exonuclease i  (Thermo Fisher)


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    Exonuclease I 20 U µL
    Description:
    Thermo Scientific Exonuclease I ExoI degrades single stranded DNA in a 3 →5 direction It releases deoxyribonucleoside 5 monophosphates in a stepwise manner and leaves 5 terminal dinucleotides intact It does not cleave DNA strands with terminal 3 OH groups blocked by phosphoryl or acetyl groups Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures • prior to PCR product sequencing• for one tube megaprimer PCR mutagenesis• Removal of single stranded DNA containing a 3 hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single stranded DNA with a 3 hydroxyl terminusNoteThe enzyme is not suitable for removing 3 overhangs of dsDNA
    Catalog Number:
    en0581
    Price:
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    Applications:
    Cloning|PCR Cloning|Mutagenesis
    Category:
    Proteins Enzymes Peptides
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    Structured Review

    Thermo Fisher exonuclease i
    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, <t>Exonuclease</t> I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P
    Thermo Scientific Exonuclease I ExoI degrades single stranded DNA in a 3 →5 direction It releases deoxyribonucleoside 5 monophosphates in a stepwise manner and leaves 5 terminal dinucleotides intact It does not cleave DNA strands with terminal 3 OH groups blocked by phosphoryl or acetyl groups Highlights• Active in PCR buffersApplications• Primer removal from PCR mixtures • prior to PCR product sequencing• for one tube megaprimer PCR mutagenesis• Removal of single stranded DNA containing a 3 hydroxyl terminus from nucleic acid mixtures• Assay for the presence of single stranded DNA with a 3 hydroxyl terminusNoteThe enzyme is not suitable for removing 3 overhangs of dsDNA
    https://www.bioz.com/result/exonuclease i/product/Thermo Fisher
    Average 99 stars, based on 828 article reviews
    Price from $9.99 to $1999.99
    exonuclease i - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates"

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa160

    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P
    Figure Legend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Techniques Used: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

    2) Product Images from "Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings"

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky769

    Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.
    Figure Legend Snippet: Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Techniques Used:

    3) Product Images from "Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects"

    Article Title: Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20151618

    Mutations in STN1 result in abnormal telomere phenotypes. (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P
    Figure Legend Snippet: Mutations in STN1 result in abnormal telomere phenotypes. (A) DNA samples, prepared from PBLs of patient P1, her heterozygous father (F1), and a noncarrier sibling (S1) and patient P2, his heterozygous mother (M2), and two independent control samples (C), were analyzed by in-gel hybridization. Duplicated lanes were electrophoresed in the same gel, and then separated and hybridized to a G-rich or C-rich telomeric probe, as indicated above the panels. After native hybridization to detect single-stranded telomeric DNA (top), the gels were denatured and rehybridized with the same probes to detect the overall duplex telomeric DNA (bottom). Treatment with exonuclease I is indicated above the lanes. (B) Graphic illustration of the mean telomere length for the patients and their family members, calculated based on the following number of independent measurements of four in-gels and two Southern analyses: P1:6, M1:3, F1:3, S1:3, P2:9, M2:3, F2:1, C1:2, and C2:3. (C) Graphic illustration of the relative native (single strand) per denatured (total) telomeric signal, normalized to the controls. The values represent the mean of four independent measurements for P1 and four for P2. (D, top) A metaphase spread from P2 PBL after CO-FISH. Bar, 5 µm. The area designated by the white frame in enlarged in the bottom panel. Arrowheads point to chromosome ends with hybridization signals on both sister chromatids, indicating that T-SCE occurred at that chromosome end. (right) The frequency of T-SCE in P2 PBLs is compared with PBLs from four age-matched controls (con-BL1-4). For P2 PBLs, 1,150 chromosome ends were analyzed. For control PBLs, between 600 and 930 telomeres were analyzed (*, P

    Techniques Used: Hybridization, Fluorescence In Situ Hybridization

    4) Product Images from "MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy"

    Article Title: MHC Multimer-Guided and Cell Culture-Independent Isolation of Functional T Cell Receptors from Single Cells Facilitates TCR Identification for Immunotherapy

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0061384

    Optimized RACE-PCR-based approach for TCR sequencing from single cells. (A) Sketch of the basic principle of the novel single-TCR sequencing strategy. Reverse transcription and exonuclease digest were consecutively done on-slide. Complete reactions were transferred to 96 well plates for tailing, anchor PCR and nested PCR round I and II. Alpha and beta chains were pre-amplified together during anchor PCR and in separate reactions for nested PCRs I and II. To change the reaction conditions for the first four steps of the protocol, the volume was increased to create optimal conditions for the subsequent enzyme. For nested PCR round I and II 1 µl of the previous reation volume was transferred to the next step. (B) Different priming strategies (standard oligo-dT/random hexamers, one gene-specific primer and three gene-specific primers per TCR chain) were tested with and without extra exonuclease-I digest of residual RT-primers. Decreasing numbers of human T cells, from 1000 to 1 as indicated served as template. (C)The anchor PCR step is used to prolong the oligo-dG overhang from the tailing step. PCR was tested without reverse primer in “linear” mode and with reverse primer in “exponential” mode of amplification. (D) Temperature switch during reverse transcription was tested. RT at a constant temperature of 51°C (upper row) was compared with a temperature increase form 51°C for 30 min to 70°C for 20 min (bottom row). (E) In seven independent single-cell PCR experiments a total number of 266 samples were processed. Numbers of samples yielding α- and/or β- chain products above the evaluated full-length cut-off size were calculated as a percentage of total samples per experiment. Mean values for all experiments taken together are indicated by horizontal lines.
    Figure Legend Snippet: Optimized RACE-PCR-based approach for TCR sequencing from single cells. (A) Sketch of the basic principle of the novel single-TCR sequencing strategy. Reverse transcription and exonuclease digest were consecutively done on-slide. Complete reactions were transferred to 96 well plates for tailing, anchor PCR and nested PCR round I and II. Alpha and beta chains were pre-amplified together during anchor PCR and in separate reactions for nested PCRs I and II. To change the reaction conditions for the first four steps of the protocol, the volume was increased to create optimal conditions for the subsequent enzyme. For nested PCR round I and II 1 µl of the previous reation volume was transferred to the next step. (B) Different priming strategies (standard oligo-dT/random hexamers, one gene-specific primer and three gene-specific primers per TCR chain) were tested with and without extra exonuclease-I digest of residual RT-primers. Decreasing numbers of human T cells, from 1000 to 1 as indicated served as template. (C)The anchor PCR step is used to prolong the oligo-dG overhang from the tailing step. PCR was tested without reverse primer in “linear” mode and with reverse primer in “exponential” mode of amplification. (D) Temperature switch during reverse transcription was tested. RT at a constant temperature of 51°C (upper row) was compared with a temperature increase form 51°C for 30 min to 70°C for 20 min (bottom row). (E) In seven independent single-cell PCR experiments a total number of 266 samples were processed. Numbers of samples yielding α- and/or β- chain products above the evaluated full-length cut-off size were calculated as a percentage of total samples per experiment. Mean values for all experiments taken together are indicated by horizontal lines.

    Techniques Used: Polymerase Chain Reaction, Sequencing, Nested PCR, Amplification

    5) Product Images from "Improved Molecular Typing Assay for Rhinovirus Species A, B, and C"

    Article Title: Improved Molecular Typing Assay for Rhinovirus Species A, B, and C

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.00075-14

    Elimination of nonspecific amplification of human sequences in the RV 5′-UTR assay. (A) Effect of exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT) treatment of the first PCR products on nonspecific amplification of human sequences, using
    Figure Legend Snippet: Elimination of nonspecific amplification of human sequences in the RV 5′-UTR assay. (A) Effect of exonuclease I and shrimp alkaline phosphatase (ExoSAP-IT) treatment of the first PCR products on nonspecific amplification of human sequences, using

    Techniques Used: Amplification, Polymerase Chain Reaction

    6) Product Images from "Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination"

    Article Title: Crystal structure of Hop2–Mnd1 and mechanistic insights into its role in meiotic recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv172

    The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.
    Figure Legend Snippet: The C-terminal portion of Hop2–Mnd1 interacts with Dmc1 nucleofilament. ( A ) Fitting of the coiled coil of Hop2–Mnd1 into the helical groove of the Dmc1-ssDNA filament (EBI entry: EMD-1492). Surface of both Hop2–Mnd1 (blue) and human Dmc1-ssDNA filament (gray) are shown in mesh representation. ( B ) LZ3wCH of Hop2–Mnd1 is necessary for binding to Dmc1 nucleofilament. ( Left ) Schematic representation of the exonuclease I protection assay. (Right, top) Wild-type Hop2–Mnd1 and the indicated deletion mutants (2.5 μM) were individually incubated with Dmc1 nucleofilament and their ssDNA protection ability was analyzed by electrophoresis on a 15% native gel. (Right, bottom) The intensities of unreacted ssDNA relative to input ssDNA are shown. The experiment was performed in triplicate. ( C ) Mapping of conserved residues on the surface of Hop2–Mnd1.

    Techniques Used: Binding Assay, Incubation, Electrophoresis

    7) Product Images from "RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA"

    Article Title: RNase H1 promotes replication fork progression through oppositely transcribed regions of Drosophila mitochondrial DNA

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA118.007015

    Proposed interpretations of mtDNA species detected by gel electrophoresis in  rnh 1 knockdown cells.  One of many possible scenarios is illustrated.  A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand.  B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR.  C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by  blue arrow  in   Fig. 6 C ).  D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products.  E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows  in   Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow  in   Fig. 6 B ).
    Figure Legend Snippet: Proposed interpretations of mtDNA species detected by gel electrophoresis in rnh 1 knockdown cells. One of many possible scenarios is illustrated. A , portion of a hypothetical replication intermediate with uncompleted lagging strand approaching the unidirectional replication origin ( O R ). A short residual RNA primer remains at the 5′ end of the leading strand. B , the lagging strand proceeds beyond the leading-strand initiation point as far as specific, reiterated termination signals in the repeat II elements of the NCR. C , impaired fork progression around the genome causes the origin structure to persist with eventual regression to form a chicken-foot structure that can branch-migrate (arc denoted by blue arrow in Fig. 6 C ). D , upon treatment with RusA, the four-way junctions resulting from these regressed forks are cut, generating effectively linear products. E , HindIII digestion liberates linear fragments with lagging-strand 3′ ssDNA extensions derived from the regressed forks ( green arrows in Fig. 6 B ). These are digestible with S1 nuclease or exonuclease I, leaving a residual double-stranded species ( purple arrow in Fig. 6 B ).

    Techniques Used: Nucleic Acid Electrophoresis, Derivative Assay

    Related Articles

    Hydrolysis Assay:

    Article Title: An exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules
    Article Snippet: .. Exonuclease I hydrolysis assay Oligonucleotides were labeled at the 5′ end with [γ-32 P]ATP using T4 polynucleotide kinase (Fermentas, Lithuania). ..

    Southern Blot:

    Article Title: Defects in coding joint formation in vivo in developing ATM-deficient B and T lymphocytes
    Article Snippet: .. For exonuclease V digestion, 15 μg of thymocyte DNA was treated with increasing concentrations of exonuclease V, using the manufacturer's buffer conditions (USB Corporation), for 1 h at 37°C before restriction enzyme digestion and Southern blot analyses. .. For mung bean nuclease assays, 15 μg of thymocyte DNA was digested with 12.5 U of mung bean nuclease (New England Biolabs, Inc.) in 200 μl using the manufacturer's buffer supplemented with 0.4 mM ZnSO4 at 25°C for 1 h before treatment with exonuclease V, as described in this section.

    Ethanol Precipitation:

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing
    Article Snippet: .. A total of 6 μl 10 mM ATP, 1 μl 10x CutSmart, 1 μl Exonuclease V, (RecBCD, M0345) and 1 μl Exonuclease I (M0293) was added to each digest and incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation in the presence of 1 μl GlycoBlue (AM9516 ThermoFisher Scientific, UK). .. DNA pellets were dissolved in 45 μl 0.1x TE, then 6 μl 10x CutSmart, 6 μl 10 mM ATP, 1 μl Exonuclease V, 1 μl Exonuclease I, and 1 μl same restriction enzyme as previous day were added and samples again incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation.

    Incubation:

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing
    Article Snippet: .. A total of 6 μl 10 mM ATP, 1 μl 10x CutSmart, 1 μl Exonuclease V, (RecBCD, M0345) and 1 μl Exonuclease I (M0293) was added to each digest and incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation in the presence of 1 μl GlycoBlue (AM9516 ThermoFisher Scientific, UK). .. DNA pellets were dissolved in 45 μl 0.1x TE, then 6 μl 10x CutSmart, 6 μl 10 mM ATP, 1 μl Exonuclease V, 1 μl Exonuclease I, and 1 μl same restriction enzyme as previous day were added and samples again incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation.

    Labeling:

    Article Title: An exonuclease I hydrolysis assay for evaluating G-quadruplex stabilization by small molecules
    Article Snippet: .. Exonuclease I hydrolysis assay Oligonucleotides were labeled at the 5′ end with [γ-32 P]ATP using T4 polynucleotide kinase (Fermentas, Lithuania). ..

    SYBR Green Assay:

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings
    Article Snippet: .. T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA). .. EvaGreen with the concentration of 20× was purchased from Biotium (Fremont, CA, USA).

    Generated:

    Article Title: A deep learning approach to identify new gene targets of a novel therapeutic for human splicing disorders
    Article Snippet: .. Characterization of the assay has shown that RLuc is expressed each time a transcript is generated from the reporter plasmid, while FLuc is only expressed if exon 20 is included in the transcript, thereby keeping FLuc in-frame. .. Evaluation of FLuc/RLuc expression yields the percent exon inclusion in the splicing assays .

    Plasmid Preparation:

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates
    Article Snippet: .. Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific). ..

    Article Title: A deep learning approach to identify new gene targets of a novel therapeutic for human splicing disorders
    Article Snippet: .. Characterization of the assay has shown that RLuc is expressed each time a transcript is generated from the reporter plasmid, while FLuc is only expressed if exon 20 is included in the transcript, thereby keeping FLuc in-frame. .. Evaluation of FLuc/RLuc expression yields the percent exon inclusion in the splicing assays .

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    Thermo Fisher exonuclease v
    Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of  CUP1  eccDNA and rDNA-derived ERCs in wild-type,  mus81 Δ,  yen1 Δ, and  slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in   Fig 1B . (B) Quantification of  CUP1  eccDNA and rDNA-derived ERCs in wild-type and  mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in   Fig 1B ,  n  = 4. (C) REC-seq analysis of  mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in   Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy  P GAL1 -3HA cup1  tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications.  P GAL1 -3HA  wild-type,  sae2 Δ, and  mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with  Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1  plasmid to complement the loss of active chromosomal  CUP1  genes, labelled as  CUP1  plasmid. This plasmid contains an  Xho I site and is hence linearised by  Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA;  n  = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on  P GAL1 -3HA  wild-type and  rad52 Δ cells along with BY4741 wild-type and  rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4  to 10 1  cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in   S1 Data  and   S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.
    Exonuclease V, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 834 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease v/product/Thermo Fisher
    Average 99 stars, based on 834 article reviews
    Price from $9.99 to $1999.99
    exonuclease v - by Bioz Stars, 2020-09
    99/100 stars
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    Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of  CUP1  eccDNA and rDNA-derived ERCs in wild-type,  mus81 Δ,  yen1 Δ, and  slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in   Fig 1B . (B) Quantification of  CUP1  eccDNA and rDNA-derived ERCs in wild-type and  mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in   Fig 1B ,  n  = 4. (C) REC-seq analysis of  mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in   Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy  P GAL1 -3HA cup1  tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications.  P GAL1 -3HA  wild-type,  sae2 Δ, and  mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with  Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1  plasmid to complement the loss of active chromosomal  CUP1  genes, labelled as  CUP1  plasmid. This plasmid contains an  Xho I site and is hence linearised by  Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA;  n  = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on  P GAL1 -3HA  wild-type and  rad52 Δ cells along with BY4741 wild-type and  rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4  to 10 1  cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in   S1 Data  and   S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.

    Journal: PLoS Biology

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

    doi: 10.1371/journal.pbio.3000471

    Figure Lengend Snippet: Mus81 is required for eccDNA formation in old and young cells. (A) Southern blot analysis of CUP1 eccDNA and rDNA-derived ERCs in wild-type, mus81 Δ, yen1 Δ, and slx4 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed as in Fig 1B . (B) Quantification of CUP1 eccDNA and rDNA-derived ERCs in wild-type and mus81 Δ cells aged for 24 hours in the presence or absence of 1 mM CuSO 4 , performed and analysed as in Fig 1B , n = 4. (C) REC-seq analysis of mus81 Δ cells compared to wild type in the absence (left) or presence (right) of 1 mM CuSO 4 . Experiment and analysis as in Fig 4D . (D) Southern blot analysis of eccDNA from the 17 copy P GAL1 -3HA cup1 tandem repeat in non–age-selected BY4741 haploid cell background lacking MEP modifications. P GAL1 -3HA wild-type, sae2 Δ, and mus81 Δ cells were pregrown on YP Raffinose before a 6 hour induction with 2% galactose or 2% glucose. Genomic DNA was digested with Xho I; then 95% of the sample was further digested with ExoV and ExoI; 5% total DNA (lanes 1–6) and 95% ExoV digested material (lanes 7–12) are shown. These cells contain an additional pRS316- CUP1 plasmid to complement the loss of active chromosomal CUP1 genes, labelled as CUP1 plasmid. This plasmid contains an Xho I site and is hence linearised by Xho I and degraded by ExoV. Signals from same membrane stripped and reprobed for rDNA show ERC species. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (E) Colony formation assay performed on P GAL1 -3HA wild-type and rad52 Δ cells along with BY4741 wild-type and rad52 Δ controls. Cells were pregrown as above on YP raffinose, then serial dilutions from 10 4 to 10 1 cells spotted on YPD and YPGal plates, which were grown at 30°C until control cells had formed equivalent sized colonies (2–3 days). The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; rDNA, ribosomal DNA; REC-seq, restriction-digested extrachromosomal circular DNA sequencing.

    Article Snippet: A total of 6 μl 10 mM ATP, 1 μl 10x CutSmart, 1 μl Exonuclease V, (RecBCD, M0345) and 1 μl Exonuclease I (M0293) was added to each digest and incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation in the presence of 1 μl GlycoBlue (AM9516 ThermoFisher Scientific, UK).

    Techniques: Southern Blot, Derivative Assay, Plasmid Preparation, Transformation Assay, Colony Assay, DNA Sequencing

    Transcription of the  CUP1  locus causes eccDNA accumulation. (A) Schematic representation of cell labelling, induction, and ageing in the presence or absence of copper. (B) Southern blot analysis of  CUP1  eccDNA and rDNA-derived eccDNA (ERCs) in yeast cells aged for 48 hours in the presence or absence of 1 mM CuSO 4 , along with young cells maintained in log phase in the presence or absence of 1 mM CuSO 4  for an equivalent time. Large linear fragments of chromosomal DNA (Chr.) migrate at the resolution limit of the gel, whereas circular DNA species (eccDNA) migrate more slowly. Abundances of eccDNA and ERCs were compared by one-way ANOVA;  n  = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (C) Southern blot analysis of  CUP1  eccDNA in cells aged for 0, 7.5, 24, or 48 hours in 1 mM CuSO 4 . Chromosomal DNA was removed with ExoV to improve sensitivity, endogenous circular 2μ DNA is shown as a loading control. (D) Southern blot analysis of eccDNA in a heterozygous strain bearing one  P GAL1 -3HA cup1  locus modified by replacing all  CUP1  promoters and ORFs with  P GAL1  promoters and 3HA ORFs, and 1 wild-type  P CUP1 -CUP1  allele. eccDNA is detected with allele specific probes; the additional band in the left panel is from hybridisation to the endogenous  GAL1  locus on Chromosome II. Quantification and analysis performed as in panel B,  n  = 3. The data underlying this figure may be found in   S1 Data  and   S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; ORF, open reading frame; rDNA, ribosomal DNA.

    Journal: PLoS Biology

    Article Title: Transcription-induced formation of extrachromosomal DNA during yeast ageing

    doi: 10.1371/journal.pbio.3000471

    Figure Lengend Snippet: Transcription of the CUP1 locus causes eccDNA accumulation. (A) Schematic representation of cell labelling, induction, and ageing in the presence or absence of copper. (B) Southern blot analysis of CUP1 eccDNA and rDNA-derived eccDNA (ERCs) in yeast cells aged for 48 hours in the presence or absence of 1 mM CuSO 4 , along with young cells maintained in log phase in the presence or absence of 1 mM CuSO 4 for an equivalent time. Large linear fragments of chromosomal DNA (Chr.) migrate at the resolution limit of the gel, whereas circular DNA species (eccDNA) migrate more slowly. Abundances of eccDNA and ERCs were compared by one-way ANOVA; n = 4 biological replicates; data were log transformed for testing to fulfil the assumptions of a parametric test. (C) Southern blot analysis of CUP1 eccDNA in cells aged for 0, 7.5, 24, or 48 hours in 1 mM CuSO 4 . Chromosomal DNA was removed with ExoV to improve sensitivity, endogenous circular 2μ DNA is shown as a loading control. (D) Southern blot analysis of eccDNA in a heterozygous strain bearing one P GAL1 -3HA cup1 locus modified by replacing all CUP1 promoters and ORFs with P GAL1 promoters and 3HA ORFs, and 1 wild-type P CUP1 -CUP1 allele. eccDNA is detected with allele specific probes; the additional band in the left panel is from hybridisation to the endogenous GAL1 locus on Chromosome II. Quantification and analysis performed as in panel B, n = 3. The data underlying this figure may be found in S1 Data and S1 Raw Images . eccDNA, extrachromosomal circular DNA; ERC, extrachromosomal ribosomal DNA circle; ExoV, exonuclease V; ORF, open reading frame; rDNA, ribosomal DNA.

    Article Snippet: A total of 6 μl 10 mM ATP, 1 μl 10x CutSmart, 1 μl Exonuclease V, (RecBCD, M0345) and 1 μl Exonuclease I (M0293) was added to each digest and incubated over night at 37°C before extraction with phenol chloroform and ethanol precipitation in the presence of 1 μl GlycoBlue (AM9516 ThermoFisher Scientific, UK).

    Techniques: Southern Blot, Derivative Assay, Transformation Assay, Modification, Hybridization

    Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Journal: Nucleic Acids Research

    Article Title: DIRS retrotransposons amplify via linear, single-stranded cDNA intermediates

    doi: 10.1093/nar/gkaa160

    Figure Lengend Snippet: Features of extrachromosomal DIRS-1 bsr cDNA. Extrachromosomal cDNA samples were extracted from rrpC– strains transformed with DIRS-1 bsr ( A ) or DIRS-1 bsr* ( B ), and were cultivated in G418/BS10 medium that selects for strains with mobilized retrotransposon. The samples were treated separately with RNase A, DNase I, Exonuclease I (Exo I), Exonuclease III (Exo III) and S1 nuclease (S1). After digestion and heat inactivation of enzymes, the samples were analyzed by PCR with the primers #2212 and #2213 binding within the region of mbsrI cassette (binding position in Figure 2A ). Quantification of endogenous ( C and D ) and mbsrI -tagged versions ( E ) of DIRS-1 extrachromosomal cDNA after treatment with Exonuclease I and S1 nuclease, relative to the non-digested sample. Samples were extracted from rrpC– strains transformed with DIRS-1 bsr and DIRS-1 bsr* upon cultivation in G418/BS10 medium that selects for strains with mobilized retrotransposons. The cDNA abundance was monitored by qPCR using primers targeting positions qP1, qP2 and qBS (Figures 2A and 3A ) and is shown in logarithmic scale relative to the non-digested sample (set to 1). Each qPCR reaction was run in triplicate. Error bars : mean with S.D. Statistics: paired t test: P

    Article Snippet: Nuclease treatment and detection of DIRS-1bsr extrachromosomal cDNA The extracted extrachromosomal cDNA sample (plasmid miniprep kit, as described before) was subjected to digestion with RNase A (Sigma-Aldrich), DNase I, exonuclease I, exonuclease III or S1 nuclease (Thermo Scientific).

    Techniques: Transformation Assay, Polymerase Chain Reaction, Binding Assay, Real-time Polymerase Chain Reaction

    Identification of the small molecule splicing modulator BPN-15477. ( a ) Molecular structure of BPN-15477 compared to kinetin, the northern heterocycle and C-2 substitution are indicated in red. ( b ) Schematic representation of the dual-reporter minigene used to measure splicing. Rluc and Fluc indicate Renilla and Firefly luciferase, respectively. A/C indicates the start codon mutation in Fluc and taagC indicates the location of the FD mutation. Dose response curves for kinetin and BPN-15477. Rluc-FD-Fluc transfected HEK293T cells treated for 24 hours. Normalized relative luciferase units (RLU), which measure exon 20 inclusion, are plotted as a function of compound concentration. Assays were run in triplicate and curves were created by nonlinear regression using Prism4 (GraphPad Software Inc.). (c) Validation of BPN-15477 splicing correction in FD fibroblasts. Cells were treated in duplicate for 24 hours at the concentrations indicated.

    Journal: bioRxiv

    Article Title: A deep learning approach to identify new gene targets of a novel therapeutic for human splicing disorders

    doi: 10.1101/2020.02.03.932103

    Figure Lengend Snippet: Identification of the small molecule splicing modulator BPN-15477. ( a ) Molecular structure of BPN-15477 compared to kinetin, the northern heterocycle and C-2 substitution are indicated in red. ( b ) Schematic representation of the dual-reporter minigene used to measure splicing. Rluc and Fluc indicate Renilla and Firefly luciferase, respectively. A/C indicates the start codon mutation in Fluc and taagC indicates the location of the FD mutation. Dose response curves for kinetin and BPN-15477. Rluc-FD-Fluc transfected HEK293T cells treated for 24 hours. Normalized relative luciferase units (RLU), which measure exon 20 inclusion, are plotted as a function of compound concentration. Assays were run in triplicate and curves were created by nonlinear regression using Prism4 (GraphPad Software Inc.). (c) Validation of BPN-15477 splicing correction in FD fibroblasts. Cells were treated in duplicate for 24 hours at the concentrations indicated.

    Article Snippet: Characterization of the assay has shown that RLuc is expressed each time a transcript is generated from the reporter plasmid, while FLuc is only expressed if exon 20 is included in the transcript, thereby keeping FLuc in-frame.

    Techniques: Northern Blot, Luciferase, Mutagenesis, Transfection, Concentration Assay, Software

    Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Journal: Nucleic Acids Research

    Article Title: Terminal hairpin in oligonucleotide dominantly prioritizes intramolecular cyclization by T4 ligase over intermolecular polymerization: an exclusive methodology for producing ssDNA rings

    doi: 10.1093/nar/gky769

    Figure Lengend Snippet: Dominant cyclization of l-DNA using hairpins as internal promoters. ( A ) The solution structures of L64 3-4,24-4 , L64 16-4,37-4 and L64 3-4 , determined by Mfold calculation under the conditions of [Mg 2+ ] = 10 mM and 25°C. ( B ) Treatments of these l-DNAs with T4 DNA ligase. Lane 1, L64 3-4,24-4 without the T4 ligase treatment; lane 2, L64 3-4,24-4 treated with T4 DNA ligase in the presence of 12-nt splint which is complementary with the 6-nt sequences in the 3′- and 5′-ends of L64 3-4,24–4 ; lane 4, L64 16-4,37-4 without the treatment; lane 5, L64 16-4,37-4 treated with T4 DNA ligase in the presence of 12-nt splint. Lane 7, L64 3-4 without the treatment; lane 8, L64 3-4 treated with T4 DNA ligase in the presence of 12-nt splint. In lanes 3, 6 and 9, the products in lanes 2, 5 and 8 were further treated with Exonuclease I to remove non-cyclic products. The conditions for the T4 ligase reactions: [l-DNA] 0 = 5 μM, [splint] 0 = 10 μM and 10 U T4 DNA ligase in 1× T4 DNA ligase buffer at 25°C for 12 h.

    Article Snippet: T4 DNA ligase, Exonuclease I and SYBR Green II were also obtained from Thermo Scientific (Pittsburgh, PA, USA).

    Techniques: