exonuclease iii  (Thermo Fisher)


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  • 97
    Name:
    Exonuclease III
    Description:
    Thermo Scientific Exonuclease III (ExoIII) exhibits four catalytic activities. The 3'→5' exodeoxyribonuclease activity of ExoIII is specific for double-stranded DNA. ExoIII degrades dsDNA from blunt ends, 5'-overhangs or nicks, releases 5'-mononucleotides from the 3'-ends of DNA strands and produces stretches of single-stranded DNA. It is not active on 3'-overhang ends of DNA that are at least four-bases long and do not carry a 3'-terminal C-residue on single-stranded DNA, or on phosphorothioate-linked nucleotides.ExoIII 3'-phosphatase activity removes the 3'-terminal phosphate, generating a 3'-OH group. ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA-DNA hybrids. ExoIII apurinic/apyrimidinic-endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free deoxyribose 5'-phosphate residues.Highlights• Active in restriction enzyme buffersApplications• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease• Generation of a single-stranded template for dideoxy-sequencing of DNA• Site-directed mutagenesis• Cloning of PCR products• Preparation of strand-specific probesNoteThe rate of DNA digestion by ExoIII depends upon temperature, salt concentration, and the molar ratio of DNA to enzyme in the reaction mixture . Optimal reaction conditions should be determined experimentally.
    Catalog Number:
    EN0191
    Price:
    None
    Applications:
    Cloning|PCR Cloning|Mutagenesis
    Size:
    4 000 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher exonuclease iii
    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease <t>III</t> or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, <t>Klenow</t> fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Thermo Scientific Exonuclease III (ExoIII) exhibits four catalytic activities. The 3'→5' exodeoxyribonuclease activity of ExoIII is specific for double-stranded DNA. ExoIII degrades dsDNA from blunt ends, 5'-overhangs or nicks, releases 5'-mononucleotides from the 3'-ends of DNA strands and produces stretches of single-stranded DNA. It is not active on 3'-overhang ends of DNA that are at least four-bases long and do not carry a 3'-terminal C-residue on single-stranded DNA, or on phosphorothioate-linked nucleotides.ExoIII 3'-phosphatase activity removes the 3'-terminal phosphate, generating a 3'-OH group. ExoIII Rnase H activity exonucleolytically degrades the RNA strand in RNA-DNA hybrids. ExoIII apurinic/apyrimidinic-endonuclease activity cleaves phosphodiester bonds at apurinic or apyrimidinic sites to produce 5'-termini that are base-free deoxyribose 5'-phosphate residues.Highlights• Active in restriction enzyme buffersApplications• Creation of unidirectional deletions in DNA fragments in conjunction with S1 Nuclease• Generation of a single-stranded template for dideoxy-sequencing of DNA• Site-directed mutagenesis• Cloning of PCR products• Preparation of strand-specific probesNoteThe rate of DNA digestion by ExoIII depends upon temperature, salt concentration, and the molar ratio of DNA to enzyme in the reaction mixture . Optimal reaction conditions should be determined experimentally.
    https://www.bioz.com/result/exonuclease iii/product/Thermo Fisher
    Average 97 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2019-10
    97/100 stars

    Images

    1) Product Images from "Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ"

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052584

    Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.
    Figure Legend Snippet: Copper(I) treatment produces short gaps with phosphate groups at the 3′ end. A ) TdT was used to incorporate Alexa-dUTP at the 3′ end of the gaps. A strong signal is observed only after the pre-incubation of cells with exonuclease III or SAP. The model shows the situation after the action of SAP in the case of double-stranded DNA with several gaps. Although the phosphate groups are shown also at the 5′ end of the gaps, it is not clear whether they are present there. Therefore, the action of SAP is shown for 3′ phosphate groups exclusively. Bar: 20 µm. B ) DNA polymerase I, Klenow fragment and Klenow fragment Exo- were used to incorporate Alexa-dUTP at the gap sites produced by monovalent copper. Only DNA polymerase I produced a strong signal. When incubation with exonuclease III preceded the polymerase step, a strong signal was observed also in the case of both Klenow fragments. The model shows the action of DNA polymerase I at the sites of created gaps. Both 3′-5′ proofreading activity enabling hydroxyl group formation and 5′-3′ exonuclease activity (for the sake of simplicity, the excised nucleotides are not shown in the model) enabling nick translation are necessary. As no ligase activity was present, nicks at the ends of the labeled chains persisted (arrows in the model picture), although it is not apparent. Bar: 20 µm.

    Techniques Used: Incubation, Produced, Activity Assay, Nick Translation, Labeling

    2) Product Images from "Potential implication of new torque teno mini viruses in parapneumonic empyema in children"

    Article Title: Potential implication of new torque teno mini viruses in parapneumonic empyema in children

    Journal: The European Respiratory Journal

    doi: 10.1183/09031936.00107212

    Replication of torque teno mini virus (TTMV) isolates TTMV-LY genomes after transfection produce more replicative TTMV in human embryonic kidney (HEK) 293T cells than in A549 cells as quantified by real-time qPCR. Histograms represent the mean± sd rate of replication of TTMV-LY in the cells transfected with the linearised full-length genomes. Replication of TTMV-LY was a) measured at day 3 post-transfection or b) monitored over a week following transfection. The curves in b) indicate the quantification of TTMV-LY1 in the culture medium of cells at days 0–7 post-transfection after DNaseI (Thermo Scientific, Illkirch, France) treatment and nucleic acid extraction. Results are expressed as the ratio of quantified TTMV-LY1 at day 1–7 post-transfection to quantified TTMV-LY1 at day 0. Data are shown only for TTMV-LY1, because the replication profile among the three viruses is similar.
    Figure Legend Snippet: Replication of torque teno mini virus (TTMV) isolates TTMV-LY genomes after transfection produce more replicative TTMV in human embryonic kidney (HEK) 293T cells than in A549 cells as quantified by real-time qPCR. Histograms represent the mean± sd rate of replication of TTMV-LY in the cells transfected with the linearised full-length genomes. Replication of TTMV-LY was a) measured at day 3 post-transfection or b) monitored over a week following transfection. The curves in b) indicate the quantification of TTMV-LY1 in the culture medium of cells at days 0–7 post-transfection after DNaseI (Thermo Scientific, Illkirch, France) treatment and nucleic acid extraction. Results are expressed as the ratio of quantified TTMV-LY1 at day 1–7 post-transfection to quantified TTMV-LY1 at day 0. Data are shown only for TTMV-LY1, because the replication profile among the three viruses is similar.

    Techniques Used: Transfection, Real-time Polymerase Chain Reaction

    3) Product Images from "Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III"

    Article Title: Cell cycle profiling by image and flow cytometry: The optimised protocol for the detection of replicational activity using 5-Bromo-2′-deoxyuridine, low concentration of hydrochloric acid and exonuclease III

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0175880

    Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.
    Figure Legend Snippet: Optimising the protocol for BrdU detection. (A) HeLa cells were incubated with BrdU for 30 min, fixed with formaldehyde and BrdU was detected in DNA using 40 mM HCl. BrdU was detected either by the B44 or Bu20a anti-BrdU antibody with exonuclease III. The effect of formaldehyde post-fixation on the signal is shown as well. The data are presented as the mean ± SD. ( B-E) A comparison of various HCl concentrations on BrdU signal in HeLa cells labelled for 30 min with BrdU and fixed either with formaldehyde (B, D) or ethanol (C, E). The incorporated BrdU was detected using either B44 (B, C) or Bu20a (D, E) antibody clone with exonuclease III. The data are presented as the mean ± SD. ( F, G) A comparison of five monoclonal anti-BrdU antibody clones and one polyclonal antibody is shown. The HeLa cells were labelled with BrdU for 30 min and fixed either with formaldehyde (F) or ethanol (G). The impact of the post-fixation step is shown as well. The data are presented as the mean ± SD. ( H) The effect of the length of the washing step on the BrdU-derived signal. The HeLa cells were labelled with BrdU for 30 min and fixed with formaldehyde. After incubation with the primary antibody, the cells were washed for 5 s (0 min) or 5 or 25 min in 1× PBS and then post-fixed with formaldehyde. The data are normalised to the % of the average signal in samples washed for 5 s in 1× PBS and then immediately post-fixed with formaldehyde. The data are presented as the mean ± SD.

    Techniques Used: Incubation, Clone Assay, Derivative Assay

    The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.
    Figure Legend Snippet: The effect of optimized procedure on the localisation of cellular proteins. HeLa cells were incubated with BrdU for 30 minutes and fixed with formaldehyde. BrdU was revealed using 20 mM HCl. The proteins SC35, mitochondrial protein MTCO2, histone H1.2 and coilin were concurrently detected with the incorporated BrdU. BrdU was detected using either chicken polyclonal antibody or B44 monoclonal antibody depending on the host producing the antibody for the particular cellular protein. The control cells were not labelled with BrdU and were not treated with HCl and exonuclease III. Proteins are in green, BrdU is in red and DAPI is in blue. Scale bar = 20 μm.

    Techniques Used: Incubation

    4) Product Images from "Quantitative Microplate Assay for Real-Time Nuclease Kinetics"

    Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0154099

    Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
    Figure Legend Snippet: Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Techniques Used: Concentration Assay, Phosphate Release Assay, Incubation, Fluorescence, De-Phosphorylation Assay, Binding Assay, Derivative Assay

    Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.
    Figure Legend Snippet: Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.

    Techniques Used: Phosphate Release Assay, Fluorescence, Binding Assay, Concentration Assay

    5) Product Images from "Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma"

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    Journal: Journal of Extracellular Vesicles

    doi: 10.1080/20013078.2018.1505403

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.
    Figure Legend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Techniques Used: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis

    6) Product Images from "Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions"

    Article Title: Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions

    Journal: Nature Communications

    doi: 10.1038/s41467-019-08673-5

    mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation
    Figure Legend Snippet: mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation

    Techniques Used: Next-Generation Sequencing, Standard Deviation, Generated

    Related Articles

    other:

    Article Title: Atomic Scissors: A New Method of Tracking the 5-Bromo-2?-Deoxyuridine-Labeled DNA In Situ
    Article Snippet: These enzymes and condition were used: Terminal deoxynucleotidyl transferase (TdT; 2 U/µl, 10 minutes, 37°C, Fermentas), buffer for TdT, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa Fluor® 555-aha-2′-deoxyuridine-5′-triphosphate (Alexa-dUTP); DNA polymerase I (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for DNA polymerase I, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Klenow fragment Exo- (0.2 U/µl, 10 minutes, RT, Fermentas), buffer for the Klenow fragment Exo-, 0.05 mM dATP, dGTP, dCTP and 0.05 mM Alexa-dUTP; Exonuclease III (1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease III; Exonuclease λ (0.1 U/µl, 30 minutes, RT, Fermentas), buffer for exonuclease λ; Shrimp alkaline phosphomonoesterase (phosphatase; SAP; 1 U/µl, 20 minutes, 37°C, Fermentas), buffer for SAP.

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    Thermo Fisher exonuclease iii
    Exonuclease <t>III</t> steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM <t>MDCC-PBP,</t> 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
    Exonuclease Iii, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease iii/product/Thermo Fisher
    Average 94 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2019-10
    94/100 stars
      Buy from Supplier

    82
    Thermo Fisher snp ehmt1 c 134213 10
    Exonuclease <t>III</t> steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM <t>MDCC-PBP,</t> 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.
    Snp Ehmt1 C 134213 10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 82/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/snp ehmt1 c 134213 10/product/Thermo Fisher
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    Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Journal: PLoS ONE

    Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    doi: 10.1371/journal.pone.0154099

    Figure Lengend Snippet: Exonuclease III steady state kinetics with increasing concentration of substrate by phosphate release assay. 1.5 nM ExoIII was incubated with 1μM MDCC-PBP, 0.0004u/μl FastAP and varying concentrations (5, 10, 20, 40, 60, 100 and 200 nM) of dsDNA substrate in 66mM Tris-HCl (pH 8.0) and 0.66mM MgCl 2 at 37°C. (A) Fluorescence increase was measured over time from the ExoIII reaction coupled to FastAP dephosphorylation of products and subsequent P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S5 Fig ). Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of substrate in the different data sets. (B) Michaelis-Menten saturation curve by plotting initial velocity (v 0 ) obtained from (A) against 3’-end concentration. Constants derived from plot were V max = 0.5947 ± 0.0380 nM s -1 and K M = 140.9 ± 20.3 nM.

    Article Snippet: MDCC-PBP (commercial name Phosphate Sensor), T4 polynucleotide kinase, FastAP and Exonuclease III (ThermoFisher).

    Techniques: Concentration Assay, Phosphate Release Assay, Incubation, Fluorescence, De-Phosphorylation Assay, Binding Assay, Derivative Assay

    Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.

    Journal: PLoS ONE

    Article Title: Quantitative Microplate Assay for Real-Time Nuclease Kinetics

    doi: 10.1371/journal.pone.0154099

    Figure Lengend Snippet: Phosphate release assay for ‘10–23’ DNAzyme steady state kinetics. Reactions were set up containing 2 μM RNA substrate, 1 μM MDCC-PBP, 0.3u/μl T4PNK and varying concentrations (50, 100, 150 and 200 nM) of DzSJ in 40 mM Tris-HCl (pH 7.5), 20 mM MgCl2 and 50 mM NaCl at room temperature. As a control 200 nM inactive DzSJ was used instead of DzSJ. Cleavage of RNA substrate exposes a 2’, 3’-cyclic phosphate ( > P). The cyclic phosphate is released by T4PNK and quantified by fluorescence increase caused by P i binding to MDCC-PBP. Background measured in parallel of a reaction without enzyme was subtracted from each data set. Fluorescence increase was converted to [P i ] by interpolation from standard curve ( S6 Fig ). (A) Inorganic phosphate concentration plotted over time. Data points are shown as bars of standard error of the mean of three independent experiments. Arrow (↑) refers to the increasing concentration of DzSJ in the different data sets. (B) Initial velocities (v 0 ) obtained from (A) plotted against DzSJ concentration showing a linear relation. R 2 = 0.98.

    Article Snippet: MDCC-PBP (commercial name Phosphate Sensor), T4 polynucleotide kinase, FastAP and Exonuclease III (ThermoFisher).

    Techniques: Phosphate Release Assay, Fluorescence, Binding Assay, Concentration Assay

    mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation

    Journal: Nature Communications

    Article Title: Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions

    doi: 10.1038/s41467-019-08673-5

    Figure Lengend Snippet: mtDNA deletions identified in patients with mutations in POLγ. a Strand-displacement DNA replication is continuous on both strands and initiated from two separate origins. b mtDNA deletions (blue) and duplications (red) predicted using high-throughput sequencing of skeletal muscle DNA from three patients with pathogenic, compound heterozygous POLG variants as well as two healthy control patients. The orange bars in the outermost circle show breakpoint frequency at a given base position. c Frequencies of exact direct repeats overlapping or flanking each pair of breakpoints, considering the longest match for each deletion and pooling the three patients (red bars). Randomized breakpoints are shown for comparison (grey bars), with error bars indicating the standard deviation (100 randomizations). d Frequencies for the most commonly observed repeat patterns. e Analysis of 5′ vs. 3′ retention of imperfect repeats. A subset of deletions where the breakpoint positions could be safely determined to within 1 bp were analyzed for the presence of imperfect direct repeats at both sides of the deleted segment and were classified as either 5′ or 3′ based on the longest discovered repeat having at most 1 mismatch. Results are shown for unique deletions as well as the complete set of deletions, pooled from the three POLG patients. Results from 100 sets of randomly generated deletions, each being similar in size as the observed data, are included. Error bars indicate the standard deviation

    Article Snippet: Following desalting by dialysis, the DNA containing the intact H-strand sequences was treated with exonuclease III (Thermo Fisher Scientific) and again dialyzed.

    Techniques: Next-Generation Sequencing, Standard Deviation, Generated

    Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Journal: Journal of Extracellular Vesicles

    Article Title: Large extracellular vesicles carry most of the tumour DNA circulating in prostate cancer patient plasma

    doi: 10.1080/20013078.2018.1505403

    Figure Lengend Snippet: Most extracellular DNA is packaged into L-EVs . (a) Tunable resistive pulse sensing (TRPS, qNano) using two different pore membranes (NP4000 and NP200) identified as L-EVs (left) and S-EVs (right) derived from PC3 cells. NP4000 membrane, which can detect particles with a diameter between 1.0 and 6.0 μm, was used for quantitation of L-EVs, while NP200 membrane, which can detect particles with a diameter between 60 and 400 nm, was used for quantitation of S-EVs. (b) Protein lysates from L-EVs and S-EVs purified by iodixanol density gradient (at 1.10 and 1.15 g/ml) were blotted with LO markers HSPA5 and CK18, and with Exo marker CD81. (c) Total DNA was quantified by Qubit Fluorometer in L-EVs and S-EVs isolated from PC3 and U87 cell lines. The plot shows the DNA ratio between L-EVs and S-EVs. (d) Double stranded (ds)DNA was quantified by High Sensitivity (HS) dsDNA Qubit Assay in L-EVs and S-EVs isolated from 1 ml of plasma from patients with mCRPC ( n = 40) and cancer-free individuals ( n = 6). (e) Quantification of both protein and DNA content in L-EVs and S-EVs isolated from conditioned media of 12.6 × 10 7 PC3 cells. (f) Single stranded (ss) and dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Exonuclease III, were quantified by Qubit. (g) Chip-based capillary electrophoresis (Bioanalyzer) showing the presence of dsDNA in PC3-derived L-EVs and S-EVs, with or without treatment with DNase I and Endonuclease III. L-EVs contain abundant DNA with a large peak around 10 kbp. Conversely, the amount of DNA in S-EVs is negligible. (h) ss- and dsDNA in PC3-derived L-EVs and S-EVs were quantified by Qubit after treatment with nucleases (DNase I and Exonuclease III) with or without addition of a detergent (Triton X-100) prior to nuclease treatment. (i) Chip-based capillary electrophoresis (Bioanalyzer) showing that only miniscule amounts of dsDNA could be detected after EV lysis using a detergent prior to treatment with nucleases.

    Article Snippet: L- and S-EVs were treated with rDNase I (2 U/μl, DNA-Free Kit, Ambion) and Exonuclease III (200 U/μl, Thermofisher).

    Techniques: Tunable Resistive Pulse Sensing, Derivative Assay, Quantitation Assay, Purification, Marker, Isolation, HS DSDNA Qubit Assay, Chromatin Immunoprecipitation, Electrophoresis, Lysis