t7 exo  (New England Biolabs)


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    Structured Review

    New England Biolabs t7 exo
    The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts.  (A)  Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of  32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.
    T7 Exo, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
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    Images

    1) Product Images from "The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair"

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw274

    The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts.  (A)  Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of  32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.
    Figure Legend Snippet: The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts. (A) Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of 32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.

    Techniques Used: Activity Assay, Avidin-Biotin Assay, Incubation, Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Staining

    2) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    3) Product Images from "A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity"

    Article Title: A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

    Journal: bioRxiv

    doi: 10.1101/548628

    Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p
    Figure Legend Snippet: Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p

    Techniques Used: Methylation

    4) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    5) Product Images from "ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells"

    Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

    Journal: bioRxiv

    doi: 10.1101/2022.06.01.494308

    Optimization of long ssDNA production using T7 exonuclease and restriction enzymes. a , Schematic of long ssDNA production using T7 exonuclease (one-step method). dsDNA is amplified by PCR with a pair of long primers containing a 90 nt HA, one of which bears five consecutive PS bonds at the 5’ end. The strand with the non-modified 5’ end of the dsDNA is selectively digested by T7 exonuclease to produce long ssDNA donors. b , T7 exonuclease reaction on dsDNA amplified using three different combinations of primers (PS-modified (PS) or non-modified (noPS) for the forward and reverse primers). The bottom image is of the same gel as the top one, with higher brightness and contrast. ssDNA shows higher mobility than dsDNA of the same length. Asterisks show undigested dsDNA remnants. c , Schematic of ssDNA production by two-step PCR and T7 exonuclease (T7 method). The first PCR uses long non-modified primers to add the HAs, and the second uses a combination of short primers, one of which is PS-modified. d , “PS-PS” dsDNA was prepared with one-step or two-step PCR and subsequently subjected to the T7 exonuclease reaction. Plot profiles for each lane are shown below the gel electrophoresis images. e , Production of long ssDNA donors using the one-step and the two-step methods. The bottom image is of the same gel as the top one, with higher brightness and contrast. f , Schematic of ssDNA production using T7 exonuclease and restriction enzymes (T7RE method). After two-step PCR and T7 exonuclease reaction, the indicated four restriction enzymes digest dsDNA remnants to produce short dsDNA fragments which can be further degraded by T7 exonuclease. g , ssDNA production by the T7 and the T7RE methods. The last two lanes contain column-purified DNA products of both reactions. Plot profiles for the last two lanes are shown below the gel electrophoresis image.
    Figure Legend Snippet: Optimization of long ssDNA production using T7 exonuclease and restriction enzymes. a , Schematic of long ssDNA production using T7 exonuclease (one-step method). dsDNA is amplified by PCR with a pair of long primers containing a 90 nt HA, one of which bears five consecutive PS bonds at the 5’ end. The strand with the non-modified 5’ end of the dsDNA is selectively digested by T7 exonuclease to produce long ssDNA donors. b , T7 exonuclease reaction on dsDNA amplified using three different combinations of primers (PS-modified (PS) or non-modified (noPS) for the forward and reverse primers). The bottom image is of the same gel as the top one, with higher brightness and contrast. ssDNA shows higher mobility than dsDNA of the same length. Asterisks show undigested dsDNA remnants. c , Schematic of ssDNA production by two-step PCR and T7 exonuclease (T7 method). The first PCR uses long non-modified primers to add the HAs, and the second uses a combination of short primers, one of which is PS-modified. d , “PS-PS” dsDNA was prepared with one-step or two-step PCR and subsequently subjected to the T7 exonuclease reaction. Plot profiles for each lane are shown below the gel electrophoresis images. e , Production of long ssDNA donors using the one-step and the two-step methods. The bottom image is of the same gel as the top one, with higher brightness and contrast. f , Schematic of ssDNA production using T7 exonuclease and restriction enzymes (T7RE method). After two-step PCR and T7 exonuclease reaction, the indicated four restriction enzymes digest dsDNA remnants to produce short dsDNA fragments which can be further degraded by T7 exonuclease. g , ssDNA production by the T7 and the T7RE methods. The last two lanes contain column-purified DNA products of both reactions. Plot profiles for the last two lanes are shown below the gel electrophoresis image.

    Techniques Used: Amplification, Polymerase Chain Reaction, Modification, Nucleic Acid Electrophoresis, Purification

    6) Product Images from "Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection"

    Article Title: Physiological protein blocks direct the Mre11–Rad50–Xrs2 and Sae2 nuclease complex to initiate DNA end resection

    Journal: Genes & Development

    doi: 10.1101/gad.308254.117

    Exo1 efficiently and specifically functions downstream from the MRX–Sae2 nuclease in the processing of dsDNA with blocked ends. ( A ) A representative experiment showing the processing of streptavidin-blocked dsDNA by MRX–Sae2 in the absence (lane 2 ) or presence (lane 3 ) of Exo1. ( B ) Representative DNA clipping reactions with MRX–Sae2 with various concentrations of Exo1, as indicated. ( C – F ) Exo1 degrades the MRX–Sae2 nuclease products in a specific manner. Representative kinetic nuclease assays with MRX–Sae2 and either no additional exonuclease ( C ), Exo1 ( D ), Exo1 D173A (nuclease-dead; E ), or T7 exonuclease ( F ). The DNA was either blocked by streptavidin ( left ) or bound by 30 nM Ku ( right ). ( G ) Quantitation of experiments such as those from C – F showing overall substrate utilization in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM. ( H ) Quantitation of experiments such as those from C – F showing the fraction of endonuclease products versus total DNA in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM.
    Figure Legend Snippet: Exo1 efficiently and specifically functions downstream from the MRX–Sae2 nuclease in the processing of dsDNA with blocked ends. ( A ) A representative experiment showing the processing of streptavidin-blocked dsDNA by MRX–Sae2 in the absence (lane 2 ) or presence (lane 3 ) of Exo1. ( B ) Representative DNA clipping reactions with MRX–Sae2 with various concentrations of Exo1, as indicated. ( C – F ) Exo1 degrades the MRX–Sae2 nuclease products in a specific manner. Representative kinetic nuclease assays with MRX–Sae2 and either no additional exonuclease ( C ), Exo1 ( D ), Exo1 D173A (nuclease-dead; E ), or T7 exonuclease ( F ). The DNA was either blocked by streptavidin ( left ) or bound by 30 nM Ku ( right ). ( G ) Quantitation of experiments such as those from C – F showing overall substrate utilization in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM. ( H ) Quantitation of experiments such as those from C – F showing the fraction of endonuclease products versus total DNA in reactions with Ku-blocked dsDNA. Averages are shown. n ≥ 3. Error bars indicate SEM.

    Techniques Used: Quantitation Assay

    7) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    8) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    9) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    10) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    11) Product Images from "Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection"

    Article Title: Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection

    Journal: Talanta

    doi: 10.1016/j.talanta.2022.123393

    Gold Nanoparticles based sensors’ functionality targeting the gene regions E, R or S mutation D614G. For the detection, ssDNA purified amplicons were prepared by RT-PCR followed by T7 exonuclease digestion and confronted to the complementary loops included in MB E, R or S G614 (loop 25 nt) used functionalize 23 nm AuNPs. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). The concentration of targets used (from left to right) were 0, 50 nM, 100 and 250 nM. Statistical analysis was performed using one-way ANOVA Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Gold Nanoparticles based sensors’ functionality targeting the gene regions E, R or S mutation D614G. For the detection, ssDNA purified amplicons were prepared by RT-PCR followed by T7 exonuclease digestion and confronted to the complementary loops included in MB E, R or S G614 (loop 25 nt) used functionalize 23 nm AuNPs. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). The concentration of targets used (from left to right) were 0, 50 nM, 100 and 250 nM. Statistical analysis was performed using one-way ANOVA Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Mutagenesis, Purification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Concentration Assay

    Gold Nanoparticles based sensors’ functionality targeting the S gene mutation D614G region from non-infected and COVID-19 patient's samples with different viral loads. For the detection, crude reactions prepared by RT-PCR followed by T7 exonuclease digestion were confronted with the S G614 sensor. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). Statistical analysis was performed using one-way ANOVA with Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Gold Nanoparticles based sensors’ functionality targeting the S gene mutation D614G region from non-infected and COVID-19 patient's samples with different viral loads. For the detection, crude reactions prepared by RT-PCR followed by T7 exonuclease digestion were confronted with the S G614 sensor. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). Statistical analysis was performed using one-way ANOVA with Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Mutagenesis, Infection, Reverse Transcription Polymerase Chain Reaction, Negative Control

    12) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    13) Product Images from "Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *"

    Article Title: Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.067439

    Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π.  B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown.  C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as  error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.
    Figure Legend Snippet: Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π. B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown. C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.

    Techniques Used: Clone Assay, Plasmid Preparation, Construct, End Labeling, Incubation, Ligation, Binding Assay, Mutagenesis

    14) Product Images from "5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs"

    Article Title: 5?CAG and 5?CTG Repeats Create Differential Impediment to the Progression of a Minimal Reconstituted T4 Replisome Depending on the Concentration of dNTPs

    Journal: Molecular Biology International

    doi: 10.4061/2011/213824

    Preparation of leading and lagging strand templates, p-t junctions and miniforks. The DNA fragments in the 100 base pair (bp) ladder are 100, 200, 300, 400, 500/517, 600, 700, 800, 900, and 1000 pbs. (a) The agarose gels stained by ethidium bromide (EtBr) show the products of the PCR obtained with the four plasmids (p-Empty, p-5′GTT16, p-5′CTG17, and p-5′CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand. The name of the plasmids used for the PCR is indicated at the top of the gels. (b) The agarose gels stained by EtBr show the products of the T7 exonuclease digestion. The PCR products obtained with p-5′CTG17 (CTG17) and p-5′CAG23 (CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand were treated (+) or not (−) by T7 exonuclease. After treatment with T7 exonuclease, the appearance of a DNA band with a slower electrophoretic migration and a weaker intensity (indicated by a backward arrow) than the ds DNA is indicative of ss DNA production. (c) The ss leading strand templates containing either 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) are mixed with increasing amounts of radiolabelled p821 (p821*) to generate the p-t junctions. Species are resolved on a native gel. Free p821 migrates faster than the p-t junctions. (d) The p-t junctions containing 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) on their leading strand are mixed with increasing amounts of ss lagging strand template to assemble the miniforks. Species are resolved on a native gel. The miniforks migrate more slowly than the p-t junctions.
    Figure Legend Snippet: Preparation of leading and lagging strand templates, p-t junctions and miniforks. The DNA fragments in the 100 base pair (bp) ladder are 100, 200, 300, 400, 500/517, 600, 700, 800, 900, and 1000 pbs. (a) The agarose gels stained by ethidium bromide (EtBr) show the products of the PCR obtained with the four plasmids (p-Empty, p-5′GTT16, p-5′CTG17, and p-5′CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand. The name of the plasmids used for the PCR is indicated at the top of the gels. (b) The agarose gels stained by EtBr show the products of the T7 exonuclease digestion. The PCR products obtained with p-5′CTG17 (CTG17) and p-5′CAG23 (CAG23) and the oligonucleotide couple specific of the leading (“PCR leading”; left) or the lagging (“PCR lagging”; right) strand were treated (+) or not (−) by T7 exonuclease. After treatment with T7 exonuclease, the appearance of a DNA band with a slower electrophoretic migration and a weaker intensity (indicated by a backward arrow) than the ds DNA is indicative of ss DNA production. (c) The ss leading strand templates containing either 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) are mixed with increasing amounts of radiolabelled p821 (p821*) to generate the p-t junctions. Species are resolved on a native gel. Free p821 migrates faster than the p-t junctions. (d) The p-t junctions containing 17 repeats of CTG (CTG17) or 23 repeats of (CAG23) on their leading strand are mixed with increasing amounts of ss lagging strand template to assemble the miniforks. Species are resolved on a native gel. The miniforks migrate more slowly than the p-t junctions.

    Techniques Used: Staining, Polymerase Chain Reaction, Migration, CTG Assay

    Strategy of the preparation of model replication miniforks. (a) The left and right sides of the figure correspond to the strategy used to build the ss leading and lagging strand templates of the minifork, respectively. The ss leading and lagging strand templates are combined with the radiolabelled p821 primer to assemble the minifork by strand hybridization. Replication miniforks are prepared in three consecutive steps. The first step (Step 1: PCR) consists of a PCR using plasmids containing a random or a TNR sequence (shown as a black rectangle) and oligonucleotides that flank the random sequence or the TNR unit. For each PCR, one of the oligonucleotides (p1033/4ps (colored in blue) for the preparation of the ss leading strand template, and (50T/4ps)/p867 (colored in red) for the preparation of the ss lagging strand template) carries 4 phosphorothioate linkages (represented as filled blue and red spheres for p1033/4ps and (50T/4ps)/p867, resp.) at its 5′ end. After PCR, the ds PCR products are digested by the T7 exonuclease that specifically degrades the DNA strand (colored in green or orange for the preparation of the ss leading or lagging strand template, resp.) that does not contain the phosphorothioate linkages (Step 2: T7 exonuclease digestion). The minifork (shown in a rounded rectangle) is assembled by hybridization of the ss leading and lagging strand templates and the radiolabelled p821 primer (in green) (Step 3: Hybridization). A gap of 15 nts exists between the 3′ end of the p821 primer and the base of the ss tail of the lagging strand template to facilitate the assembly of the DNA polymerase at the p-t junction. (b) A minifork containing n repeats of 5′CTG is shown. The repeats are located on the leading strand template.
    Figure Legend Snippet: Strategy of the preparation of model replication miniforks. (a) The left and right sides of the figure correspond to the strategy used to build the ss leading and lagging strand templates of the minifork, respectively. The ss leading and lagging strand templates are combined with the radiolabelled p821 primer to assemble the minifork by strand hybridization. Replication miniforks are prepared in three consecutive steps. The first step (Step 1: PCR) consists of a PCR using plasmids containing a random or a TNR sequence (shown as a black rectangle) and oligonucleotides that flank the random sequence or the TNR unit. For each PCR, one of the oligonucleotides (p1033/4ps (colored in blue) for the preparation of the ss leading strand template, and (50T/4ps)/p867 (colored in red) for the preparation of the ss lagging strand template) carries 4 phosphorothioate linkages (represented as filled blue and red spheres for p1033/4ps and (50T/4ps)/p867, resp.) at its 5′ end. After PCR, the ds PCR products are digested by the T7 exonuclease that specifically degrades the DNA strand (colored in green or orange for the preparation of the ss leading or lagging strand template, resp.) that does not contain the phosphorothioate linkages (Step 2: T7 exonuclease digestion). The minifork (shown in a rounded rectangle) is assembled by hybridization of the ss leading and lagging strand templates and the radiolabelled p821 primer (in green) (Step 3: Hybridization). A gap of 15 nts exists between the 3′ end of the p821 primer and the base of the ss tail of the lagging strand template to facilitate the assembly of the DNA polymerase at the p-t junction. (b) A minifork containing n repeats of 5′CTG is shown. The repeats are located on the leading strand template.

    Techniques Used: Hybridization, Polymerase Chain Reaction, Sequencing, CTG Assay

    15) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    16) Product Images from "A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity"

    Article Title: A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

    Journal: bioRxiv

    doi: 10.1101/548628

    Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p
    Figure Legend Snippet: Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p

    Techniques Used: Methylation

    17) Product Images from "Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks"

    Article Title: Enhanced bacterial immunity and mammalian genome editing via RNA polymerase-mediated dislodging of Cas9 from double strand DNA breaks

    Journal: bioRxiv

    doi: 10.1101/300962

    The Cas9-DSB complex precludes DNA repair activities  in vitro. A,  Agarose gel electrophoresis of linear dsDNA cut by Cas9, then treated with Proteinase K. B, E. coli  colony formation from circular plasmid DNA undergoing the indicated digestion and ligation conditions. Cas9 was denatured at 75C for 10m before addition of phage T4 DNA ligase. Values represent mean +/- s.d., n=3. C,  Agarose gel displaying plasmid DNA digested with PmeI restriction endonuclease or Cas9 before incubation with phage T7 exonuclease. D,  Schematic depicting the experiment in  E  testing if Ku70/80 can displace Cas9 from a DSB. The Cas9-DSB complex is formed on an immobilized, fluorescent substrate then challenged with purified Ku70/80. Disruption of the Cas9-DSB complex causes release of the fluorescent DNA end into the soluble fraction. E,  Release of fluorescently labeled DNA ends into the soluble fraction after challenging the immobilized target DNA with indicated conditions. NcoI is a restriction endonuclease that cuts the DNA substrate. Values represent mean +/- s.d., n=3.
    Figure Legend Snippet: The Cas9-DSB complex precludes DNA repair activities in vitro. A, Agarose gel electrophoresis of linear dsDNA cut by Cas9, then treated with Proteinase K. B, E. coli colony formation from circular plasmid DNA undergoing the indicated digestion and ligation conditions. Cas9 was denatured at 75C for 10m before addition of phage T4 DNA ligase. Values represent mean +/- s.d., n=3. C, Agarose gel displaying plasmid DNA digested with PmeI restriction endonuclease or Cas9 before incubation with phage T7 exonuclease. D, Schematic depicting the experiment in E testing if Ku70/80 can displace Cas9 from a DSB. The Cas9-DSB complex is formed on an immobilized, fluorescent substrate then challenged with purified Ku70/80. Disruption of the Cas9-DSB complex causes release of the fluorescent DNA end into the soluble fraction. E, Release of fluorescently labeled DNA ends into the soluble fraction after challenging the immobilized target DNA with indicated conditions. NcoI is a restriction endonuclease that cuts the DNA substrate. Values represent mean +/- s.d., n=3.

    Techniques Used: In Vitro, Agarose Gel Electrophoresis, Plasmid Preparation, Ligation, Incubation, Purification, Labeling

    18) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    19) Product Images from "BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *"

    Article Title: BRCA1 Localization to the Telomere and Its Loss from the Telomere in Response to DNA Damage *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.025825

    BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.
    Figure Legend Snippet: BRCA1 regulates 3′ G-strand overhang length; hybridization protection assay. A , standard curves of luminescence (relative units) versus genomic DNA input were obtained using an AE-labeled telomeric probe ( left ) or an AE-labeled AluDNA probe ( right ). Data are shown for DNA treated with or without Exo I, which removes single-stranded DNA. B–G , cells were treated with the indicated siRNAs and/or transfected overnight with wild-type ( wt ) BRCA1 or empty pcDNA3 vector, and genomic DNA (5 μg) was assayed to determine the ratio of luminescence (arbitrary units ( a.u. )) obtained using the telomeric and Alu probes. Controls using Exo I and, in some cases, negative controls ( no DNA ) are provided. C , a Western blot to document overexpression of BRCA1 in cells transfected with wild-type BRCA1. H , the telomeric probe signal for genomic DNA (5 μg) treated with T7 exonuclease (which digests duplex DNA, but not single-stranded DNA, in a 5′ to 3′ direction) for different time intervals. All data are means ± S.E. of three independent experiments.

    Techniques Used: Hybridization, Labeling, Transfection, Plasmid Preparation, Western Blot, Over Expression

    20) Product Images from "Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed"

    Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

    Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).
    Figure Legend Snippet: Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).

    Techniques Used: Purification, Plasmid Preparation, Migration, Concentration Assay, Activity Assay, Incubation, Produced

    21) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    22) Product Images from "Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection"

    Article Title: Development of colorimetric sensors based on gold nanoparticles for SARS-CoV-2 RdRp, E and S genes detection

    Journal: Talanta

    doi: 10.1016/j.talanta.2022.123393

    Gold Nanoparticles based sensors’ functionality targeting the gene regions E, R or S mutation D614G. For the detection, ssDNA purified amplicons were prepared by RT-PCR followed by T7 exonuclease digestion and confronted to the complementary loops included in MB E, R or S G614 (loop 25 nt) used functionalize 23 nm AuNPs. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). The concentration of targets used (from left to right) were 0, 50 nM, 100 and 250 nM. Statistical analysis was performed using one-way ANOVA Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Gold Nanoparticles based sensors’ functionality targeting the gene regions E, R or S mutation D614G. For the detection, ssDNA purified amplicons were prepared by RT-PCR followed by T7 exonuclease digestion and confronted to the complementary loops included in MB E, R or S G614 (loop 25 nt) used functionalize 23 nm AuNPs. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). The concentration of targets used (from left to right) were 0, 50 nM, 100 and 250 nM. Statistical analysis was performed using one-way ANOVA Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Mutagenesis, Purification, Reverse Transcription Polymerase Chain Reaction, Negative Control, Concentration Assay

    Gold Nanoparticles based sensors’ functionality targeting the S gene mutation D614G region from non-infected and COVID-19 patient's samples with different viral loads. For the detection, crude reactions prepared by RT-PCR followed by T7 exonuclease digestion were confronted with the S G614 sensor. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). Statistical analysis was performed using one-way ANOVA with Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Gold Nanoparticles based sensors’ functionality targeting the S gene mutation D614G region from non-infected and COVID-19 patient's samples with different viral loads. For the detection, crude reactions prepared by RT-PCR followed by T7 exonuclease digestion were confronted with the S G614 sensor. Data are presented as mean ± SD (n = 3) in terms of relative absorbance (%) vs negative control (without target). Statistical analysis was performed using one-way ANOVA with Tukey's test (pairwise comparison). Signif. Codes: 0 ‚***’ 0.001 ‚**’ 0.01 ‚*’ 0.05 ‚.’ 0.1 ‚ ‚ 1. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Mutagenesis, Infection, Reverse Transcription Polymerase Chain Reaction, Negative Control

    23) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    24) Product Images from "Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed"

    Article Title: Effective and robust plasmid topology analysis and the subsequent characterization of the plasmid isoforms thereby observed

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gnh124

    Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).
    Figure Legend Snippet: Fraction D analysis by AGE. ( A ) Separation of fraction D and open-circle species by both 0.6 and 0.4% AGE (left and right hand gel images, respectively). For 0.6% AGE, both the open-circle standard (lane 1) and parental purified plasmid (lane 2) were analysed. For 0.4% AGE, the AEC-collected fraction D (lane 1), the open-circle standard (lane 2) and the parental purified plasmid (lane 3) were analysed. The arrow doublet indicates the positions of fraction D and open-circle species resolved during 0.4% AGE. Such resolution is not achieved during 0.6% AGE and the position of species co-migration is indicated by a single arrow. ( B ) Analysis by chloroquine gel. AEC-collected fraction D (lane 1), open-circle standard (lane 2) and purified plasmid (lane 3) were analysed by 1D chloroquine-AGE. Brackets indicate the migration pattern and size of the differently linked sub-species existing in fraction D and the purified plasmid. As expected, the open-circle (nicked) derivative species migrates as a single, non-coiled species. Due to the low concentration of the fraction D collected by AEC combined with the reduced sensitivity of chloroquine-AGE (M. Uden, unpublished data), the separated, differently linked forms observed are of low image intensity. ( C ) Fraction D resistance to T7 exonuclease activity. A purified plasmid sample was incubated without (lane 2) or with (lane 3) T7 endonuclease prior to subsequent 0.4% AGE-based analysis. The arrow indicates the position of the open-circle species selectively degraded by T7 exonuclease. Also included (lane 1) is a supercoiled DNA ladder (Sigma), with visible markers (from top to bottom) of 16, 14, 12, 10, 8, 7, 6 and 5 kb. ( D ) Fraction D resolution by AGE in differently sized plasmids. Quadruplicate mini-preps of a 5.0 kb plasmid, a 4.5 kb plasmid and a parental 6.5 kb plasmid were made and then analysed by 0.6% AGE. An arrow doublet indicates the positions of fraction D and open-circle species in the 4.5 kb plasmid samples. These species are more readily resolved in the smaller 4.5 and 5.0 kb plasmids. A single arrow indicates the position of the open-circle/fraction D co-migration observed in the 6.5 kb plasmid samples. ( E ) Restriction enzyme mediated linearization of the plasmid. Aliquots of 800 ng of the parental 6.5 kb plasmid were digested with a linearizing enzyme for 0, 1, 2, 4, 8, 16, 32, 64 and 128 min in lanes 3–11, respectively. Also included is a 1 kb linear DNA ladder (lane1: with visible markers, from bottom to top, of 3–12 kb) and a −70°C stored plasmid reference standard (lane 2). Indicated are the positions of the supercoiled (SC) and linear (L) species. Note that general smearing is observed because of overloading (800 ng per lane). Such overloading is required so as to observe the faint linear species (indicated by an arrow) produced during the time-course and migrating as an estimated 13 kb species (if linear).

    Techniques Used: Purification, Plasmid Preparation, Migration, Concentration Assay, Activity Assay, Incubation, Produced

    25) Product Images from "Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes"

    Article Title: Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2009.06.009

    Backtracking of Trailing Polymerase Caused by Collision with Leading Polymerase (A) Experimental approach. A second G stop at position −27 relative to the first G stop was introduced. Drawn to scale. Numbers corresponding to each substrate also apply in (B) and (C). (B) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C–E) T7 exonuclease mapping of elongation complexes and models, as in Figure 3 .
    Figure Legend Snippet: Backtracking of Trailing Polymerase Caused by Collision with Leading Polymerase (A) Experimental approach. A second G stop at position −27 relative to the first G stop was introduced. Drawn to scale. Numbers corresponding to each substrate also apply in (B) and (C). (B) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C–E) T7 exonuclease mapping of elongation complexes and models, as in Figure 3 .

    Techniques Used: Polyacrylamide Gel Electrophoresis

    Exonuclease Mapping of Trailing Polymerase after Collision (A) (Box) Experimental approach. (Lower) Mono- and dielongation complexes with 3′ end-labeled, nontranscribed strand DNA were incubated with NTPs (or with NTPs and TFIIS), digested with T7 exonuclease, and analyzed by denaturing PAGE. DNA fragment sizes are indicated to the left. The position of the elongation complexes relative to the active site of the leading polymerase (−82, −79, etc.) is indicated on the right. Grey arrow indicates distance between the active site of the elongation complex at the G stop and the position of that same elongation complex as marked by T7 exonuclease. Double arrows (black) mark distances between elongation complexes, and thin lines between lanes indicate major exonuclease stops (see text for details). (B) (Box) Experimental design. (Right) Transcription products resolved by denaturing PAGE as in  Figure 2 C. (C) Relationship between lengths of transcripts and positions of trailing elongation complex as marked by exonuclease. Angled line indicates forward translocation toward the point of collision. Horizontal lines with arrows indicate backtracking, and gray, vertical lines with arrows indicate transcript cleavage and the return of RNAPII to forward translocation. (D) Summary of results, drawn to scale. Distances between elongation complexes are indicated.
    Figure Legend Snippet: Exonuclease Mapping of Trailing Polymerase after Collision (A) (Box) Experimental approach. (Lower) Mono- and dielongation complexes with 3′ end-labeled, nontranscribed strand DNA were incubated with NTPs (or with NTPs and TFIIS), digested with T7 exonuclease, and analyzed by denaturing PAGE. DNA fragment sizes are indicated to the left. The position of the elongation complexes relative to the active site of the leading polymerase (−82, −79, etc.) is indicated on the right. Grey arrow indicates distance between the active site of the elongation complex at the G stop and the position of that same elongation complex as marked by T7 exonuclease. Double arrows (black) mark distances between elongation complexes, and thin lines between lanes indicate major exonuclease stops (see text for details). (B) (Box) Experimental design. (Right) Transcription products resolved by denaturing PAGE as in Figure 2 C. (C) Relationship between lengths of transcripts and positions of trailing elongation complex as marked by exonuclease. Angled line indicates forward translocation toward the point of collision. Horizontal lines with arrows indicate backtracking, and gray, vertical lines with arrows indicate transcript cleavage and the return of RNAPII to forward translocation. (D) Summary of results, drawn to scale. Distances between elongation complexes are indicated.

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Translocation Assay

    TFIIS Induces Oscillation of Trailing Polymerase upon Collision with Leading Polymerase (A) (Box) Experimental design. (Right) Unlabeled elongation complexes were incubated with ATP/UTP/GTP and α- 32 P GTP. After 5 min, TFIIS was added (0.2 and 1 molar ratios to RNAPII). Products were analyzed by 20% denaturing PAGE. Length of transcripts generated by leading polymerase (L, on left side) and trailing polymerase (T, on the right), and the expected number of guanines in these transcripts (in brackets), are indicated. (B) Depiction of the situation with a 26 nt (upper, T26), and with a 5 nt (lower, T5) long cleaved nascent RNA, an alternative view to that shown in Figure 5 D. Numbers indicate positions relative to that of the leading polymerase's active site. Long arrows, position of cleavage induced by TFIIS. Short arrows, position of T7 exonuclease footprint.
    Figure Legend Snippet: TFIIS Induces Oscillation of Trailing Polymerase upon Collision with Leading Polymerase (A) (Box) Experimental design. (Right) Unlabeled elongation complexes were incubated with ATP/UTP/GTP and α- 32 P GTP. After 5 min, TFIIS was added (0.2 and 1 molar ratios to RNAPII). Products were analyzed by 20% denaturing PAGE. Length of transcripts generated by leading polymerase (L, on left side) and trailing polymerase (T, on the right), and the expected number of guanines in these transcripts (in brackets), are indicated. (B) Depiction of the situation with a 26 nt (upper, T26), and with a 5 nt (lower, T5) long cleaved nascent RNA, an alternative view to that shown in Figure 5 D. Numbers indicate positions relative to that of the leading polymerase's active site. Long arrows, position of cleavage induced by TFIIS. Short arrows, position of T7 exonuclease footprint.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Generated

    Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.
    Figure Legend Snippet: Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.

    Techniques Used: Labeling, Marker, Incubation, Modification, Polyacrylamide Gel Electrophoresis, Isolation, Sequencing, Footprinting

    Transcription Bubbles Remain Intact and Separated upon Collision (A) Experimental design. (B) Elongation complexes (end-labeled NTS) were incubated with NTPs and TFIIS, digested with T7 exonuclease, and modified with increasing amounts of KMnO 4  (12, 18, and 24 mM) as indicated and resolved by denaturing PAGE. Major T7 exonuclease-marked bands as seen in  Figure 3 , lane 17, line, were isolated, cleaved at modified thymines with piperidine, and analyzed by denaturing PAGE. The positions of isolated bands are indicated at the top. Thin lines between lanes on the autoradiograph indicate transcription bubbles. Naked NTS (6 mM KMnO 4  footprinting) is shown in lane 16. Important distances and positions are indicated on the right. Arrowheads indicate the respective active sites. (C) As in (B), but both in the absence and presence of TFIIS, modified with increasing amounts of permanganate (4, 8, and 18 mM). (D) Summary of results obtained in the absence (upper) and presence (lower) of TFIIS, drawn to scale.
    Figure Legend Snippet: Transcription Bubbles Remain Intact and Separated upon Collision (A) Experimental design. (B) Elongation complexes (end-labeled NTS) were incubated with NTPs and TFIIS, digested with T7 exonuclease, and modified with increasing amounts of KMnO 4 (12, 18, and 24 mM) as indicated and resolved by denaturing PAGE. Major T7 exonuclease-marked bands as seen in Figure 3 , lane 17, line, were isolated, cleaved at modified thymines with piperidine, and analyzed by denaturing PAGE. The positions of isolated bands are indicated at the top. Thin lines between lanes on the autoradiograph indicate transcription bubbles. Naked NTS (6 mM KMnO 4 footprinting) is shown in lane 16. Important distances and positions are indicated on the right. Arrowheads indicate the respective active sites. (C) As in (B), but both in the absence and presence of TFIIS, modified with increasing amounts of permanganate (4, 8, and 18 mM). (D) Summary of results obtained in the absence (upper) and presence (lower) of TFIIS, drawn to scale.

    Techniques Used: Labeling, Incubation, Modification, Polyacrylamide Gel Electrophoresis, Isolation, Autoradiography, Footprinting

    26) Product Images from "Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA"

    Article Title: Single-strand RPA for rapid and sensitive detection of SARS-CoV-2 RNA

    Journal: medRxiv

    doi: 10.1101/2020.08.17.20177006

    ssRPA assay design, workflow, and characterization. (a) Key to ssRPA design is the rapid generation of many ssDNA copies from a single RNA target. ssDNA output offers straightforward specific readout by fluorescence or colorimetric/visual methods such as lateral flow devices. (b) Step 1: Target viral RNA region (of domains a-b-c-d ) is reverse transcribed into cDNA via extension of the reverse primer ( d* ) by the Reverse Transcriptase in the reaction mixture 48 . Subsequently, the cDNA is amplified via isothermal RPA at 42 °C by templated extension of the forward ( a ) and reverse primers ( d* ). The forward primer has a 6-nucleotide long poly-T segment with phosphorothioate bonds. Step 2a: Products of RPA are amended with SDS and transferred to the exo/LFD buffer that contains T7 exonuclease (a dsDNA-specific 5′ to 3′ exonuclease) and detection probes. The resulting mixture is incubated for 1 min at ambient temperature and reverse strand of the dsDNA amplicon products get preferentially digested yielding ssDNA amplicon ( a-b-c-d ) homologous to the target RNA sequence. Step 2b: The 3′ biotin ( b* ) and 5′ FAM ( c* ) modified detection probes, already added, hybridize to target sequences. These probes make the assay directly compatible with commercially available test strips that feature a biotin-binding test line and gold nanoparticles conjugated to rabbit anti-FAM IgG on the conjugate pad. Step 3: The test strip is vertically inserted into the resulting 100 µl mixture. The right ssDNA amplicon acts as a bridge that binds both the biotin-probe and the FAM-probe independently resulting in immobilization of the complex at the test line, where formation of a colored line indicates a positive result. The control line formed of rabbit secondary antibodies captures the remaining gold nanoparticle conjugates by binding to rabbit anti-FAM IgG. (c) Timeline of the assay shows the incubation conditions and duration of the 3 main steps in ssRPA: (1) RT-RPA, (2) exonuclease digestion and (3) lateral flow. The test line and control line can be visualized as early as 1-2 min or as late as 60 + min without false positives. (d) Sensitivity of ssRPA-LFD demonstrated by serial dilution of synthetic SARS-CoV-2 full genome standard (Twist) from ∼ 1,000,000 copies down to ∼ 2 copies per reaction. A 5 µl volume of genomic viral RNA in DNase/RNase-free water was used as input for a 50 µl reaction volume. After RT-RPA for 5 minutes at 42 °C, 8 µl product was mixed with 12 µl of 10% SDS and then transferred into 80 µl of exo/LFD buffer. Following 1 min T7 exonuclease digestion at room temperature, samples were applied to commercial HybriDetect strips for ≥ 1 min. A time series for the same strip is shown in each column. Note the zero test line signal in negative controls, even at 60 min. (e) Using the same procedure as in (d), but spiking 8 copies cultured, heat inactivated SARS-CoV-2 virus (BEI) into 5 µl of SARS-CoV-2-negative human saliva in 20 repeats. No-template negative controls are also shown. ( f ) Specificity was shown by testing 8 other respiratory virus genomic samples, including genomically similar coronaviruses 229E, MERS, (2003) SARS-CoV, and NL63, and alternative diagnoses of influenza B, influenza A, respiratory syncytial virus (RSV), and rhinovirus 17, each at > 10 5 copies per assay and spiked in DNase/RNase-free water. A sample with virus-derived SARS-CoV-2 RNA at low copy is shown as a positive control. Strips show the readout at 10 min of lateral flow. (g) Clinical samples were taken as NP swabs in VTM, NP swabs in water, or saliva, from SARS-CoV-2 positive and negative patients alike, and heat inactivated at vendor for safety. Sample order was randomized prior to ssRPA, and technician was blinded as to patient type or qPCR status to avoid experimental bias. During assay, samples further underwent a 2 min extraction protocol with Lucigen DNA extraction buffer at 50% dilution and 95 °C, and were tested at 10% v/v into ssRPA for 5′ spike targets (see Methods). Non-extracted samples yielded little or no signal ( Fig. S6 ). Presumptive diagnosis from supplier testing is noted. Concomitant qPCR quantification in our lab, using CDC N2 primers from buffer-exchanged samples, is shown with each result (See also Fig. S7 ).
    Figure Legend Snippet: ssRPA assay design, workflow, and characterization. (a) Key to ssRPA design is the rapid generation of many ssDNA copies from a single RNA target. ssDNA output offers straightforward specific readout by fluorescence or colorimetric/visual methods such as lateral flow devices. (b) Step 1: Target viral RNA region (of domains a-b-c-d ) is reverse transcribed into cDNA via extension of the reverse primer ( d* ) by the Reverse Transcriptase in the reaction mixture 48 . Subsequently, the cDNA is amplified via isothermal RPA at 42 °C by templated extension of the forward ( a ) and reverse primers ( d* ). The forward primer has a 6-nucleotide long poly-T segment with phosphorothioate bonds. Step 2a: Products of RPA are amended with SDS and transferred to the exo/LFD buffer that contains T7 exonuclease (a dsDNA-specific 5′ to 3′ exonuclease) and detection probes. The resulting mixture is incubated for 1 min at ambient temperature and reverse strand of the dsDNA amplicon products get preferentially digested yielding ssDNA amplicon ( a-b-c-d ) homologous to the target RNA sequence. Step 2b: The 3′ biotin ( b* ) and 5′ FAM ( c* ) modified detection probes, already added, hybridize to target sequences. These probes make the assay directly compatible with commercially available test strips that feature a biotin-binding test line and gold nanoparticles conjugated to rabbit anti-FAM IgG on the conjugate pad. Step 3: The test strip is vertically inserted into the resulting 100 µl mixture. The right ssDNA amplicon acts as a bridge that binds both the biotin-probe and the FAM-probe independently resulting in immobilization of the complex at the test line, where formation of a colored line indicates a positive result. The control line formed of rabbit secondary antibodies captures the remaining gold nanoparticle conjugates by binding to rabbit anti-FAM IgG. (c) Timeline of the assay shows the incubation conditions and duration of the 3 main steps in ssRPA: (1) RT-RPA, (2) exonuclease digestion and (3) lateral flow. The test line and control line can be visualized as early as 1-2 min or as late as 60 + min without false positives. (d) Sensitivity of ssRPA-LFD demonstrated by serial dilution of synthetic SARS-CoV-2 full genome standard (Twist) from ∼ 1,000,000 copies down to ∼ 2 copies per reaction. A 5 µl volume of genomic viral RNA in DNase/RNase-free water was used as input for a 50 µl reaction volume. After RT-RPA for 5 minutes at 42 °C, 8 µl product was mixed with 12 µl of 10% SDS and then transferred into 80 µl of exo/LFD buffer. Following 1 min T7 exonuclease digestion at room temperature, samples were applied to commercial HybriDetect strips for ≥ 1 min. A time series for the same strip is shown in each column. Note the zero test line signal in negative controls, even at 60 min. (e) Using the same procedure as in (d), but spiking 8 copies cultured, heat inactivated SARS-CoV-2 virus (BEI) into 5 µl of SARS-CoV-2-negative human saliva in 20 repeats. No-template negative controls are also shown. ( f ) Specificity was shown by testing 8 other respiratory virus genomic samples, including genomically similar coronaviruses 229E, MERS, (2003) SARS-CoV, and NL63, and alternative diagnoses of influenza B, influenza A, respiratory syncytial virus (RSV), and rhinovirus 17, each at > 10 5 copies per assay and spiked in DNase/RNase-free water. A sample with virus-derived SARS-CoV-2 RNA at low copy is shown as a positive control. Strips show the readout at 10 min of lateral flow. (g) Clinical samples were taken as NP swabs in VTM, NP swabs in water, or saliva, from SARS-CoV-2 positive and negative patients alike, and heat inactivated at vendor for safety. Sample order was randomized prior to ssRPA, and technician was blinded as to patient type or qPCR status to avoid experimental bias. During assay, samples further underwent a 2 min extraction protocol with Lucigen DNA extraction buffer at 50% dilution and 95 °C, and were tested at 10% v/v into ssRPA for 5′ spike targets (see Methods). Non-extracted samples yielded little or no signal ( Fig. S6 ). Presumptive diagnosis from supplier testing is noted. Concomitant qPCR quantification in our lab, using CDC N2 primers from buffer-exchanged samples, is shown with each result (See also Fig. S7 ).

    Techniques Used: Fluorescence, Amplification, Recombinase Polymerase Amplification, Incubation, Sequencing, Modification, Binding Assay, Stripping Membranes, Serial Dilution, Cell Culture, Derivative Assay, Positive Control, Real-time Polymerase Chain Reaction, DNA Extraction

    Gel and full-length LFDs of main text Fig. 1g . ( a ) A denaturing PAGE gel showing the results of a 5 min RT-RPA and subsequent SDS addition, 1 min T7 exonuclease digestions, and addition of LFD biotin and FAM probes, for the clinical samples shown in the first of each triplicate of main text Fig. 1g . ( b ) Full length LFD strips from main text Fig. 1g .
    Figure Legend Snippet: Gel and full-length LFDs of main text Fig. 1g . ( a ) A denaturing PAGE gel showing the results of a 5 min RT-RPA and subsequent SDS addition, 1 min T7 exonuclease digestions, and addition of LFD biotin and FAM probes, for the clinical samples shown in the first of each triplicate of main text Fig. 1g . ( b ) Full length LFD strips from main text Fig. 1g .

    Techniques Used: Polyacrylamide Gel Electrophoresis, Recombinase Polymerase Amplification

    Gels and full-length images of strips in main text Figure 1d . ( a ) A denaturing PAGE gel showing the results of a 5 min RT-RPA for the series dilution of synthetic SARS-CoV-2 RNA shown in main text Figure 1d . Results show strong product bands over a large dynamic range of copy number. ( b , next page) Full length LFD strips from main text Figure 1d , in which RT-RPA amplicons were subsequently treated via SDS addition, 1 minute T7 exonuclease digestion, and addition of LFD biotin and FAM probes.
    Figure Legend Snippet: Gels and full-length images of strips in main text Figure 1d . ( a ) A denaturing PAGE gel showing the results of a 5 min RT-RPA for the series dilution of synthetic SARS-CoV-2 RNA shown in main text Figure 1d . Results show strong product bands over a large dynamic range of copy number. ( b , next page) Full length LFD strips from main text Figure 1d , in which RT-RPA amplicons were subsequently treated via SDS addition, 1 minute T7 exonuclease digestion, and addition of LFD biotin and FAM probes.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Recombinase Polymerase Amplification

    27) Product Images from "DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway"

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv969

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.
    Figure Legend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Techniques Used: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining

    28) Product Images from "ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells"

    Article Title: ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid cells

    Journal: bioRxiv

    doi: 10.1101/2022.06.01.494308

    Optimization of long ssDNA production using T7 exonuclease and restriction enzymes. a , Schematic of long ssDNA production using T7 exonuclease (one-step method). dsDNA is amplified by PCR with a pair of long primers containing a 90 nt HA, one of which bears five consecutive PS bonds at the 5’ end. The strand with the non-modified 5’ end of the dsDNA is selectively digested by T7 exonuclease to produce long ssDNA donors. b , T7 exonuclease reaction on dsDNA amplified using three different combinations of primers (PS-modified (PS) or non-modified (noPS) for the forward and reverse primers). The bottom image is of the same gel as the top one, with higher brightness and contrast. ssDNA shows higher mobility than dsDNA of the same length. Asterisks show undigested dsDNA remnants. c , Schematic of ssDNA production by two-step PCR and T7 exonuclease (T7 method). The first PCR uses long non-modified primers to add the HAs, and the second uses a combination of short primers, one of which is PS-modified. d , “PS-PS” dsDNA was prepared with one-step or two-step PCR and subsequently subjected to the T7 exonuclease reaction. Plot profiles for each lane are shown below the gel electrophoresis images. e , Production of long ssDNA donors using the one-step and the two-step methods. The bottom image is of the same gel as the top one, with higher brightness and contrast. f , Schematic of ssDNA production using T7 exonuclease and restriction enzymes (T7RE method). After two-step PCR and T7 exonuclease reaction, the indicated four restriction enzymes digest dsDNA remnants to produce short dsDNA fragments which can be further degraded by T7 exonuclease. g , ssDNA production by the T7 and the T7RE methods. The last two lanes contain column-purified DNA products of both reactions. Plot profiles for the last two lanes are shown below the gel electrophoresis image.
    Figure Legend Snippet: Optimization of long ssDNA production using T7 exonuclease and restriction enzymes. a , Schematic of long ssDNA production using T7 exonuclease (one-step method). dsDNA is amplified by PCR with a pair of long primers containing a 90 nt HA, one of which bears five consecutive PS bonds at the 5’ end. The strand with the non-modified 5’ end of the dsDNA is selectively digested by T7 exonuclease to produce long ssDNA donors. b , T7 exonuclease reaction on dsDNA amplified using three different combinations of primers (PS-modified (PS) or non-modified (noPS) for the forward and reverse primers). The bottom image is of the same gel as the top one, with higher brightness and contrast. ssDNA shows higher mobility than dsDNA of the same length. Asterisks show undigested dsDNA remnants. c , Schematic of ssDNA production by two-step PCR and T7 exonuclease (T7 method). The first PCR uses long non-modified primers to add the HAs, and the second uses a combination of short primers, one of which is PS-modified. d , “PS-PS” dsDNA was prepared with one-step or two-step PCR and subsequently subjected to the T7 exonuclease reaction. Plot profiles for each lane are shown below the gel electrophoresis images. e , Production of long ssDNA donors using the one-step and the two-step methods. The bottom image is of the same gel as the top one, with higher brightness and contrast. f , Schematic of ssDNA production using T7 exonuclease and restriction enzymes (T7RE method). After two-step PCR and T7 exonuclease reaction, the indicated four restriction enzymes digest dsDNA remnants to produce short dsDNA fragments which can be further degraded by T7 exonuclease. g , ssDNA production by the T7 and the T7RE methods. The last two lanes contain column-purified DNA products of both reactions. Plot profiles for the last two lanes are shown below the gel electrophoresis image.

    Techniques Used: Amplification, Polymerase Chain Reaction, Modification, Nucleic Acid Electrophoresis, Purification

    29) Product Images from "ParB spreading on DNA requires cytidine triphosphate in vitro"

    Article Title: ParB spreading on DNA requires cytidine triphosphate in vitro

    Journal: bioRxiv

    doi: 10.1101/2019.12.11.865972

    Dual biotin-labeled DNA fragments form a closed substrate on the surface of the BLI probe. (A) A schematic of a double digestion assay using Exonuclease T7 + Exonuclease VII and PCR. PCR was performed using M13F, M13R oligos, and DNA attached to the BLI surface as a template. (B) The BLI probe was severed from the plastic adaptor and immerged into a PCR master mix. (C) Dual biotin-labeled DNA fragments on the BLI surface were resistant to Exo T7 + Exo VII digestion while single biotin-labeled DNA fragments on the BLI surface were not.
    Figure Legend Snippet: Dual biotin-labeled DNA fragments form a closed substrate on the surface of the BLI probe. (A) A schematic of a double digestion assay using Exonuclease T7 + Exonuclease VII and PCR. PCR was performed using M13F, M13R oligos, and DNA attached to the BLI surface as a template. (B) The BLI probe was severed from the plastic adaptor and immerged into a PCR master mix. (C) Dual biotin-labeled DNA fragments on the BLI surface were resistant to Exo T7 + Exo VII digestion while single biotin-labeled DNA fragments on the BLI surface were not.

    Techniques Used: Labeling, Polymerase Chain Reaction

    30) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    31) Product Images from "Impact of DNA ligase IV on the fidelity of end joining in human cells"

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    Journal: Nucleic Acids Research

    doi:

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.
    Figure Legend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Techniques Used: Incubation, Labeling, Electrophoresis, Autoradiography

    32) Product Images from "TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA"

    Article Title: TIN2 is an architectural protein that facilitates TRF2-mediated trans- and cis-interactions on telomeric DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkab1142

    TIN2 facilitates TRF2-mediated T-loop formation. ( A ) Blocking and marking of the 3′ overhang at the genomic DNA end on the linear T270 DNA substrate (5.4 kb). Position distribution of strep-QDs on the linear T270 DNA after treatment with T7 exonuclease and annealing of a biotinylated primer to the 3′ overhang at the genomic DNA end. Insert: an AFM image of T7 exonuclease-treated linear T270 DNA with a strep-QD at one DNA end; XY scale bar = 100 nm. ( B ) AFM images of T-loops formed on the linear T270 DNA with the nontelomeric end labeled with QDs in the presence of TRF2 (25 nM dimer); XY scale bar = 100 nm. ( C ) Percentages of T-loop formation on the linear T270 DNA with a 3′ overhang at different TRF2 concentrations. TRF2 dimer concentrations tested: 12.5 nM ( N = 832, 3.1%), 18 nM ( N = 1114, 3.5%), 20 nM ( N = 445, 7.0%), 25 nM ( N = 332, 10.2%), 30 nM ( N = 276, 5.4%), 35.5 nM ( N = 269, 6.7%), 40 nM ( N = 88, 4.5%). ( D ) AFM images of T-loop formed in the presence of TRF2-TIN2L on the linear T270 DNA with a 3′ overhang; XY scale bar = 100 nm. ( E ) Percentages of T270 DNA molecules with loop formation at a fixed TRF2 concentration (25 nM dimer) and increasing concentrations of TIN2L. No overhang control DNA: T270 DNA without T7 exonuclease treatment. All other data sets: T270 DNA with T7 exonuclease treatment. The data were pooled from at least three independent experiments. N = 912–2463 DNA molecules for each condition.
    Figure Legend Snippet: TIN2 facilitates TRF2-mediated T-loop formation. ( A ) Blocking and marking of the 3′ overhang at the genomic DNA end on the linear T270 DNA substrate (5.4 kb). Position distribution of strep-QDs on the linear T270 DNA after treatment with T7 exonuclease and annealing of a biotinylated primer to the 3′ overhang at the genomic DNA end. Insert: an AFM image of T7 exonuclease-treated linear T270 DNA with a strep-QD at one DNA end; XY scale bar = 100 nm. ( B ) AFM images of T-loops formed on the linear T270 DNA with the nontelomeric end labeled with QDs in the presence of TRF2 (25 nM dimer); XY scale bar = 100 nm. ( C ) Percentages of T-loop formation on the linear T270 DNA with a 3′ overhang at different TRF2 concentrations. TRF2 dimer concentrations tested: 12.5 nM ( N = 832, 3.1%), 18 nM ( N = 1114, 3.5%), 20 nM ( N = 445, 7.0%), 25 nM ( N = 332, 10.2%), 30 nM ( N = 276, 5.4%), 35.5 nM ( N = 269, 6.7%), 40 nM ( N = 88, 4.5%). ( D ) AFM images of T-loop formed in the presence of TRF2-TIN2L on the linear T270 DNA with a 3′ overhang; XY scale bar = 100 nm. ( E ) Percentages of T270 DNA molecules with loop formation at a fixed TRF2 concentration (25 nM dimer) and increasing concentrations of TIN2L. No overhang control DNA: T270 DNA without T7 exonuclease treatment. All other data sets: T270 DNA with T7 exonuclease treatment. The data were pooled from at least three independent experiments. N = 912–2463 DNA molecules for each condition.

    Techniques Used: Blocking Assay, Labeling, Concentration Assay

    33) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    34) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    35) Product Images from "Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *"

    Article Title: Investigations of ? Initiator Protein-mediated Interaction between Replication Origins ? and ? of the Plasmid R6K *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.067439

    Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π.  B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown.  C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as  error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.
    Figure Legend Snippet: Determination of the frequency of DNA looping by enhancement of ligase-catalyzed DNA circularization. A , schematic depiction of the circularization assay showing the pUC19-based substrate that contained γ-γ or α-γ iterons cloned at the indicated locations on either side of a unique EcoR1 site. A similar plasmid having a single γ iteron on one side and the 7 γ iterons on the other was also constructed. The assay steps including linearization by EcoR1, 5′-end labeling, incubation with either π cop, or with a 1:1 mixture of wt π and π cop, ligation with T4 DNA ligase at 16 °C for various periods of time, and digestion with T7 gene 6 exonuclease are shown; 20 fmol of DNA were incubated with a total of 128 pmol of π. B , DNA circularization kinetics showing that there was a small difference between γ-γ and γ-7 γ iteron interactions; the control experiment with a solo γ iteron is also shown. C , DNA circularization kinetics with various combinations of DNA substrates and π proteins as indicated. The α-γ plasmid incubated with a 1:1 mixture of wt π and π cop had a small but distinct and reproducible enhancement in ligation kinetics over the same incubated with π cop only. The γ-γ plasmid incubated with π monomer-dimer mixture had a much higher rate of DNA circularization over that when only π cop was used. Data were collected from six separate sets of experiments and are presented with the S.D. as error bars. D , ligation experiments showing that π D226A (non-DNA binding mutant dimers) did not complement π cop to loop two γ-γ iterons. Controls show that under identical conditions, wt π complemented π cop in promoting γ-γ interaction, and there was no interaction as expected when π was withheld from the reaction.

    Techniques Used: Clone Assay, Plasmid Preparation, Construct, End Labeling, Incubation, Ligation, Binding Assay, Mutagenesis

    36) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    37) Product Images from "A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity"

    Article Title: A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

    Journal: bioRxiv

    doi: 10.1101/548628

    Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p
    Figure Legend Snippet: Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p

    Techniques Used: Methylation

    38) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

    39) Product Images from "Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes"

    Article Title: Stability, Flexibility, and Dynamic Interactions of Colliding RNA Polymerase II Elongation Complexes

    Journal: Molecular Cell

    doi: 10.1016/j.molcel.2009.06.009

    Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.
    Figure Legend Snippet: Elongation Complex Footprint and Mapping of the Transcription Bubble (A) (Upper) Summary of elongation complex preparation, drawn to scale. (Lower) Elongation complexes with 5′ end-labeled, 131-mer transcribed DNA were digested with increasing amounts of DNase I, as indicated. The footprint region where DNase I cleavage is repressed (bold line) and a hypersensitive site (sphere) are indicated on the right. M, thymine-specific cleavage marker. Elongation complex (oval) and RNA (bold line) are indicated on the left. Position of the active site is taken as +1. (B) (Upper) Schematic diagram of the locations of the RNA primer (bold text) and G stop (gray box) in the elongation complex. The 150-mer transcribed strand (TS), the corresponding end-labeled nontranscribed strand (NTS), and the direction of exonuclease digestion are indicated. (Lower) Elongation complexes incubated alone, or with ATP/UTP/GTP and TFIIS, were digested with T7 exonuclease to mark their position(s), modified with increasing amounts of KMnO 4 (2 and 8 mM), and analyzed by denaturing PAGE (lanes 1–4). Bands corresponding to the major elongation complexes (2 mM KMnO 4 experiment shown) were isolated, cleaved at modified thymines with piperidine, and resolved by denaturing PAGE (lanes 5 and 6). DNA sequence is shown on the left. Spheres, modified thymines. Arrowheads, positions of active sites. (C) Crystal structure of the elongation complex with modeled DNA paths (dashed lines). Space-filling model shows the DNA (color) engulfed in protein bulk. (D) Area protected by RNAPII (DNase I footprinting, solid line; crystallography model, dashed line) and their transcription bubbles. Only reconstituted elongation complex was mapped by DNase I footprinting. However, T7 exonuclease digests to the same distance from the edge of the two footprints (±1 base; positions indicated by vertical arrow) before and after transcription, showing that RNAPII moved forward upon NTP addition.

    Techniques Used: Labeling, Marker, Incubation, Modification, Polyacrylamide Gel Electrophoresis, Isolation, Sequencing, Footprinting

    40) Product Images from "Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *"

    Article Title: Anti-apoptotic Protein BCL2 Down-regulates DNA End Joining in Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.140350

    T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.
    Figure Legend Snippet: T7 exonuclease digestion to determine the topology of end-joined products. A , diagrammatic representation of plausible end joined products by cell-free extracts. B , T7 exonuclease and XhoI digestion followed by T7 exonuclease digestion pattern of end-joined products of K562 cell-free extracts. Multiple end-joining reactions were carried out using 5′ compatible substrates, and products were incubated with either T7 exonuclease (5 units/10-μl reaction for 2 h), XhoI, or both. The products were resolved on an 8% denaturing PAGE. M , 50-nt ladder.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis

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    New England Biolabs t7 exo
    The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts.  (A)  Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of  32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.
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    The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts.  (A)  Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of  32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.

    Journal: Nucleic Acids Research

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair

    doi: 10.1093/nar/gkw274

    Figure Lengend Snippet: The Mre11 nuclease activity is critical for the resection of DNA with 5′ bulky adducts. (A) Effect of Mre11 depletion on the resection of 5′ p-Tyr and 5′ avidin DNA. The substrates were incubated in mock-depleted or Mre11-depleted extracts and the products were analyzed on a 1% TAE-agarose gel. ( B ) Plots of the amounts of 32 P on the remaining substrates at the indicated times. ( C ) Detection of 5′ biotin on the resection intermediates of 5′ avidin DNA. Control: untreated substrate. Mock and –Mre11: intermediates isolated after 30 min in the indicated extracts. They were pre-incubated with buffer or avidin, and then treated with T7 Exo. (T7 Exo falls off DNA once the two enzyme molecules meet in the middle, resulting in the accumulation of ss-DNA of the 3′ half). The reactions also contained a plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, and dried for exposure to X-ray film.

    Article Snippet: To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min.

    Techniques: Activity Assay, Avidin-Biotin Assay, Incubation, Agarose Gel Electrophoresis, Isolation, Plasmid Preparation, Staining

    Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Journal: Nucleic Acids Research

    Article Title: Impact of DNA ligase IV on the fidelity of end joining in human cells

    doi:

    Figure Lengend Snippet: Ku and DNA ligase IV–XRCC4 separately and in combination can protect against T7 exonuclease digestion. T7 exonuclease (5 U) in the absence or presence of Ku and/or DNA ligase IV–XRCC4, as indicated, was incubated with an end-labeled substrate having cohesive ends (top) or blunt ends (bottom), and the products were analyzed by electrophoresis and autoradiography. The results shown are representive of three experiments undertaken, and the percentage recovery of the label represents the mean of the three experiments. Lane 1, control substrate; lane 2, substrate + 5 U of T7 exonuclease; lanes 3–8, substrate with T7 exonuclease and Ku and/or DNA ligase IV–XRCC4 as indicated.

    Article Snippet: We also show that DNA ligase IV–XRCC4 can contribute to the protection of ends from degradation by T7 exonuclease.

    Techniques: Incubation, Labeling, Electrophoresis, Autoradiography

    Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p

    Journal: bioRxiv

    Article Title: A universal fluorescence-based toolkit for real-time quantification of DNA and RNA nuclease activity

    doi: 10.1101/548628

    Figure Lengend Snippet: Increased methylcytosine content delays ExoIII-mediated resection; but does not affect the rate of resection of either ExoIII or T7 Exonuclease a, 5 nM and b, 10 nM ExoIII was added to a non-methylated substrate, a substrate containing one methylated cytosine, and a substrate containing four methylated cytosines (*S, * S 1M and * S 4M , respectively). Standard curve is represented by the grey dotted lines. c, Time (seconds) until the ExoIII reaction reaches completion on the methylated and unmethylated substrates based on the point at which the graphs plateau in (a) and (b). d, Calculated resection rate of ExoIII based on maximum gradients in (a) and (b). e, 6 nM and f, 12 nM T7 Exo on non-methylated and differentially methylated substrates. Standard curve is represented by the grey dotted lines. g, Time (seconds) until the T7 Exo reaction reaches completion on the methylated and unmethylated substrates based on point at which the graphs plateau in (e) and (f). h, Calculated resection rate of ExoIII. Error bars represent SEM; n=3 in all cases; *p

    Article Snippet: Nucleases and PG preparationThe nucleases used were RQ1 RNase-Free DNase I (Promega), T7 exonuclease (New England Biolabs), Exonuclease III (New England Biolabs), Trex2 (Stratech), Klenow Fragment (3’ → 5’ exo-; New England Biolabs), RNase A (Thermo Fisher) and RNase H (New England Biolabs).

    Techniques: Methylation

    DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Journal: Nucleic Acids Research

    Article Title: DNA double-strand breaks with 5′ adducts are efficiently channeled to the DNA2-mediated resection pathway

    doi: 10.1093/nar/gkv969

    Figure Lengend Snippet: DNA2 does not affect the removal of avidin from the 5′ end. The resection intermediates were isolated after 30 min in the mock or DNA2-depleted extracts. They and the substrate were treated with T7 Exo in the presence of absence of avidin. The reactions also contained a linear plasmid (pUC) to serve as a control for digestion. The products were analyzed on a 1% TAE-agarose gel, stained with SYBR Gold, dried and exposed to X-ray film.

    Article Snippet: Notably, the linear pBS DNA with no biotin at the end in the same reactions was equally sensitive to T7 Exo with or without avidin, indicating that it's the avidin at the 5′ end that is blocking T7 Exo.

    Techniques: Avidin-Biotin Assay, Isolation, Plasmid Preparation, Agarose Gel Electrophoresis, Staining