Schematic representation of hCas9 mRNA, sgRNA and lssDNA co-injection into mouse embryos to generate founders (F0) with the knock-in mutation. (D) Conventional PCR analysis of genomic DNA from F0, F1 and F2 generations by using each primer pair. Black arrows show the parent-offspring relationships. (E) Family trees of numbers 5 and 24 of the F0 generation.
    Figure Legend Snippet: CRISPR-Cas9-mediated knock-in strategy at the prolactin releasing-peptide receptor (Prlhr) locus and analysis of Venus integration by using conventional PCR. (A) Schematic representation of wild-type mouse Prlhr genomic locus, sgRNA targeting site, targeting vector containing the Venus - polyadenylation signal and primer sets (internal primer pair, Internal-1and Internal-2: primer external to the targeting vector and internal primer pairs, 5’ext-1, 3’ext-1, 5’ext-2: primer pair external to the targeting vector, Full ext-1). Red and black arrows show the primer external to the targeting vector and internal primer, respectively. (B) Target sequence of Prlhr sgRNA on the C57BL6/N genome. (C) Schematic representation of hCas9 mRNA, sgRNA and lssDNA co-injection into mouse embryos to generate founders (F0) with the knock-in mutation. (D) Conventional PCR analysis of genomic DNA from F0, F1 and F2 generations by using each primer pair. Black arrows show the parent-offspring relationships. (E) Family trees of numbers 5 and 24 of the F0 generation.

    Techniques Used: CRISPR, Knock-In, Polymerase Chain Reaction, Plasmid Preparation, Sequencing, Injection, Mutagenesis

    Analysis of conventional PCR and genomic sequencing in number 1 of the F1 generation. (A) Schematic representation of designed Prlhr knock-in locus and primer sets for PCR amplification (primer external to the targeting vector pairs, Full ext-2 and -3: internal primer and primer external to the targeting vector pair, 3’ ext-2). Red and black arrows show the primer external to the targeting vector and internal primer, respectively. (B) Conventional PCR analysis of genomic DNA from number 1 of the F1 generation by using each primer pair. Sequence analysis for PCR products of Full ext-2 and -3 (C) and 3’ ext-2 (D).
    Figure Legend Snippet: Analysis of conventional PCR and genomic sequencing in number 1 of the F1 generation. (A) Schematic representation of designed Prlhr knock-in locus and primer sets for PCR amplification (primer external to the targeting vector pairs, Full ext-2 and -3: internal primer and primer external to the targeting vector pair, 3’ ext-2). Red and black arrows show the primer external to the targeting vector and internal primer, respectively. (B) Conventional PCR analysis of genomic DNA from number 1 of the F1 generation by using each primer pair. Sequence analysis for PCR products of Full ext-2 and -3 (C) and 3’ ext-2 (D).

    Techniques Used: Polymerase Chain Reaction, Genomic Sequencing, Knock-In, Amplification, Plasmid Preparation, Sequencing

    17) Product Images from "Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage"

    Article Title: Nick-seq for single-nucleotide resolution genomic maps of DNA modifications and damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa473

    Nick-seq validation. ( A ) Mapping single-strand breaks produced by Nb.BsmI in E. coli genomic DNA. Middle panel : Representative view of sequencing reads distributed in one genomic region. Red and green peaks mark reads mapped to forward and reverse strands of the genome, respectively. Lower panel : Amplification of the genomic region surrounding one peak, with read pile ups for TdT and NT sequencing converging on the site of the strand-break. ( B ) Nb.BsmI mapping data were used to define data processing parameters for accuracy and sensitivity of Nick-seq. Coverage ratios (the ratio of the peaks relative to corresponding sites in an untreated DNA control) were calculated for sequencing data performed with TdT alone (blue line) or the combination of TdT and NT (orange line). The sensitivity and specificity for detection of site-specific strand-breaks was then plotted for ratios ranging from 2 to 7. In general, higher coverage ratios yield greater accuracy but lower sensitivity, and the combination of TdT and NT provided significantly greater specificity. ( C ) With a coverage ratio of 2, Nick-seq identified 2462 (97.5%) of the 2681 predicted Nb.BsmI sites. Among the 62 (2.5%) ‘false-positive’ sites, 27 (1%) of them occurred in sequences differing from the consensus by one nucleotide. These sites showed lower average sequencing coverage (75 versus 1318) and likely represent Nb.BsmI ‘star’ activity.
    Figure Legend Snippet: Nick-seq validation. ( A ) Mapping single-strand breaks produced by Nb.BsmI in E. coli genomic DNA. Middle panel : Representative view of sequencing reads distributed in one genomic region. Red and green peaks mark reads mapped to forward and reverse strands of the genome, respectively. Lower panel : Amplification of the genomic region surrounding one peak, with read pile ups for TdT and NT sequencing converging on the site of the strand-break. ( B ) Nb.BsmI mapping data were used to define data processing parameters for accuracy and sensitivity of Nick-seq. Coverage ratios (the ratio of the peaks relative to corresponding sites in an untreated DNA control) were calculated for sequencing data performed with TdT alone (blue line) or the combination of TdT and NT (orange line). The sensitivity and specificity for detection of site-specific strand-breaks was then plotted for ratios ranging from 2 to 7. In general, higher coverage ratios yield greater accuracy but lower sensitivity, and the combination of TdT and NT provided significantly greater specificity. ( C ) With a coverage ratio of 2, Nick-seq identified 2462 (97.5%) of the 2681 predicted Nb.BsmI sites. Among the 62 (2.5%) ‘false-positive’ sites, 27 (1%) of them occurred in sequences differing from the consensus by one nucleotide. These sites showed lower average sequencing coverage (75 versus 1318) and likely represent Nb.BsmI ‘star’ activity.

    Techniques Used: Produced, Sequencing, Amplification, Activity Assay

    Overview of Nick-seq and data analysis workflow. ( A ) Nick-seq library preparation. Briefly, genomic DNA is first subjected to sequencing-compatible fragmentation; the resulting 3′-OH ends are blocked with dideoxyNTPs; the DNA modification is converted to a strand-break by enzymatic or chemical treatment; capture of the 3′- and 5′-ends of resulting strand-breaks using two complementary strategies: one portion of DNA is subjected to nick translation (NT) with α-thio-dNTPs to generate phosphorothioate (PT)-containing oligonucleotides that are resistant to subsequent hydrolysis of the bulk of the genomic DNA by exonuclease III and RecJ f . The purified PT-protected fragments are used to generate an NGS library with the modification of interest positioned at the 5′-end of the PT-labeled fragment. A second portion of the same DNA sample is used for terminal transferase (TdT)-dependent poly(dT) tailing of the 3′-end of the strand-break, with the tail used to create a sequencing library by reverse transcriptase template switching ( 9 ). Subsequent NGS positions the modification of interest 5′-end of the poly(dT) tail. ( B ) Processing of the Nick-seq data includes: raw NGS reads are aligned to the reference genome for read coverage calculation; the genome sites with reads coverage ≥5 are then filtered for nick site calling with three parameters: x = the read coverage at position N/coverage at N – 1; y = coverage at position N/coverage at N + 1; z = coverage at position N /coverage at N of negative control sample. The site N is defined as a nick site if its x > 1, y > 1, z > 1 for NT reads and x > 2, y > 2, z > 2 for TdT reads.
    Figure Legend Snippet: Overview of Nick-seq and data analysis workflow. ( A ) Nick-seq library preparation. Briefly, genomic DNA is first subjected to sequencing-compatible fragmentation; the resulting 3′-OH ends are blocked with dideoxyNTPs; the DNA modification is converted to a strand-break by enzymatic or chemical treatment; capture of the 3′- and 5′-ends of resulting strand-breaks using two complementary strategies: one portion of DNA is subjected to nick translation (NT) with α-thio-dNTPs to generate phosphorothioate (PT)-containing oligonucleotides that are resistant to subsequent hydrolysis of the bulk of the genomic DNA by exonuclease III and RecJ f . The purified PT-protected fragments are used to generate an NGS library with the modification of interest positioned at the 5′-end of the PT-labeled fragment. A second portion of the same DNA sample is used for terminal transferase (TdT)-dependent poly(dT) tailing of the 3′-end of the strand-break, with the tail used to create a sequencing library by reverse transcriptase template switching ( 9 ). Subsequent NGS positions the modification of interest 5′-end of the poly(dT) tail. ( B ) Processing of the Nick-seq data includes: raw NGS reads are aligned to the reference genome for read coverage calculation; the genome sites with reads coverage ≥5 are then filtered for nick site calling with three parameters: x = the read coverage at position N/coverage at N – 1; y = coverage at position N/coverage at N + 1; z = coverage at position N /coverage at N of negative control sample. The site N is defined as a nick site if its x > 1, y > 1, z > 1 for NT reads and x > 2, y > 2, z > 2 for TdT reads.

    Techniques Used: Sequencing, Modification, Nick Translation, Purification, Next-Generation Sequencing, Labeling, Negative Control

    18) Product Images from "Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells"

    Article Title: Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-018-0465-4

    Identification of chromosome breaks in BA-treated TWO4 cells. Genomic DNA was extracted from BA-treated TWO4 cells for nested IPCR as described in “Methods” section. a Representative gel picture showing the AF9 gene cleavages in BA-treated TWO4 cells detected within: ( a i ) SAR region ( a ii ) Non-SAR region. TWO4 cells were left untreated (Lanes 1–6) or treated for 3 h with 0.5 mM of BA at pH 7.4 (Lanes 7–12) and pH 5.8 (Lanes 13–18). The IPCR bands derived from the AF9 cleaved fragments were indicated by the side bracket. M: 100 bp DNA ladder. N: Negative control for IPCR. b The average number of AF9 gene cleavages detected by IPCR. Data represents means and SDs of three independent experiments. Each experiment consisted of at least two sets of IPCR assays performed in five to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p
    Figure Legend Snippet: Identification of chromosome breaks in BA-treated TWO4 cells. Genomic DNA was extracted from BA-treated TWO4 cells for nested IPCR as described in “Methods” section. a Representative gel picture showing the AF9 gene cleavages in BA-treated TWO4 cells detected within: ( a i ) SAR region ( a ii ) Non-SAR region. TWO4 cells were left untreated (Lanes 1–6) or treated for 3 h with 0.5 mM of BA at pH 7.4 (Lanes 7–12) and pH 5.8 (Lanes 13–18). The IPCR bands derived from the AF9 cleaved fragments were indicated by the side bracket. M: 100 bp DNA ladder. N: Negative control for IPCR. b The average number of AF9 gene cleavages detected by IPCR. Data represents means and SDs of three independent experiments. Each experiment consisted of at least two sets of IPCR assays performed in five to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p

    Techniques Used: Derivative Assay, Negative Control

    Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p
    Figure Legend Snippet: Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p

    Techniques Used: DNA Extraction, Derivative Assay, Marker, Negative Control

    19) Product Images from "A Universal Proximity CRISPR Cas12a Assay for Ultrasensitive Detection of Nucleic Acids and Proteins"

    Article Title: A Universal Proximity CRISPR Cas12a Assay for Ultrasensitive Detection of Nucleic Acids and Proteins

    Journal: bioRxiv

    doi: 10.1101/734582

    Optimization of the concentrations of DNA polymerase (Klenow Fragment, unit) for protein analysis. (A) The detection of protein was achieved using a binding-induced primer extension and then CRISPR Cas12a ampification. (B-D) Detection of anti-biotin antibody using varying concentrations of DNA polymerase. The optimal amount of Klenow Fragment was found to be 5 units, as it maximizes detection signals and kinetics while maintains a reasonably low background. [Anti-biotin] = 5 nM, [P1’] = [P2’] = 5 nM.
    Figure Legend Snippet: Optimization of the concentrations of DNA polymerase (Klenow Fragment, unit) for protein analysis. (A) The detection of protein was achieved using a binding-induced primer extension and then CRISPR Cas12a ampification. (B-D) Detection of anti-biotin antibody using varying concentrations of DNA polymerase. The optimal amount of Klenow Fragment was found to be 5 units, as it maximizes detection signals and kinetics while maintains a reasonably low background. [Anti-biotin] = 5 nM, [P1’] = [P2’] = 5 nM.

    Techniques Used: Binding Assay, CRISPR

    20) Product Images from "Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes"

    Article Title: Metal transcription factor-1 regulation via MREs in the transcribed regions of selenoprotein H and other metal- responsive genes

    Journal: Biochimica et biophysica acta

    doi: 10.1016/j.bbagen.2009.11.003

    Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.
    Figure Legend Snippet: Metal response elements in Selh genes from nine species A. Position of MREs predicted in the Selh genes from nine species. Genomic DNA from the indicated species (dashed lines) was analyzed over the region spanning 1000bp upstream and downstream from the translational start (TrSS) sites, indicated by bent arrows below the dashed lines. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. Experimentally verified MTF-1 binding sites are marked by asterisks. MRE type a through f indicates the mouse metallothionein MRE with which the given MRE shows identity in the core sequence. MREx indicates that those sequences do not have a corresponding counterpart among the mouse metallothionein MREs. The conserved MREs located 200–350bp downstream of the TSS are encircled in the vertical box. Scale is shown at the lower left. B. Predicted MRE sequences in the human Selh gene. MRE1 through 4 predicted in the human Selh gene share identical core sequences (in bold) with MREs from the mouse metallothionein gene. MRE types are given in parentheses.

    Techniques Used: Binding Assay, Sequencing

    Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.
    Figure Legend Snippet: Mouse and human Selh genes are new targets of MTF-1 A. MTF-1 binding to the MRE-1 located in the 5′ UTR of the human Selh gene. ChIP assays were performed on MSTO-211H and HEK-293T cell extracts in the absence or presence of heavy metal treatment. Black bars represent immunoprecipitation with MTF-1 specific antibody (MTF-1 Ab) and white bars represent negative control (normal goat serum), indicated as no Ab. DNA fold enrichment units are normalized against input of total DNA used for immunoprecipitation and no Ab control. Three independent immunoprecipitations were carried out, immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. B. MTF-1 binding to MREs of mouse Selh coding region was assessed in MEF cells, as described in the legend for Fig 3A. Quantitation of MTF-1 binding for mouse MRE1 was evaluated by real time PCR (n=3) and data are plotted as mean ± SD. C . Gel electrophoresis analysis (4% polyacrylamide -TBE) of the amplification products from HEK-293T cell ChIP assays in the absence or presence Zn ++ addition. Band intensities from the gel were quantified using Adobe Photoshop software. Percent MTF-1 binding (signal minus background (no Ab) relative to input) is shown in the table below the gel pictures. Three independent immunoprecipitations were carried out and a representative of the output data is shown in this figure.

    Techniques Used: Binding Assay, Chromatin Immunoprecipitation, Immunoprecipitation, Negative Control, Real-time Polymerase Chain Reaction, Quantitation Assay, Nucleic Acid Electrophoresis, Amplification, Software

    Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.
    Figure Legend Snippet: Mouse Txnrd2 gene is a new target of MTF-1 A. In-silico analysis of the mouse selenoprotein gene Txnrd 2 identified 3 putative MRE sequences. We analyzed the region spanning 1000bp upstream and downstream from the translational start site. Gray ovals above and below the dashed lines represent predicted MREs on the plus and minus strand, respectively. MRE1 through 3 share identical core sequences (in bold) with MREs from the mouse metallothionein gene. The MRE types are given in parentheses. B. MTF-1 binding was assessed in MEF cells using primers specific for MRE2+3 in the absence or presence of heavy metal treatment as described in the text and in the legend for Fig. 3A. Three independent immunoprecipitations were carried. Immunoprecipitated MRE-containing DNA was quantified by real time PCR (n=3) and data are plotted as mean ± SD. C. Mouse Txnrd2 mRNA expression was analyzed in MTF-1-KO and MTF-1FLAG cells in the absence of added metal. Three independent experiments were carried out. mRNAs levels relative to Hprt were analyzed by real time PCR ( n = 3) and are plotted as mean ± SD. Student’s t-test was used to evaluate statistical significance (indicated by asterisk) between the two groups.

    Techniques Used: In Silico, Binding Assay, Immunoprecipitation, Real-time Polymerase Chain Reaction, Expressing

    21) Product Images from "Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35"

    Article Title: Disclosing early steps of protein-primed genome replication of the Gram-positive tectivirus Bam35

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw673

    Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.
    Figure Legend Snippet: Deoxynucleotide specificity for Bam35 TP initiation reaction. B35DNAP and TP were incubated in template-independent ( A ) or TP-DNA directed ( B ) initiation assays. Template sequence determination of Bam35 TP initiation reaction is shown in ( C ). Initiation assays either in the absence of template (panel C, lanes 1, 8, 15 and 22) or with single stranded 29-mer oligonucleotide template containing the sequence of the genome left end or variants of this sequence (Supplementary Table S1). The deoxynucleotide used as well as the first six nucleotides of the template oligonucleotide sequence (in the 3′-5′ direction) are indicated above the gels. Reactions were triggered with MnCl 2 (see Materials and Methods). Longer autoradiography exposition times, related to the dATP assays, are indicated for each provided deoxynucleotide.

    Techniques Used: Incubation, Sequencing, Autoradiography

    22) Product Images from "Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿"

    Article Title: Unique Substrate Spectrum and PCR Application of Nanoarchaeum equitans Family B DNA Polymerase ▿

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00624-08

    Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.
    Figure Legend Snippet: Amino acid sequence alignment, corresponding to residues 1 to 147 of Neq DNA polymerase of archaeal family B DNA polymerases. Multiple alignments were produced using the AlignX software (Invitrogen): Tko, Thermococcus kodakarensis KOD1 (GenBank accession number TK0001); Tfu, Thermococcus fumicolans (CAA93738); Tgo, Thermococcus gorgonarius (P56689); Tli, Thermococcus litoralis (AAA72101); Pfu, Pyrococcus furiosus (PF0212); Pwo, Pyrococcus woesei (P61876); Neq, Nanoarchaeum equitans (NEQ068). Shaded amino acid residues indicate identical and conserved residues in those DNA polymerases. The amino acid residues indicated by asterisks comprise the uracil-binding pocket of Tgo ). To assist in recognizing obvious differences of amino acids concerning the uracil-binding pocket, nonidentical residues of Neq DNA polymerase are rounded with rectangle borders.

    Techniques Used: Sequencing, Produced, Software, Binding Assay

    23) Product Images from "RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae"

    Article Title: RAD54 controls access to the invading 3?-OH end after RAD51-mediated DNA strand invasion in homologous recombination in Saccharomyces cerevisiae

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn980

    Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.
    Figure Legend Snippet: Rad51–K191R requires higher Rad54 concentrations for efficient D-loop extension. ( A ) Rad51 titration in the D-loop extension assay with Pst I-95-mer: 0.17 µM (lanes 2–4), 0.34 µM (lanes 5–7), 0.5 µM (lanes 8–10), 0.67 µM (lanes 11–13), 1 µM (lanes 14–16), and 2 µM (lanes 18–20). Reactions contain 72 nM Rad54, as determined to be the optimum in Supplementary Figure 4 . Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker. ( B ) Rad51–K191R titration, otherwise as in (A). The signals labeled by asterisk in A and B are generated by the proofreading activity (3′–5′ exonuclease) of Klenow polymerase as determined in reconstruction experiments and verified using a proofreading-deficient version of Klenow polymerase (data not shown). This signal disappears when Rad51 fully occupies the 95-mer, further validating that Klenow polymerase has no access to the 3′-OH end with Rad51 bound to it. ( C ) Quantitation of the Rad51 and Rad51–K191R protein titration results in (a, b; 20 min time points). A higher stoichiometry (1/2 nt) is optimal for the Rad51–K191R protein compared to the 1/4 nt stoichiometry for the wild-type Rad51 protein. This was expected from previous results showing a DNA-binding defect for the Rad51–K191R protein, requiring higher protein to DNA ratios to assemble saturated protein filaments ( 7 ). ( D ) Titration of Rad54 in D-loop extension assay with Rad51 at optimal 1/4 stoichiometry. ( E ) Titration of Rad54 in D-loop extension assay with Rad51–K191R at optimal 1/2 stoichiometry. ( F ) Quantitation of results in (D, E; 20 min time points). The results were normalized for the amount of linear D-loops captured by psoralen crosslinking under each assay condition. D-loop extension with wild-type Rad51 reaches an optimum at 36 nM Rad54, whereas the optimum with Rad51–K191R is reached at 54 nM. Shown are the means from three determinations; error bars represent 1 SD.

    Techniques Used: Titration, Labeling, Marker, Generated, Activity Assay, Quantitation Assay, Binding Assay

    Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.
    Figure Legend Snippet: Rad54 is required for D-loop extension. D-loop extension assays. The Pst I-95-mer is homologous to the terminal sequence of the Pst I-linearized pUC19 DNA (2686 bp). Reaction products are identified either by end-labeling the 95-mer ( A , B , F ) or by incorporation of α- 32 P-dGTP ( C – E , G ). (A) D-loop extension assay with end-labeled Pst I-95-mer. Rad51 nucleoprotein filaments were incubated either in the presence of Rad54 (72 nM, lanes 9–11), or absence of Rad54 (lanes 6–8), or absence of DNA polymerase I (Klenow fragment 24 nM, lanes 12–14). Protein-free 95-mer was also incubated either in the presence of Rad54 (72 nM, lanes 3–5), or absence of Rad54 (lanes 15–17). Lane 1 shows end-labeled Pst I-linearized pUC19 as a size marker, and lane 2 the end-labeled 95-mer. (B) Quantification of the results for D-loop extension in (A). (C) D-loop extension assay with α- 32 P-dGTP and unlabeled Pst I-95-mer. Reactions were as in (A), except lane 2 contains unlabeled Pst I-95-mer and lanes 18–20 show Pst I-linearized pUC19 with DNA polymerase I (Klenow fragment, 24 nM). (D) Quantification of the stable extension product from (C). Stable extension products are D-loops of sufficient length to be stable under electrophoresis conditions. (E) Quantification of the unstable extension product from (C). Unstable extension products are extended 95-mers with insufficient length to result in stable D-loops under electrophoresis conditions. For (B)–(E) shown are means from three determinations; error bars represent 1 SD. (F) Analysis of extension products on denaturing gel from reactions with end-labeled 95-mer and (G) with α- 32 P-dGTP. The signals labeled by asterisk are due to a combination of 3′–5′ exonuclease (proofreading) and/or polymerase activity of Klenow polymerase on the 95-mer or the linear dsDNA.

    Techniques Used: Sequencing, End Labeling, Labeling, Incubation, Marker, Electrophoresis, Activity Assay

    24) Product Images from "The Repressor Function of the Chlamydia Late Regulator EUO Is Enhanced by the Plasmid-Encoded Protein Pgp4"

    Article Title: The Repressor Function of the Chlamydia Late Regulator EUO Is Enhanced by the Plasmid-Encoded Protein Pgp4

    Journal: Journal of Bacteriology

    doi: 10.1128/JB.00793-19

    EUO binding and repression are enhanced by Pgp4. (A) EMSAs with radiolabeled DNA probes containing promoter regions of C. trachomatis glgA , ctl338 , omcB (EUO positive control), and groEL (negative control) in the presence of 0 or 30 nM rEUO and increasing concentrations of rPgp4 (0, 0.5, 1.0, or 2.0 μM). (B) In vitro transcription assays in which the C. trachomatis glgA , ctl338 , omcB , ctl305 , ctl397 , and groEL promoters were transcribed by RNA polymerase in the absence or presence of 0 or 0.5 μM rEUO and 0, 0.5, 1.0, or 2.0 μM rPgp4. Transcript levels were quantified with a phosphorimager: for each promoter, baseline transcription in the absence of Pgp4 and EUO was defined as 100%, and other transcript levels were normalized to this value and reported as relative transcription. Results are the averages from three independent experiments with standard deviation indicated by an error bar. Asterisks indicate statistically significant differences compared to rEUO alone (*, P
    Figure Legend Snippet: EUO binding and repression are enhanced by Pgp4. (A) EMSAs with radiolabeled DNA probes containing promoter regions of C. trachomatis glgA , ctl338 , omcB (EUO positive control), and groEL (negative control) in the presence of 0 or 30 nM rEUO and increasing concentrations of rPgp4 (0, 0.5, 1.0, or 2.0 μM). (B) In vitro transcription assays in which the C. trachomatis glgA , ctl338 , omcB , ctl305 , ctl397 , and groEL promoters were transcribed by RNA polymerase in the absence or presence of 0 or 0.5 μM rEUO and 0, 0.5, 1.0, or 2.0 μM rPgp4. Transcript levels were quantified with a phosphorimager: for each promoter, baseline transcription in the absence of Pgp4 and EUO was defined as 100%, and other transcript levels were normalized to this value and reported as relative transcription. Results are the averages from three independent experiments with standard deviation indicated by an error bar. Asterisks indicate statistically significant differences compared to rEUO alone (*, P

    Techniques Used: Binding Assay, Positive Control, Negative Control, In Vitro, Standard Deviation

    EUO binds to and represses Pgp4 target promoters. (A) Electrophoretic mobility shift assays (EMSAs) measuring binding of recombinant EUO (rEUO) to the C. trachomatis promoter regions of two Pgp4 target genes, glgA (–100 to +5) and ctl338 (–100 to +5), as well as a known EUO target, omcB (−60 to +5) as a positive control and a nontarget, groEL (−60 to +5), as a negative control. 32 P-radiolabeled DNA probes were incubated with 0, 30, or 120 nM rEUO and subjected to electrophoresis on an acrylamide gel. The locations of free and bound probes are indicated on the right. (B) In vitro transcription assays showing transcription of these four promoters by C. trachomatis RNA polymerase in the presence of 0, 0.5, or 1.0 μM rEUO. 32 P-radiolabeled transcripts were subjected to electrophoresis on an acrylamide gel and detected with a phosphorimager. (C) EMSA measuring binding of 0 or 2 μM recombinant Pgp4 (rPgp4) to the promoter regions of C. trachomatis glgA (–100 to +5) and ctl338 (–100 to +5). (D) In vitro transcription of the glgA and ctl338 promoters by C. trachomatis RNA polymerase in the absence or presence of 2 μM rPgp4.
    Figure Legend Snippet: EUO binds to and represses Pgp4 target promoters. (A) Electrophoretic mobility shift assays (EMSAs) measuring binding of recombinant EUO (rEUO) to the C. trachomatis promoter regions of two Pgp4 target genes, glgA (–100 to +5) and ctl338 (–100 to +5), as well as a known EUO target, omcB (−60 to +5) as a positive control and a nontarget, groEL (−60 to +5), as a negative control. 32 P-radiolabeled DNA probes were incubated with 0, 30, or 120 nM rEUO and subjected to electrophoresis on an acrylamide gel. The locations of free and bound probes are indicated on the right. (B) In vitro transcription assays showing transcription of these four promoters by C. trachomatis RNA polymerase in the presence of 0, 0.5, or 1.0 μM rEUO. 32 P-radiolabeled transcripts were subjected to electrophoresis on an acrylamide gel and detected with a phosphorimager. (C) EMSA measuring binding of 0 or 2 μM recombinant Pgp4 (rPgp4) to the promoter regions of C. trachomatis glgA (–100 to +5) and ctl338 (–100 to +5). (D) In vitro transcription of the glgA and ctl338 promoters by C. trachomatis RNA polymerase in the absence or presence of 2 μM rPgp4.

    Techniques Used: Electrophoretic Mobility Shift Assay, Binding Assay, Recombinant, Positive Control, Negative Control, Incubation, Electrophoresis, Acrylamide Gel Assay, In Vitro

    25) Product Images from "Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog"

    Article Title: Synergistic Activation of the Pathogenicity-Related Proline Iminopeptidase Gene in Xanthomonas campestris pv. campestris by HrpX and a LuxR Homolog

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01726-12

    Binding of HrpX to potential PIP box by EMSA. The interaction of the DNA probe with purified HrpX-MBP is shown. Each lane contains 0.65 μg isotope-labeled probe. Lanes 1 to 3 contain 12.28 μg, 24.56 μg, and 49.12 μg HrpX-MBP, respectively; lane 4 contains MBP (44.48 μg) as the negative control; lane 5 contains the free probe as the positive control; and lanes 6 to 10 contain the same concentration of HrpX-MBP (49.12 μg) and various amounts of unlabeled probe (0.65 μg, 3.23 μg, 13 μg, and 19.5 μg, respectively) as competitors.
    Figure Legend Snippet: Binding of HrpX to potential PIP box by EMSA. The interaction of the DNA probe with purified HrpX-MBP is shown. Each lane contains 0.65 μg isotope-labeled probe. Lanes 1 to 3 contain 12.28 μg, 24.56 μg, and 49.12 μg HrpX-MBP, respectively; lane 4 contains MBP (44.48 μg) as the negative control; lane 5 contains the free probe as the positive control; and lanes 6 to 10 contain the same concentration of HrpX-MBP (49.12 μg) and various amounts of unlabeled probe (0.65 μg, 3.23 μg, 13 μg, and 19.5 μg, respectively) as competitors.

    Techniques Used: Binding Assay, Purification, Labeling, Negative Control, Positive Control, Concentration Assay

    26) Product Images from "Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay"

    Article Title: Rapid and Sensitive Detection of Didymella bryoniae by Visual Loop-Mediated Isothermal Amplification Assay

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.01372

    Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.
    Figure Legend Snippet: Optimal reaction temperatures of LAMP. (A) Detecting LAMP products by adding fluorescence metal indicator (calcein); the assessment was based on visualization of a color change from brown to yellowish-green. (B) Agarose gel electrophoresis analysis of the LAMP products. In (A,B) , lane 1: 61°C, lane 2: 62°C, lane 3: 63°C, lane 4: 64°C, lane 5: 65°C, lane 6: 66°C, lane 7: 68°C. M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.
    Figure Legend Snippet: LAMP detection of D. bryoniae (DBJSJY2). Assessment is based on (A) LAMP for detection of D. bryoniae was using a fluorescence metal indicator (calcein) as a visual indicator. The positive reaction becomes yellowish-green, and the negative is still brown; (B) LAMP product was manifested as a ladder-like pattern on a 2.0% agarose gel. M: Trans DNA Marker II (Transgen Biotech, Beijing). In (A,B) , 1: Negative reaction (without target DNA), 2: Positive reaction (with target DNA). The same results were obtained in all three replicates.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Marker

    Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Specificity of LAMP detection of D. bryoniae . Assessment was based on (A) fluorescence metal indicator calcein visualization of color change, (B) the turbidity analysis of the LAMP products or (C) agarose gel electrophoresis analysis of the LAMP products. Lane 1, Didymella bryoniae (strain DBJSJY2) RGI; lane 2, Didymella bryoniae (strain DBAHHF2,) RGI; lane 3, Didymella bryoniae (strain DBZJNB5) RGI; lane 4, Didymella bryoniae (strain DBJSNJ60) RGI; lane 5, Didymella bryoniae (strain DBZJNB7) RGII; lane 6, Ascochyta pinodes ZJ-1; lane 7, Colletotrichum orbiculare NJ-1; lane 8, Pythium paroecandrum Drechsler ; lane 9, Alternaria alternata LH1401; lane 10, Fusarium verticillioide ; lane 11, Fusarium oxysporum f.sp. niveum Race 0; lane 12, Fusarium oxysporum f.sp. niveum Race 1; lane 13, Fusarium oxysporum f.sp. niveum Race 2; lane 14, positive control; lane 15, negative control. M, Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Fluorescence, Agarose Gel Electrophoresis, Positive Control, Negative Control, Marker

    Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Optimal reaction time of LAMP. (A) Agarose gel electrophoresis analysis of the LAMP products. (B) Detecting LAMP products by adding a fluorescence metal indicators (calcein). In (A,B) , lane 1: 60 min, lane 2: 45 min, lane 3: 30 min, lane 4: 15 min, M: Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Agarose Gel Electrophoresis, Fluorescence, Marker

    Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.
    Figure Legend Snippet: Sensitivity of the LAMP and conventional PCR. LAMP and conventional PCR assays using 10-fold serial dilutions of purified target DNA from D. bryoniae genomic DNA (strain DBJSJY2). (A) Detecting LAMP products by adding a fluorescence metal indicator (calcein). (B) Agarose gel electrophoresis analysis of the LAMP products. (C) Conventional PCR. Concentrations of template DNA (fg μL -1 ) per reaction in (A,B) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1, lane 7 = 10 -1 and lane 8 = 10 -2 . Concentrations of template DNA (fg μL -1 ) per reaction in (C) were: lane 1 = 10 5 , lane 2 = 10 4 , lane 3 = 10 3 , lane 4 = 10 2 , lane 5 = 10, lane 6 = 1 and lane 7 = 10 -1 . In (B,C) , M indicates a Trans DNA Marker II (Transgen Biotech, Beijing). The same results were obtained in two repeat assessments.

    Techniques Used: Polymerase Chain Reaction, Purification, Fluorescence, Agarose Gel Electrophoresis, Marker

    27) Product Images from "The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1"

    Article Title: The cytotoxic T lymphocyte protease granzyme A cleaves and inactivates poly(adenosine 5?-diphosphate-ribose) polymerase-1

    Journal: Blood

    doi: 10.1182/blood-2008-12-195768

    DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)
    Figure Legend Snippet: DNA repair by PARP-1 is disrupted by GzmA. (A) GzmA-induced DNA nicks, radiolabeled with Klenow, in HeLa cells are enhanced by inhibiting PARP-1 using the chemical inhibitor 1,5 dihydroxyisoquinoline (DIQ) and reduced by overexpressing WT PARP-1. (B)

    Techniques Used:

    28) Product Images from "Landscape of DNA binding signatures of myocyte enhancer factor-2B reveals a unique interplay of base and shape readout"

    Article Title: Landscape of DNA binding signatures of myocyte enhancer factor-2B reveals a unique interplay of base and shape readout

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa642

    Sequence variations for MEF2B binding sites. ( A ) Major MEF2B–DNA interactions based on co-crystal structure of MEF2B in complex with DNA (PDB ID: 1N6J) ( 27 ) are observed at the DNA minor groove and backbone. Amino acids that interact with DNA, including positively charged residues in the vicinity of DNA, are shown in stick representation. Different views are shown to depict binding recognition by helix H1 and N-terminal tail regions. ( B ) PWM obtained from SELEX-seq data using MEME Suite ( 45 ). Analyzed sequences were obtained after two rounds of selection for top 10-mers with relative affinity > 0.7. ( C ) Strip chart showing relative binding affinities for 10-mers displaying full or partial matches to MEF2B consensus motif, represented by YTAW 4 TAR (Y = C or T; W = A or T; R = A or G). Variations in core motif are represented by underlined regions, with W4 , YTA and TAR denoting regions that deviate from W 4 , YTA and TAR, respectively. ( D ) Heat map of relative affinities for triplet variations at 5′ peripheral half-site ( YTA W 4 TAR) based on central core (W 4 ) preferences.
    Figure Legend Snippet: Sequence variations for MEF2B binding sites. ( A ) Major MEF2B–DNA interactions based on co-crystal structure of MEF2B in complex with DNA (PDB ID: 1N6J) ( 27 ) are observed at the DNA minor groove and backbone. Amino acids that interact with DNA, including positively charged residues in the vicinity of DNA, are shown in stick representation. Different views are shown to depict binding recognition by helix H1 and N-terminal tail regions. ( B ) PWM obtained from SELEX-seq data using MEME Suite ( 45 ). Analyzed sequences were obtained after two rounds of selection for top 10-mers with relative affinity > 0.7. ( C ) Strip chart showing relative binding affinities for 10-mers displaying full or partial matches to MEF2B consensus motif, represented by YTAW 4 TAR (Y = C or T; W = A or T; R = A or G). Variations in core motif are represented by underlined regions, with W4 , YTA and TAR denoting regions that deviate from W 4 , YTA and TAR, respectively. ( D ) Heat map of relative affinities for triplet variations at 5′ peripheral half-site ( YTA W 4 TAR) based on central core (W 4 ) preferences.

    Techniques Used: Sequencing, Binding Assay, Selection, Stripping Membranes

    29) Product Images from "Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor"

    Article Title: Regulation of the sol Locus Genes for Butanol and Acetone Formation in Clostridium acetobutylicum ATCC 824 by a Putative Transcriptional Repressor

    Journal: Journal of Bacteriology

    doi:

    Schematic representations (a) and results of PCR analysis (b) on wild-type (WT) C. acetobutylicum ATCC 824 and solR mutants B and H using primers (a) designed to amplify the junction between the vector portion of pO1X and the solR gene. For each gel in panel b, lane 1 contains Hin dIII-digested lambda marker, lane 2 contains the WT genomic template, lane 3 contains the solR mutant B template, and lane 4 contains the solR mutant H as the template. (Gel A) Extralong PCR using primers solR453 and solR1361 designed to amplify the complete insert. In lane 2, WT DNA shows an expected ∼0.9-kb band. This band is also seen, but much weaker, in both lanes 3 and 4. In addition, lane 4 contains a band with an apparent size of ∼7 kb (marked with an arrow), consistent with the presence of one insert of pO1X into solR of mutant H. (Gel B) PCR results using primers solR453 and Tc238. A band of ∼1.2 kb can be seen in lanes 3 and 4 with no product in lane 2. (Gel C) PCR results using primers Em373 and solR1361. A band of ∼2.1 kb is visible in lanes 3 and 4. Again, no product was observed with WT DNA (lane 2).
    Figure Legend Snippet: Schematic representations (a) and results of PCR analysis (b) on wild-type (WT) C. acetobutylicum ATCC 824 and solR mutants B and H using primers (a) designed to amplify the junction between the vector portion of pO1X and the solR gene. For each gel in panel b, lane 1 contains Hin dIII-digested lambda marker, lane 2 contains the WT genomic template, lane 3 contains the solR mutant B template, and lane 4 contains the solR mutant H as the template. (Gel A) Extralong PCR using primers solR453 and solR1361 designed to amplify the complete insert. In lane 2, WT DNA shows an expected ∼0.9-kb band. This band is also seen, but much weaker, in both lanes 3 and 4. In addition, lane 4 contains a band with an apparent size of ∼7 kb (marked with an arrow), consistent with the presence of one insert of pO1X into solR of mutant H. (Gel B) PCR results using primers solR453 and Tc238. A band of ∼1.2 kb can be seen in lanes 3 and 4 with no product in lane 2. (Gel C) PCR results using primers Em373 and solR1361. A band of ∼2.1 kb is visible in lanes 3 and 4. Again, no product was observed with WT DNA (lane 2).

    Techniques Used: Polymerase Chain Reaction, Plasmid Preparation, Marker, Mutagenesis

    30) Product Images from "Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS"

    Article Title: Cytidine triphosphate promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional spreading from parS

    Journal: bioRxiv

    doi: 10.1101/2021.02.11.430778

    DNA condensation by nanomolar ParB is parS dependent. ( A ) ParB does not condense scrambled parS DNA under standard CTP-Mg 2+ conditions. Errors are standard error of the mean of measurements on different molecules (N = 5). ( B ) The ParS-binding mutant, ParB R149G , does not condense 13x parS DNA under standard CTP-Mg 2+ conditions. Errors are standard error of the mean of measurements on different molecules (N = 14). ( C ) Schematic representation of DNA substrates containing 7, 13, and 26 copies of parS . The positions of the parS sites in the DNA cartoon are represented to scale. ( D ) Average force-extension curves of 7x parS DNA, 13x parS DNA, and 26x parS DNA obtained under standard CTP-Mg 2+ conditions. The condensation force correlates with increasing number of parS sequences. Solid lines in condensed data are guides for the eye. Errors are standard error of the mean of measurements on different molecules (N ≥ 7). ( E ) Schematic representation of DNA substrates containing 1, 2, and 4 copies of parS . The positions of the parS sites in the DNA cartoon are represented to scale. ( F ) Average force-extension curves of 1x parS DNA, 2x parS DNA, and 4x parS DNA obtained under standard CTP-Mg 2+ conditions. No condensation was observed for these three experiments. Errors are standard error of the mean of measurements on different molecules (N ≥ 7). No ParB data represent force-extension curves of DNA taken in the absence of protein and are fitted to the worm-like chain model.
    Figure Legend Snippet: DNA condensation by nanomolar ParB is parS dependent. ( A ) ParB does not condense scrambled parS DNA under standard CTP-Mg 2+ conditions. Errors are standard error of the mean of measurements on different molecules (N = 5). ( B ) The ParS-binding mutant, ParB R149G , does not condense 13x parS DNA under standard CTP-Mg 2+ conditions. Errors are standard error of the mean of measurements on different molecules (N = 14). ( C ) Schematic representation of DNA substrates containing 7, 13, and 26 copies of parS . The positions of the parS sites in the DNA cartoon are represented to scale. ( D ) Average force-extension curves of 7x parS DNA, 13x parS DNA, and 26x parS DNA obtained under standard CTP-Mg 2+ conditions. The condensation force correlates with increasing number of parS sequences. Solid lines in condensed data are guides for the eye. Errors are standard error of the mean of measurements on different molecules (N ≥ 7). ( E ) Schematic representation of DNA substrates containing 1, 2, and 4 copies of parS . The positions of the parS sites in the DNA cartoon are represented to scale. ( F ) Average force-extension curves of 1x parS DNA, 2x parS DNA, and 4x parS DNA obtained under standard CTP-Mg 2+ conditions. No condensation was observed for these three experiments. Errors are standard error of the mean of measurements on different molecules (N ≥ 7). No ParB data represent force-extension curves of DNA taken in the absence of protein and are fitted to the worm-like chain model.

    Techniques Used: Binding Assay, Mutagenesis

    31) Product Images from "Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe"

    Article Title: Ready-to-use nanopore platform for the detection of any DNA/RNA oligo at attomole range using an Osmium tagged complementary probe

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-76667-1

    Alternative approaches to testing hybridization between osmylated probes and targets. ( a ) Enzymatic elongation of osmylated primers using ssM13mp18 DNA as the template and DNA polymerase; time points obtained at 5, 10 and 20 min. No primer and M13 rev (−48) used as negative controls. With the exception of BJ1, all the other osmylated primers exhibit enzymatic elongation comparable to the positive control M13 fwd (6097). Absence of elongation with BJ1 is attributed to the presence of a T(OsBp) at the 3′end. Experiment shown at the far right of the graph, done with a 1:1 mixture of BJ1 and the positive control indicates that any components/traces present in the BJ1 preparation after osmylation were NOT responsible for inhibiting the elongation of BJ1. ( b – d ) Overlapping HPLC profiles from the analyses of different samples, with samples at about 5 μM in about 90% ONT buffer. The same HPLC method B was used for all the samples (see “Experimental section”). Intact ss oligos and ds oligos appear as sharp peaks, whereas osmylated oligos and hybrids with one osmylated strand appear as broad peaks; hybrids elute later compared to ss nucleic acids. ( b ) Sample composition: intact BJ2 (blue trace), intact complement of primerM13 for (-41) (red trace). The HPLC profile of their equimolar mixture is consistent with hybridization (green trace). ( c ) Sample composition: miRNA21 (blue trace), probe 21EXT carrying 8 dT(OsBp) moieties (red trace), and equimolar mixture of the two (green trace). HPLC profile of the mixture consistent with NO hybridization, attributed to the high number of single OsBp tags, 6 within a sequence of 22 nt, likely to distort the helical structure of the probe, and prevent ds formation. ( d ) Sample composition practically the same as under ( b ), with the exception that in these samples BJ2 carries 6 dT(OsBp) moieties (first peak with broad shape and absorbance at 312 nm). HPLC profile of the mixture is consistent with hybridization, attributed to the fact that most of the OsBp moieties are adjacent, so that the rest of the sequence can still hybridize with the target.
    Figure Legend Snippet: Alternative approaches to testing hybridization between osmylated probes and targets. ( a ) Enzymatic elongation of osmylated primers using ssM13mp18 DNA as the template and DNA polymerase; time points obtained at 5, 10 and 20 min. No primer and M13 rev (−48) used as negative controls. With the exception of BJ1, all the other osmylated primers exhibit enzymatic elongation comparable to the positive control M13 fwd (6097). Absence of elongation with BJ1 is attributed to the presence of a T(OsBp) at the 3′end. Experiment shown at the far right of the graph, done with a 1:1 mixture of BJ1 and the positive control indicates that any components/traces present in the BJ1 preparation after osmylation were NOT responsible for inhibiting the elongation of BJ1. ( b – d ) Overlapping HPLC profiles from the analyses of different samples, with samples at about 5 μM in about 90% ONT buffer. The same HPLC method B was used for all the samples (see “Experimental section”). Intact ss oligos and ds oligos appear as sharp peaks, whereas osmylated oligos and hybrids with one osmylated strand appear as broad peaks; hybrids elute later compared to ss nucleic acids. ( b ) Sample composition: intact BJ2 (blue trace), intact complement of primerM13 for (-41) (red trace). The HPLC profile of their equimolar mixture is consistent with hybridization (green trace). ( c ) Sample composition: miRNA21 (blue trace), probe 21EXT carrying 8 dT(OsBp) moieties (red trace), and equimolar mixture of the two (green trace). HPLC profile of the mixture consistent with NO hybridization, attributed to the high number of single OsBp tags, 6 within a sequence of 22 nt, likely to distort the helical structure of the probe, and prevent ds formation. ( d ) Sample composition practically the same as under ( b ), with the exception that in these samples BJ2 carries 6 dT(OsBp) moieties (first peak with broad shape and absorbance at 312 nm). HPLC profile of the mixture is consistent with hybridization, attributed to the fact that most of the OsBp moieties are adjacent, so that the rest of the sequence can still hybridize with the target.

    Techniques Used: Hybridization, Positive Control, High Performance Liquid Chromatography, Sequencing

    32) Product Images from "Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *"

    Article Title: Remodeling of Nucleoprotein Complexes Is Independent of the Nucleotide State of a Mutant AAA+ Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M111.223495

    DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated
    Figure Legend Snippet: DnaA(L366K) in either nucleotide form represses in vitro transcription from dnaA promoter similar to ATP-DnaA. The indicated amounts of ATP and ADP forms of DnaA(WT) ( A ) and DnaA(L366K) ( B ) were incubated with the template DNA, and RNA synthesis was initiated

    Techniques Used: In Vitro, Incubation

    33) Product Images from "CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS"

    Article Title: CTP promotes efficient ParB-dependent DNA condensation by facilitating one-dimensional diffusion from parS

    Journal: eLife

    doi: 10.7554/eLife.67554

    Tethered particle motion (TPM) experiments show single- parS DNA condensation by ParB. ( A ) Cartoon of the TPM setup. Essentially, the same employed for magnetic tweezers (MT) experiments but without the magnets. ( B ) Schematic representation of the TPM 1× parS DNA and TPM scrambled parS DNA substrates used for TPM experiments. ( C ) Examples of time courses of root mean squared (RMS) and RMS τ excursions (see Materials and methods) of one tether under the indicated experimental conditions. ( D ) Box plot of mean RMS τ excursions of multiple tethers for different experimental conditions. ParB is able to condense single- parS DNA in the presence of CTP-Mg 2+ .
    Figure Legend Snippet: Tethered particle motion (TPM) experiments show single- parS DNA condensation by ParB. ( A ) Cartoon of the TPM setup. Essentially, the same employed for magnetic tweezers (MT) experiments but without the magnets. ( B ) Schematic representation of the TPM 1× parS DNA and TPM scrambled parS DNA substrates used for TPM experiments. ( C ) Examples of time courses of root mean squared (RMS) and RMS τ excursions (see Materials and methods) of one tether under the indicated experimental conditions. ( D ) Box plot of mean RMS τ excursions of multiple tethers for different experimental conditions. ParB is able to condense single- parS DNA in the presence of CTP-Mg 2+ .

    Techniques Used:

    Atomic force microscopy (AFM) experiments show single- parS DNA condensation by ParB. ( A ) AFM image of control non- parS DNA (left) and histogram of contour lengths (right, n = 44). ( B ) AFM image of AFM 1× parS DNA (left) and histogram of contour lengths (right, n = 41). ( C ) Characteristic AFM image of experiment including non- parS DNA and 10 nM ParB 2 in the absence of cytidine triphosphate (CTP). No interaction between protein and DNA was observed. ( D ) Representative AFM image of experiment including 1× parS DNA and 10 nM ParB 2 in the absence of CTP. No interaction between protein and DNA was observed. ( E ) Characteristic AFM image of experiment including 1× parS DNA, 10 nm ParB 2 , and 3.3 mM CTP. 1× parS plasmids appeared partially compacted due to the interaction with ParB. ( F ) Examples of individual 1× parS DNA molecules interacting with ParB under CTP conditions.
    Figure Legend Snippet: Atomic force microscopy (AFM) experiments show single- parS DNA condensation by ParB. ( A ) AFM image of control non- parS DNA (left) and histogram of contour lengths (right, n = 44). ( B ) AFM image of AFM 1× parS DNA (left) and histogram of contour lengths (right, n = 41). ( C ) Characteristic AFM image of experiment including non- parS DNA and 10 nM ParB 2 in the absence of cytidine triphosphate (CTP). No interaction between protein and DNA was observed. ( D ) Representative AFM image of experiment including 1× parS DNA and 10 nM ParB 2 in the absence of CTP. No interaction between protein and DNA was observed. ( E ) Characteristic AFM image of experiment including 1× parS DNA, 10 nm ParB 2 , and 3.3 mM CTP. 1× parS plasmids appeared partially compacted due to the interaction with ParB. ( F ) Examples of individual 1× parS DNA molecules interacting with ParB under CTP conditions.

    Techniques Used: Microscopy

    34) Product Images from "Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis"

    Article Title: Accessing unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    Journal: Nature Communications

    doi: 10.1038/s41467-020-17061-3

    Schematic outline of TRIAD. a Generation of deletion libraries. Step 1: The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence on circular plasmid DNA. Step 2: MlyI digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a: self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 14 . Step 3b: DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b: MlyI digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b: self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. b Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by NotI and MlyI removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins3 and Ins3 (with, respectively, 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3′-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.
    Figure Legend Snippet: Schematic outline of TRIAD. a Generation of deletion libraries. Step 1: The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence on circular plasmid DNA. Step 2: MlyI digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a: self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 14 . Step 3b: DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b: MlyI digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b: self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. b Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by NotI and MlyI removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins3 and Ins3 (with, respectively, 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3′-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Techniques Used: Generated, In Vitro, Sequencing, Plasmid Preparation, Variant Assay, Ligation

    Mechanism for the generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1 bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5′, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry, respectively, 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1, 2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5′CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3′, 2-base overhangs with the target DNA (i.e., 5′N 5 T on one end and 5′TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.
    Figure Legend Snippet: Mechanism for the generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1 bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5′, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry, respectively, 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1, 2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5′CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3′, 2-base overhangs with the target DNA (i.e., 5′N 5 T on one end and 5′TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Techniques Used: Sequencing, Ligation

    35) Product Images from "Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization"

    Article Title: Restriction enzyme-free mutagenesis via the light regulation of DNA polymerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp150

    Effects of a caged thymidine nucleobase on the DNA polymerization by mesophilic DNA polymerases. ( A ) Products resulting from T4 DNA Polymerase extension. When using the non-caged template full-length product (38 nt) is obtained; however, using either D1 or D2 caged templates, polymerization is halted, leading to truncated product (26 or 22 nt, respectively). ( B ) Products resulting from T7 DNA Polymerase extension. Similar truncations are observed as with T4 DNA Polymerase. ( C ) Products resulting from DNA Polymerase I extension, demonstrating a polymerase read-through to yield full-length product (38 nt) in all cases.
    Figure Legend Snippet: Effects of a caged thymidine nucleobase on the DNA polymerization by mesophilic DNA polymerases. ( A ) Products resulting from T4 DNA Polymerase extension. When using the non-caged template full-length product (38 nt) is obtained; however, using either D1 or D2 caged templates, polymerization is halted, leading to truncated product (26 or 22 nt, respectively). ( B ) Products resulting from T7 DNA Polymerase extension. Similar truncations are observed as with T4 DNA Polymerase. ( C ) Products resulting from DNA Polymerase I extension, demonstrating a polymerase read-through to yield full-length product (38 nt) in all cases.

    Techniques Used:

    DNA polymerization through extension of a primer using a 32 nt template with a caged thymidine (blue square) 17 or 21 nt from the 3′ end of the template. A single caging group blocked polymerization by T4 and T7 DNA polymerase.
    Figure Legend Snippet: DNA polymerization through extension of a primer using a 32 nt template with a caged thymidine (blue square) 17 or 21 nt from the 3′ end of the template. A single caging group blocked polymerization by T4 and T7 DNA polymerase.

    Techniques Used:

    36) Product Images from "Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells"

    Article Title: Inhibitor of caspase-activated DNase expression enhances caspase-activated DNase expression and inhibits oxidative stress-induced chromosome breaks at the mixed lineage leukaemia gene in nasopharyngeal carcinoma cells

    Journal: Cancer Cell International

    doi: 10.1186/s12935-015-0205-1

    Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products
    Figure Legend Snippet: Flow chart showing DNA modification and IPCR. The arrow heads indicate the forward and reverse primers that were designed in opposite direction. Bam H I digestion yielded a mixture of intact chromosome and cleaved chromosome. Klenow fill-in produced blunt ended chromosome fragments which were then cyclilsed by T4 DNA ligase. The intact chromosome will become a large circle while the cleaved chromosome will become a smaller circle. Upon cyclisation, the primers are now in correct orientation for amplification. Msc I digestion cleaved both circles outside the amplification region, thus merely linearise the molecule. Amplification from intact MLL gene will produce longer PCR products while amplification from cleaved MLL gene will yield shorter PCR products

    Techniques Used: Flow Cytometry, Modification, Produced, Amplification, Polymerase Chain Reaction

    37) Product Images from "Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro"

    Article Title: Construction and characterization of mismatch-containing circular DNA molecules competent for assessment of nick-directed human mismatch repair in vitro

    Journal: Nucleic Acids Research

    doi:

    Intermediates in the ligation-based protocol. The plasmid pBR322 was used to illustrate the individual steps in the protocol for two reasons. Its smaller size (4361 bp) makes it easier to see small changes in molecular weight, and the large distance separating Eco RI and Bam HI (377 bp) is useful to illustrate the excision of such a fragment. Lane 1, 100 ng pBR322, which is primarily supercoiled (band A) after CsCl banding but contains a small amount of nicked circle (band E). Lane 2, treatment with 2 U Bam HI/µg DNA yields a linear plasmid (band B). Lane 3, addition of a 40-fold excess of heteroduplex oligo ends and T4 DNA ligase (100 U/µg plasmid DNA) yields band D. Lane 4, digestion with Eco RI, followed by size-exclusion chromatography on Sephacryl S500 resin (band C). An equivalent fraction of the total volume from each step was loaded, so that the relative staining intensities reflect relative yield.
    Figure Legend Snippet: Intermediates in the ligation-based protocol. The plasmid pBR322 was used to illustrate the individual steps in the protocol for two reasons. Its smaller size (4361 bp) makes it easier to see small changes in molecular weight, and the large distance separating Eco RI and Bam HI (377 bp) is useful to illustrate the excision of such a fragment. Lane 1, 100 ng pBR322, which is primarily supercoiled (band A) after CsCl banding but contains a small amount of nicked circle (band E). Lane 2, treatment with 2 U Bam HI/µg DNA yields a linear plasmid (band B). Lane 3, addition of a 40-fold excess of heteroduplex oligo ends and T4 DNA ligase (100 U/µg plasmid DNA) yields band D. Lane 4, digestion with Eco RI, followed by size-exclusion chromatography on Sephacryl S500 resin (band C). An equivalent fraction of the total volume from each step was loaded, so that the relative staining intensities reflect relative yield.

    Techniques Used: Ligation, Plasmid Preparation, Molecular Weight, Size-exclusion Chromatography, Staining

    Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).
    Figure Legend Snippet: Strategy for constructing nicked heteroduplexes. A mismatch-containing oligonucleotide duplex (Fig. 1) is ligated into a template plasmid molecule (1). Linearization of the plasmid (2) in the presence of the heteroduplex oligo, T4 ligase and restriction enzyme ( Bam HI) allows ligation of the small fragments onto each DNA end as a dead-end complex (3), because the Bam HI site is eliminated. Re-ligation of Bam HI-generated plasmid ends yields a molecule competent for a second digestion, returning them to the substrate pool. In the next step, digestion with Eco RI removes one ligation product and generates a ligation-competent DNA end (4). After removal of the smaller fragment, an intramolecular ligation reaction generates the nicked circular product (5). Unwanted linear molecules are removed by digestion with Exonuclease V (Materials and Methods).

    Techniques Used: Plasmid Preparation, Ligation, Generated

    Optimization of the ring closure ligation. A modified pET11aΔH intermediate (Fig. 1, structure 4) was incubated with T4 DNA ligase using DNA concentrations decreasing from 100 to 10 ng/µl (see Materials and Methods for conditions). This LS has one DNA end generated by Eco RI digestion and one contributed by the T·G-10H oligonucleotide heteroduplex. The supercoiled (SC) pET11aΔH plasmid loaded in the first and last lanes contains a small amount of NC molecules that serves as a close marker for the mobility of the desired product. At 100 ng/µl DNA, the predominant products are multimers that migrate more slowly. At 10 ng/µl template, the desired NC heteroduplex represents up to 30% of the products, and the formation of linear multimers is reduced. For each lane representing a sample of a ligation reaction, an equivalent sample was treated with Exonuclease V, which degrades linear molecules and reveals the circular products. Note that a trace amount of CD is formed in this experiment.
    Figure Legend Snippet: Optimization of the ring closure ligation. A modified pET11aΔH intermediate (Fig. 1, structure 4) was incubated with T4 DNA ligase using DNA concentrations decreasing from 100 to 10 ng/µl (see Materials and Methods for conditions). This LS has one DNA end generated by Eco RI digestion and one contributed by the T·G-10H oligonucleotide heteroduplex. The supercoiled (SC) pET11aΔH plasmid loaded in the first and last lanes contains a small amount of NC molecules that serves as a close marker for the mobility of the desired product. At 100 ng/µl DNA, the predominant products are multimers that migrate more slowly. At 10 ng/µl template, the desired NC heteroduplex represents up to 30% of the products, and the formation of linear multimers is reduced. For each lane representing a sample of a ligation reaction, an equivalent sample was treated with Exonuclease V, which degrades linear molecules and reveals the circular products. Note that a trace amount of CD is formed in this experiment.

    Techniques Used: Ligation, Modification, Incubation, Generated, Plasmid Preparation, Marker

    38) Product Images from "Plasmid replication-associated single-strand-specific methyltransferases"

    Article Title: Plasmid replication-associated single-strand-specific methyltransferases

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa1163

    The ColE1 ori orientation determines which strand is modified by M.EcoGIX. Black ticks: observed sites of m6A modification. Black arrows: coding sequences for beta-lactamase ( bla ) and the MTase ( ecoGIXM ). Thick orange arrow: the origin of replication ( ori ), determined by the rnaII nucleotide sequence. RNAII primes leading strand DNA synthesis in the direction of the thin orange arrow.
    Figure Legend Snippet: The ColE1 ori orientation determines which strand is modified by M.EcoGIX. Black ticks: observed sites of m6A modification. Black arrows: coding sequences for beta-lactamase ( bla ) and the MTase ( ecoGIXM ). Thick orange arrow: the origin of replication ( ori ), determined by the rnaII nucleotide sequence. RNAII primes leading strand DNA synthesis in the direction of the thin orange arrow.

    Techniques Used: Modification, Sequencing, DNA Synthesis

    Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.
    Figure Legend Snippet: Polymerase and MTase activities copurify when domains are fused. Panel ( A ): Size and purity of fusion proteins. For each MTase, both of the immunoreactive components of the MTase-PolI fusion proteins run at the same position, and comigrate with the Coomassie-stained purified proteins. Western blot (lanes 1, 5, 9 and 10) detected 1 μg of MTase-PolI fusion proteins; Coomassie (lanes 2, 3, 6, 7) visualized 1 μg or 20 μg of the same fractions. Western blots were probed separately with anti-Pol1 rabbit polyclonal or anti-6xHis (detecting the MTase) monoclonal antibodies and developed with horseradish peroxidase-labeled antirabbit or antimouse following kit instructions as detailed in Material and Methods. Dots on lane 1 correspond to the position of protein markers after Western blotting. The bands at the side of lane 10 are spillover from the adjacent lane, which were control 6xHis tagged proteins from a PurExpress extract. Panel ( B ): Activity copurification through two columns. Pooled HiTrapHepHP (#22–26) and HiTrapQHP (#15–19) protein fractions were tested for MTase activity on single-stranded M13mp18 DNA in the presence of [H 3 ]SAM and for DNA-polymerase activity on sonicated sperm-whale DNA in the presence of [H 3 ]TTP.

    Techniques Used: Staining, Purification, Western Blot, Labeling, Activity Assay, Copurification, Sonication

    Nicks stimulate both MTase and polymerase activities of the M.EcoGIX::PolI fusion. Panel ( A ): MTase activity was measured with [H 3 ]SAM alone (left set of bars) or with cold dNTP to enable nick translation (middle set). DNA polymerase activity was measured with [H 3 ]TTP (right set). The gDNA substrate (unmethylated ER2796 as isolated) has preexisting nicks that provide priming sites for polymerization (blue bars). Additional priming sites were added using nicking enzymes, either with a frequent site (Nt.CviPII |CCD, red bars) or with a rarer site (Nt.BspQI, GCTCTTCN|, green bars). Panel ( B ): Immunologic detection of m6A modification using anti-m6A monoclonal rabbit antibodies. A M.EcoGIX::PolI nick-translation reaction with gDNA as in Panel A was spotted and developed with antibody. (1) ER2796 gDNA, no enzyme control; (2) ER2796 gDNA nicked with Nt.BspQI (671 sites per genome); (3) nicked with Nt.CviPII (438,784 sites per genome); (4) ER2796 E.coli gDNA no added nicks; (5) m6A positive control: ER2683 E. coli gDNA (Dam + ) with no enzyme. Lanes 2–4 are in the reverse order as the bars shown in Panel A.
    Figure Legend Snippet: Nicks stimulate both MTase and polymerase activities of the M.EcoGIX::PolI fusion. Panel ( A ): MTase activity was measured with [H 3 ]SAM alone (left set of bars) or with cold dNTP to enable nick translation (middle set). DNA polymerase activity was measured with [H 3 ]TTP (right set). The gDNA substrate (unmethylated ER2796 as isolated) has preexisting nicks that provide priming sites for polymerization (blue bars). Additional priming sites were added using nicking enzymes, either with a frequent site (Nt.CviPII |CCD, red bars) or with a rarer site (Nt.BspQI, GCTCTTCN|, green bars). Panel ( B ): Immunologic detection of m6A modification using anti-m6A monoclonal rabbit antibodies. A M.EcoGIX::PolI nick-translation reaction with gDNA as in Panel A was spotted and developed with antibody. (1) ER2796 gDNA, no enzyme control; (2) ER2796 gDNA nicked with Nt.BspQI (671 sites per genome); (3) nicked with Nt.CviPII (438,784 sites per genome); (4) ER2796 E.coli gDNA no added nicks; (5) m6A positive control: ER2683 E. coli gDNA (Dam + ) with no enzyme. Lanes 2–4 are in the reverse order as the bars shown in Panel A.

    Techniques Used: Activity Assay, Nick Translation, Isolation, Modification, Positive Control

    Immunologic and enzymatic detection of PolI in active MTase fractions from in vivo expression. Panel ( A ): Anti-His-tag detects 6xHis::M.BceJIII from pAF9 in both active and further-purified inactive fractions (top row), but anti-PolI detects the polymerase only in the MTase active fraction (bottom row). Panel ( B ): MTase action and nucleotide incorporation by these fractions. Blue bars: single-stranded M13mp18 DNA modified with [H 3 ]SAM; red bars: sperm whale DNA labeled by [H 3 ] dTTP (DNA polymerase activity) measured without enzyme (1) or with active (2) or inactive (3) MTase column fractions. Panel ( C ): Reciprocal immunoprecipitation assays recover PolI and His-tagged MTases together. Top panel: Western detection of PolI by anti-PolI of MTase tagged anti-His IP. Bottom panel: Western detection of His-tagged MTase anti-His of anti-PolI IP. In vivo expression employed pAF9 (M.BceJIII), pAF10 (M.EcoGIX WT) and pAF11 (M.EcoGIX mut).
    Figure Legend Snippet: Immunologic and enzymatic detection of PolI in active MTase fractions from in vivo expression. Panel ( A ): Anti-His-tag detects 6xHis::M.BceJIII from pAF9 in both active and further-purified inactive fractions (top row), but anti-PolI detects the polymerase only in the MTase active fraction (bottom row). Panel ( B ): MTase action and nucleotide incorporation by these fractions. Blue bars: single-stranded M13mp18 DNA modified with [H 3 ]SAM; red bars: sperm whale DNA labeled by [H 3 ] dTTP (DNA polymerase activity) measured without enzyme (1) or with active (2) or inactive (3) MTase column fractions. Panel ( C ): Reciprocal immunoprecipitation assays recover PolI and His-tagged MTases together. Top panel: Western detection of PolI by anti-PolI of MTase tagged anti-His IP. Bottom panel: Western detection of His-tagged MTase anti-His of anti-PolI IP. In vivo expression employed pAF9 (M.BceJIII), pAF10 (M.EcoGIX WT) and pAF11 (M.EcoGIX mut).

    Techniques Used: In Vivo, Expressing, Purification, Modification, Labeling, Activity Assay, Immunoprecipitation, Western Blot

    MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.
    Figure Legend Snippet: MTase activity requires single strands. Panels ( A ) and ( C ): M13 substrates stained with ethidium bromide. Panels ( B ) and ( D ): fluorograms of modification reactions using [H 3 ]SAM. M13 SS: virion DNA substrate. M13 RF cut: DS replication intermediate RFI was digested following the labelling reaction for visual simplification; NdeI (Panels A and B) or NdeI+BamHI (Panels C and D). The substrates were treated with MTase proteins obtained with PURExpress in vitro transcription-translation (Panels A and B) or were partially-purified (Ni-NTA purification) proteins synthesized in vivo (Panels C and D). Lanes 1) empty pSAPv6 vector, 2) M.BceJIII WT (pAF9), 3) M.EcoGIX WT (pAF10) and 4) M.EcoGIX APPA variant (pAF11). H 3 radiolabeled markers (M) are HindIII digested lambda DNA modified at A by M.EcoGII.

    Techniques Used: Activity Assay, Staining, Modification, In Vitro, Purification, Synthesized, In Vivo, Plasmid Preparation, Variant Assay, Lambda DNA Preparation

    Conjugal DNA transfer positions the MTase gene for expression and coordinated action with PolI in the native orientation but not with inverted oriT . Blue lines are DNA; light blue carries the coding sequence for the MTase gene (green arrow); dark blue carries the F rpo promoter. ( A ) In the native orientation the relaxase (R) nicks at oriT (T), becoming covalently attached to the 5′ side of the nick. Leading strand synthesis from the 3′ side of the nick, and interaction of relaxase with the conjugal apparatus (green structure at the cell periphery), conveys the displaced single strand into the recipient. Transcription (dashed black arrow) from F rpo on the conjugal DNA strand allows translation of the MTase gene. This MTase (green hexagon) can then interact with endogenous PolI (lavender hexagon) to modify the single conjugal strand as lagging strand synthesis proceeds from F rpo transcripts (not shown). ( B ) When oriT is inverted, the MTase gene is transferred late and on the wrong strand for expression, leaving the double-stranded product (not shown) sensitive to restriction.
    Figure Legend Snippet: Conjugal DNA transfer positions the MTase gene for expression and coordinated action with PolI in the native orientation but not with inverted oriT . Blue lines are DNA; light blue carries the coding sequence for the MTase gene (green arrow); dark blue carries the F rpo promoter. ( A ) In the native orientation the relaxase (R) nicks at oriT (T), becoming covalently attached to the 5′ side of the nick. Leading strand synthesis from the 3′ side of the nick, and interaction of relaxase with the conjugal apparatus (green structure at the cell periphery), conveys the displaced single strand into the recipient. Transcription (dashed black arrow) from F rpo on the conjugal DNA strand allows translation of the MTase gene. This MTase (green hexagon) can then interact with endogenous PolI (lavender hexagon) to modify the single conjugal strand as lagging strand synthesis proceeds from F rpo transcripts (not shown). ( B ) When oriT is inverted, the MTase gene is transferred late and on the wrong strand for expression, leaving the double-stranded product (not shown) sensitive to restriction.

    Techniques Used: Expressing, Sequencing

    39) Product Images from "Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples"

    Article Title: Optimization and validation of sample preparation for metagenomic sequencing of viruses in clinical samples

    Journal: Microbiome

    doi: 10.1186/s40168-017-0317-z

    Optimized workflow for metagenomic virus sequencing. A workflow for metagenomic virus sequencing for diagnostic use was developed. Sample pre-processing included low-speed centrifugation, 0.45-μm filtration, storage at −80 °C, and DNase and RNase digestion. Random reverse transcription with an 8N primer including an anchor sequence, second strand synthesis, and anchor PCR amplification was performed separately for an RNA and DNA workflow. The two workflows were pooled in equal concentration for library preparation with NexteraXT
    Figure Legend Snippet: Optimized workflow for metagenomic virus sequencing. A workflow for metagenomic virus sequencing for diagnostic use was developed. Sample pre-processing included low-speed centrifugation, 0.45-μm filtration, storage at −80 °C, and DNase and RNase digestion. Random reverse transcription with an 8N primer including an anchor sequence, second strand synthesis, and anchor PCR amplification was performed separately for an RNA and DNA workflow. The two workflows were pooled in equal concentration for library preparation with NexteraXT

    Techniques Used: Sequencing, Diagnostic Assay, Centrifugation, Filtration, Polymerase Chain Reaction, Amplification, Concentration Assay

    Separate workflows for RNA and DNA yielded higher sequencing reads for DNA viruses. Plasma samples were spiked with four different viruses (adenovirus, HHV-4, influenzavirus, poliovirus) and processed and sequenced with the combined and the new separate workflow. In the separate workflow, random amplification products were pooled before NexteraXT library preparation in equal concentrations. The experiment was performed in triplicates. a Distribution of sequencing reads into the different taxonomic categories viral, human, bacterial, and unknown origin. b Number of reads ( upper panels ) and fraction of all quality passing reads ( lower panels ) obtained for each individual virus
    Figure Legend Snippet: Separate workflows for RNA and DNA yielded higher sequencing reads for DNA viruses. Plasma samples were spiked with four different viruses (adenovirus, HHV-4, influenzavirus, poliovirus) and processed and sequenced with the combined and the new separate workflow. In the separate workflow, random amplification products were pooled before NexteraXT library preparation in equal concentrations. The experiment was performed in triplicates. a Distribution of sequencing reads into the different taxonomic categories viral, human, bacterial, and unknown origin. b Number of reads ( upper panels ) and fraction of all quality passing reads ( lower panels ) obtained for each individual virus

    Techniques Used: Sequencing, Amplification

    40) Product Images from "Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells"

    Article Title: Matrix association region/scaffold attachment region: the crucial player in defining the positions of chromosome breaks mediated by bile acid-induced apoptosis in nasopharyngeal epithelial cells

    Journal: BMC Medical Genomics

    doi: 10.1186/s12920-018-0465-4

    Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p
    Figure Legend Snippet: Identification of chromosome breaks in BA-treated NP69 cells. IPCR was employed to identify the AF9 gene cleavages in NP69 cells after exposed to BA. a Representative gel picture showing the AF9 gene cleavages identified by IPCR within: ( a i ) SAR region ( a ii ) Non-SAR region. NP69 cells were left untreated ( a i , Lanes 1–5; a ii , Lanes 1–6) or treated for 1 h with 0.5 mM of BA at pH 7.4 ( a i , Lanes 6–10; a ii , Lanes 7–12) and pH 5.8 ( a i , Lanes 11–15; a ii , Lanes 13–18). Genomic DNA extraction and nested IPCR were performed as described in “Methods” section. The side bracket represents the IPCR bands derived from the AF9 cleaved fragments. M: 100 bp DNA marker. N: negative control for IPCR. b The average number of the AF9 gene cleavages identified in BA-treated NP69 cells. Data are expressed as means and SDs of two independent experiments. Each experiment consisted of two to four sets of IPCR carried out in three to six replicates per set for each cell sample. Values are expressed as fold change normalised to the value of the untreated control. The differences between untreated control and treated groups were compared by using Student’s t test, * p

    Techniques Used: DNA Extraction, Derivative Assay, Marker, Negative Control

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs t4 dna polymerase
    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized <t>T4</t> DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.
    T4 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/t4 dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    t4 dna polymerase - by Bioz Stars, 2022-07
    99/100 stars
      Buy from Supplier

    86
    New England Biolabs taq dna polymerases
    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp <t>DNA</t> ladder; Lane 1–2: complete PEAR reactions containing <t>Taq</t> DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.
    Taq Dna Polymerases, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq dna polymerases/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    taq dna polymerases - by Bioz Stars, 2022-07
    86/100 stars
      Buy from Supplier

    95
    New England Biolabs vent dna polymerase
    <t>RT-PCR</t> amplification of spliced B4-B5 exons. The first lane contains a <t>DNA</t> ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.
    Vent Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/vent dna polymerase/product/New England Biolabs
    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    vent dna polymerase - by Bioz Stars, 2022-07
    95/100 stars
      Buy from Supplier

    98
    New England Biolabs ts k1 dna polymerase
    Phylogenetic tree showing the evolutionary relationship between Ts <t>K1</t> DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X
    Ts K1 Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 98/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ts k1 dna polymerase/product/New England Biolabs
    Average 98 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    ts k1 dna polymerase - by Bioz Stars, 2022-07
    98/100 stars
      Buy from Supplier

    Image Search Results


    CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: CE analysis of processing synthetic DNA by soluble enzyme mix PKT and immobilized enzymes. 5′ FAM-labeled blunt-end substrates, 51-AT possessing multiple 3′ terminal A-T base pairs, and 51-GC possessing multiple 3′ terminal G-C base pairs, were incubated with PKT for end repair at 20 °C for 30 min followed by 65 °C for 30 min (PKT mix). The substrates were also treated with immobilized T4 DNA pol and PNK at 20 °C for 30 min, followed by separation of the enzymes on beads and the reaction medium (supernatant). The reaction medium was subsequently treated with immobilized Taq DNA pol for 3′ A-tailing at 37 °C for 30 min (IM PKT mix). The CE data show that incubation with PKT resulted in extensive degradation of 51-AT and little degradation of 51-GC. Treatment of 51-AT or 51-GC with the immobilized enzymes resulted in mostly 3′ A-tailing product, without detectable degradation of the 5′ FAM-labeled oligos. NC, negative control reaction performed in the absence of enzyme.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Labeling, Incubation, Negative Control

    Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: Enzyme immobilization and comparison of Illumina library preparation protocols. ( a ) A schematic of covalent conjugation of SNAP-tagged enzyme fusion proteins to magnetic beads functionalized with O 6 . ( b ) Workflow for library construction using immobilized enzymes for Illumina sequencing. A typical streamlined protocol for Illumina library construction is modified by employing immobilized enzymes to catalyze end repair and 3′ A-tailing. This method utilizes SNAP-tagged T4 DNA pol and PNK covalently conjugated to BG-functionalized magnetic beads to carry out end repair of fragmented DNA at 20°C (or 37 °C) for 30 min. The enzymes are removed by magnetic separation from the DNA pool, which is subjected to 3′ A-tailing at 37 °C for 30 min using immobilized Taq DNA pol. ( c ) Streamlined protocol for Illumina amplification-free library preparation using soluble enzymes. Typically, end repair and 3′ A-tailing of fragmented DNA are catalyzed by an enzyme mixture at 20 °C for 30 min, followed by heat treatment at 65 °C for 30 min. ( d ) The workflow of Illumina TruSeq DNA PCR-free LT Library Prep Kit with a purification step. End repair is performed for 30 min at 30 °C, followed by a bead-based step for clean up and size selection. 3′ A-tailing is carried out for 30 min at 37 °C with a subsequent treatment for 5 min at 70 °C. Each library was ligated to preannealed full-length paired-end Illumina adaptors, size-selected and analyzed, and sequenced on an Illumina sequencing platform.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Conjugation Assay, Magnetic Beads, Sequencing, Modification, Amplification, Polymerase Chain Reaction, Purification, Selection

    A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Journal: Scientific Reports

    Article Title: Solid-phase enzyme catalysis of DNA end repair and 3′ A-tailing reduces GC-bias in next-generation sequencing of human genomic DNA

    doi: 10.1038/s41598-018-34079-2

    Figure Lengend Snippet: A model for GC-related sequence coverage bias in amplification-free NGS data. ( a ) A schematic of DNA end “breathing” (or “fraying”) present in the AT-rich fraction of a DNA library. DNA thermal breathing refers to spontaneous local conformational fluctuations, leading to unpaired bases at the ends of DNA duplex. The extent of breathing is highly dependent upon temperature and DNA sequence so that AT-rich segments (AT) melt before GC-rich segments (GC). The difference of the end breathing profile relevant to GC-content leads to less efficient end-polishing of AT-rich fragments during library construction using DNA modifying enzymes, resulting in the under-representation of the AT-rich regions. ( b ) Degradation of AT-rich DNA by 3′-5′ exonuclease activity of T4 DNA pol (blue). Preferential degradation of AT-rich DNA fragments that undergo terminal base pair breathing may occur at the end repair step or during high temperature incubation. ( c , yielding unintended cleavage and primer extension products. Arrow (red) indicates the position of cleavage whereas arrow in black indicates the orientation of primer extension due to intermolecular annealing of two single-stranded 3′ terminal sequences. Primer extension may also occur from intramolecular annealing of a single-stranded 3′ terminal sequence.

    Article Snippet: Enzyme mix PKT was comprised of approximately 1,200 units/ml T4 DNA polymerase, 2,000 units/ml T4 PNK and 2,000 units/ml Taq DNA polymerase (NEB) while PK contained T4 DNA polymerase and T4 PNK only.

    Techniques: Sequencing, Amplification, Next-Generation Sequencing, Activity Assay, Incubation

    PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: PEAR reactions with complete and incomplete components. Target (X) and probe (X′R′X′R′X′) concentrations were at 1nM and 100 nM respectively. For PspGI, H and L stand for high (0.4 U/µL) and low (0.1 U/µL) concentrations respectively. Lane M: Invitrogen Trackit™ 10 bp DNA ladder; Lane 1–2: complete PEAR reactions containing Taq DNA polymerase, PspGI, the target and the probe. The lower band (shown by an arrow) represents the 20-bp duplex monomers, and the upper bands represent tandem repeats; Lane 3–9: incomplete PEAR reactions lacking one or both enzymes or the target. No product band was observed. The bands represent probe self-dimerization formed by intermolecular interactions.

    Article Snippet: Alternatively, in order to remove BSA, Taq DNA polymerases and excess dNTPs, PEAR products were phenol extracted, ethanol precipitated, washed three times with 75% ethanol, dried and resuspended in ddH2 O, and double digested by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4, and 1.0 u/µl each of Hpy99I and PspGI.

    Techniques:

    Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Journal: PLoS ONE

    Article Title: Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    doi: 10.1371/journal.pone.0008430

    Figure Lengend Snippet: Schematic description of PEAR. Sense and antisense strands are represented by solid and dashed lines respectively, the 3′ ends are indicated by arrows and the restriction sites for PspGI are indicated by solid diamonds. When a target oligonucleotide ( X ) binds to a probe in the upstream, it is elongated by the Taq DNA polymerase, and a full-duplex oligonucleotide containing tandem repeats is produced. If the repeats are cleaved by PspGI, short duplex oligos ( X/X′ ) are released; and when they are not cleaved, the number of tandem repeats increases by slipping and elongation.

    Article Snippet: Alternatively, in order to remove BSA, Taq DNA polymerases and excess dNTPs, PEAR products were phenol extracted, ethanol precipitated, washed three times with 75% ethanol, dried and resuspended in ddH2 O, and double digested by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4, and 1.0 u/µl each of Hpy99I and PspGI.

    Techniques: Produced

    RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    Article Title: A prokaryote and human tRNA synthetase provide an essential RNA splicing function in yeast mitochondria

    doi:

    Figure Lengend Snippet: RT-PCR amplification of spliced B4-B5 exons. The first lane contains a DNA ladder of markers (Stratagene). The second and third lanes, respectively, represent RNA isolated from the yeast null strain (QBY320) that contained plasmids expressing wild-type LeuRS from yeast and M. tuberculosis . The unspliced and spliced B4-B5 exon products are indicated by respective bands at about 1.5 kbp and 250 bp. The fourth and fifth lanes, respectively, show that the intron remains unspliced when genes containing either the W286C mutant or G288SΔC5 deletion of M. tuberculosis LeuRS are used in attempts to complement the yeast null strain.

    Article Snippet: The pC3 -Mtb plasmid was engineered by PCR amplification of the M. tuberculosis LeuRS gene from plasmid pLEU10 using Vent DNA polymerase (New England Biolabs) and 5′ and 3′ primers that introduced, respectively, Xba I and Bgl II sites.

    Techniques: Reverse Transcription Polymerase Chain Reaction, Amplification, Isolation, Expressing, Mutagenesis

    Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Phylogenetic tree showing the evolutionary relationship between Ts K1 DNA polymerase and other Thermus DNA polymerases based on maximum likelihood analysis. The tree with the highest likelihood (7699.30) is shown, and the bootstrap value (1000 replicates) for each clade is shown next to each branch. The final dataset contained 824 positions. All positions containing gaps or missing data were eliminated (complete deletion option). Bar indicates 0.05 substitutions per amino acid position. Evolutionary analyses were conducted using MEGA X

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques:

    Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Properties of Ts K1 DNA polymerase. (a) Effect of temperature on Ts K1 DNA polymerase activity; (b) effect of pH on Ts K1 DNA polymerase activity in MOPS‐NaOH (■), Tris‐HCl (▲), and glycine‐NaOH (●) buffers; (c) effect of KCl concentration on Ts K1 DNA polymerase activity; and (d) effect of the divalent cations Mg 2+ (■) and Mn 2+ (●) on Ts K1 DNA polymerase activity. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Activity Assay, Concentration Assay, Standard Deviation

    Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of Ts K1 DNA polymerase purification: (1) noninduced culture, (2) induced culture, (3) sonicated extract from host cells, (4) supernatant after heat treatment, 5) purified protein, M1—Full Range Rainbow molecular‐mass marker (Amersham)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polyacrylamide Gel Electrophoresis, Purification, Sonication, Marker

    Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Polymerase chain reaction products amplified using Ts K1 DNA polymerase. Lane 1, 265 bp; lane 2, 500 bp; lane 3, 1500 bp; lane 4, 1920 bp; lane 5, 2.5 kb; M, GeneRuler 1 kb Plus DNA ladder (New England BioLabs)

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Polymerase Chain Reaction, Amplification

    Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Journal: MicrobiologyOpen

    Article Title: Characteristics of DNA polymerase I from an extreme thermophile, Thermus scotoductus strain K1

    doi: 10.1002/mbo3.1149

    Figure Lengend Snippet: Ts K1 DNA polymerase thermostability. ×Represents 75°C, ▲ represents 80°C, ● represents 88°C, ♦ represents 95°C. Data are represented on a logarithmic scale. Each point represents the average of 3 measured values, and error bars represent the standard deviation between these 3 values

    Article Snippet: 3.5 PCR by Ts K1 DNA polymerase The electrophoretogram of the PCR products amplified by using the Ts K1 DNA polymerase is shown in Figure .

    Techniques: Standard Deviation