exonuclease iii  (New England Biolabs)


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    Exonuclease III E coli
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    Exonuclease III E coli 25 000 units
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    New England Biolabs exonuclease iii
    Exonuclease III E coli
    Exonuclease III E coli 25 000 units
    https://www.bioz.com/result/exonuclease iii/product/New England Biolabs
    Average 99 stars, based on 87 article reviews
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    Images

    1) Product Images from "Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation"

    Article Title: Role of Hepatitis B virus capsid phosphorylation in nucleocapsid disassembly and covalently closed circular DNA formation

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008459

    Effects of N2A on CCC DNA formation during infection. The WT or N2A mutant virus inoculum was prepared from transiently transfected Huh7 cells. The WT and N2A replicon constructs were transfected into Huh7 cells. The culture supernatant was harvested on day 5, 7 and 9 post-transfection, pooled and concentrated by PEG precipitation as described in the Methods. HepG2-NTCP and PXB cells were infected with the WT or N2A mutant virus. HBV PF DNA was extracted from the infected cells and measured by Southern blot analysis using a 32 P-labeled HBV DNA probe. The Southern blot images shown were from phosphorimaging scan. A. A representative Southern blot autoradiogram of PF DNA from HepG2-NTCP cells extracted at the indicated days post-infection. Quantitative analysis of CCC DNA levels at day three post-infection from multiple infection experiments is presented in the graph to the right, with the CCC DNA level from WT HBV infected cells set to 1.0. M, DNA m.w. markers in kbp; B. Representative Southern blot autoradiograms of PF DNA from two batches of PXB cells extracted three days post-infection. Relative CCC DNA levels are indicated at the bottom of the autoradiograms with the CCC DNA level from WT HBV infected cells set to 1.0.
    Figure Legend Snippet: Effects of N2A on CCC DNA formation during infection. The WT or N2A mutant virus inoculum was prepared from transiently transfected Huh7 cells. The WT and N2A replicon constructs were transfected into Huh7 cells. The culture supernatant was harvested on day 5, 7 and 9 post-transfection, pooled and concentrated by PEG precipitation as described in the Methods. HepG2-NTCP and PXB cells were infected with the WT or N2A mutant virus. HBV PF DNA was extracted from the infected cells and measured by Southern blot analysis using a 32 P-labeled HBV DNA probe. The Southern blot images shown were from phosphorimaging scan. A. A representative Southern blot autoradiogram of PF DNA from HepG2-NTCP cells extracted at the indicated days post-infection. Quantitative analysis of CCC DNA levels at day three post-infection from multiple infection experiments is presented in the graph to the right, with the CCC DNA level from WT HBV infected cells set to 1.0. M, DNA m.w. markers in kbp; B. Representative Southern blot autoradiograms of PF DNA from two batches of PXB cells extracted three days post-infection. Relative CCC DNA levels are indicated at the bottom of the autoradiograms with the CCC DNA level from WT HBV infected cells set to 1.0.

    Techniques Used: Countercurrent Chromatography, Infection, Mutagenesis, Transfection, Construct, Southern Blot, Labeling

    NTD phosphorylation could affect cM-RC DNA formation. The same PF-DNA samples shown in Fig 3 . were treated with the exonuclease I and III before detection by Southern blot analysis, using strand specific riboprobes to detect either the minus (-) or plus (+) strand DNA separately ( A ). M, DNA size marker in kilo-basepairs (kbp); CCC, CCC DNA; cM, closed minus strand DNA. The Southern blot images shown were from phosphorimaging scan. B and C. Quantitative results from multiple experiments. B . cM-RC DNA normalized to core RC DNA. C. CCC DNA normalized to cM-RC DNA. All normalized values from the WT HBc were set to 1.0.
    Figure Legend Snippet: NTD phosphorylation could affect cM-RC DNA formation. The same PF-DNA samples shown in Fig 3 . were treated with the exonuclease I and III before detection by Southern blot analysis, using strand specific riboprobes to detect either the minus (-) or plus (+) strand DNA separately ( A ). M, DNA size marker in kilo-basepairs (kbp); CCC, CCC DNA; cM, closed minus strand DNA. The Southern blot images shown were from phosphorimaging scan. B and C. Quantitative results from multiple experiments. B . cM-RC DNA normalized to core RC DNA. C. CCC DNA normalized to cM-RC DNA. All normalized values from the WT HBc were set to 1.0.

    Techniques Used: Southern Blot, Marker, Countercurrent Chromatography

    Effects of NTD phosphorylation mutants on core DNA and CCC DNA levels. HepG2 cells were transfected as in Fig 2 . A. Cytoplasmic lysate from the transfected cells was treated with SDS-proteinase K to release the HBV core DNA from NCs, which was then resolved on an agarose gel (1% agarose) (lanes 1–6). In addition, a portion of cytoplasmic lysate was digested first with MNase to remove input plasmid DNA (and any core DNA not protected by the capsid) before SDS-proteinase K treatment. The core DNA was then purified and resolved on an agarose gel (lanes 7–9). Core DNA was then detected by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); PI, plasmid DNA; RC, RC DNA; SS, single-stranded DNA. B. Viral particles released into the culture supernatant of the transfected HepG2 cells were concentrated by PEG precipitation and resolved on an agarose gel (1% agarose). Following transfer to nitrocellulose membrane, HBV DNA associated with virions (V) or naked capsids (Ca) was detected by Southern blot analysis using an HBV DNA probe. To facilitate a more clear visualization of the degree of N2E deficiency in DNA synthesis and virion secretion, as compared to the WT, serial dilutions (1/4 th , 1/8 th , 1/16 th ) of the WT samples were loaded ( A and B , lanes 2–4). C. HBV PF DNA was extracted from the transfected HepG2 cells. The HBc F122V mutant (lane 4) defective in DNA synthesis was included as a negative control for PF DNA analysis. The extracted DNA was digested with Dpn I (to degrade the input plasmid DNA) (lanes 1–4), Dpn I plus the exonuclease I and III (I III) (lanes 5–8), or Dpn I plus the exonuclease T5 (T5) (lanes 9–12) before resolution on an agarose gel (1.2% agarose) and detection by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); RC, RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. All Southern blot images shown in A , B , and C were from phosphorimaging scan. D. Quantitative results from multiple experiments. Left, levels of core DNA were normalized to those of RNA packaging measured in Fig 2 ; middle, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized DNA values from the WT HBc were set to 1.0.
    Figure Legend Snippet: Effects of NTD phosphorylation mutants on core DNA and CCC DNA levels. HepG2 cells were transfected as in Fig 2 . A. Cytoplasmic lysate from the transfected cells was treated with SDS-proteinase K to release the HBV core DNA from NCs, which was then resolved on an agarose gel (1% agarose) (lanes 1–6). In addition, a portion of cytoplasmic lysate was digested first with MNase to remove input plasmid DNA (and any core DNA not protected by the capsid) before SDS-proteinase K treatment. The core DNA was then purified and resolved on an agarose gel (lanes 7–9). Core DNA was then detected by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); PI, plasmid DNA; RC, RC DNA; SS, single-stranded DNA. B. Viral particles released into the culture supernatant of the transfected HepG2 cells were concentrated by PEG precipitation and resolved on an agarose gel (1% agarose). Following transfer to nitrocellulose membrane, HBV DNA associated with virions (V) or naked capsids (Ca) was detected by Southern blot analysis using an HBV DNA probe. To facilitate a more clear visualization of the degree of N2E deficiency in DNA synthesis and virion secretion, as compared to the WT, serial dilutions (1/4 th , 1/8 th , 1/16 th ) of the WT samples were loaded ( A and B , lanes 2–4). C. HBV PF DNA was extracted from the transfected HepG2 cells. The HBc F122V mutant (lane 4) defective in DNA synthesis was included as a negative control for PF DNA analysis. The extracted DNA was digested with Dpn I (to degrade the input plasmid DNA) (lanes 1–4), Dpn I plus the exonuclease I and III (I III) (lanes 5–8), or Dpn I plus the exonuclease T5 (T5) (lanes 9–12) before resolution on an agarose gel (1.2% agarose) and detection by Southern blot analysis using an HBV DNA probe. M, DNA size marker in kilo-basepairs (kbp); RC, RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. All Southern blot images shown in A , B , and C were from phosphorimaging scan. D. Quantitative results from multiple experiments. Left, levels of core DNA were normalized to those of RNA packaging measured in Fig 2 ; middle, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized DNA values from the WT HBc were set to 1.0.

    Techniques Used: Countercurrent Chromatography, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation, Purification, Southern Blot, Marker, DNA Synthesis, Mutagenesis, Negative Control

    Effects of CDK2 inhibitors on CCC DNA formation during HBV infection. The PXB cells were infected with HBV and treated with the CDK2 inhibitor K03861 at the indicated concentrations (A) or CDK2 inhibitor III (CDK2i III; 125 nM) (B) at the same time. HBV PF DNA was extracted from the cells three days after infection and measured by Southern blot analysis using a 32 P-labeled HBV DNA probe. Shown are representative Southern blot autoradiograms (phosphorimaging scan) of PF DNA, with the relative levels of CCC DNA indicated at the bottom and CCC DNA level from the mock-treated cells set to 1.0.
    Figure Legend Snippet: Effects of CDK2 inhibitors on CCC DNA formation during HBV infection. The PXB cells were infected with HBV and treated with the CDK2 inhibitor K03861 at the indicated concentrations (A) or CDK2 inhibitor III (CDK2i III; 125 nM) (B) at the same time. HBV PF DNA was extracted from the cells three days after infection and measured by Southern blot analysis using a 32 P-labeled HBV DNA probe. Shown are representative Southern blot autoradiograms (phosphorimaging scan) of PF DNA, with the relative levels of CCC DNA indicated at the bottom and CCC DNA level from the mock-treated cells set to 1.0.

    Techniques Used: Countercurrent Chromatography, Infection, Southern Blot, Labeling

    2) Product Images from "Identification of a novel proliferation-inducing determinant using lentiviral expression cloning"

    Article Title: Identification of a novel proliferation-inducing determinant using lentiviral expression cloning

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gng115

    Proliferation-inducing capacity of pPCR102-2 and derivatives in HUVECs. HUVECs were transiently transfected with either pPCR102-2 ( A ), pND-A8 ( B ), pND-A2 ( C ) or pPI1-His ( D ) in order to assess their proliferation-inducing potential. Forty-eight hours post transfection, the cell populations were transduced with a GFP-encoding oncoretroviral vector, which exclusively targets proliferating cells. Forty-eight hours post-transduction, GFP-mediated fluorescence was quantified by FACS. Fluorescence values were calculated by multiplying the number of GFP-expressing cells by the average intensity of GFP expression. The relative fluorescence units were obtained by comparison with GFP-mediated fluorescence control populations (cntrl) transfected with isogenic pcDNA3.1/V5-His-TOPO. Corresponding FACS histograms are also shown. All values are representative of at least three independent experiments. FS, forward scatter; FL, fluoresence.
    Figure Legend Snippet: Proliferation-inducing capacity of pPCR102-2 and derivatives in HUVECs. HUVECs were transiently transfected with either pPCR102-2 ( A ), pND-A8 ( B ), pND-A2 ( C ) or pPI1-His ( D ) in order to assess their proliferation-inducing potential. Forty-eight hours post transfection, the cell populations were transduced with a GFP-encoding oncoretroviral vector, which exclusively targets proliferating cells. Forty-eight hours post-transduction, GFP-mediated fluorescence was quantified by FACS. Fluorescence values were calculated by multiplying the number of GFP-expressing cells by the average intensity of GFP expression. The relative fluorescence units were obtained by comparison with GFP-mediated fluorescence control populations (cntrl) transfected with isogenic pcDNA3.1/V5-His-TOPO. Corresponding FACS histograms are also shown. All values are representative of at least three independent experiments. FS, forward scatter; FL, fluoresence.

    Techniques Used: Transfection, Transduction, Plasmid Preparation, Fluorescence, FACS, Expressing

    3) Product Images from "Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3"

    Article Title: Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm165

    HCc3 promotes ligase-mediated DNA concatenation. All images are presented as negative images. ( a ) Concatenation of 125-bp (left panel) and 2.8-kb (right panel) DNA fragments to a higher level in the presence of HCc3 at respective dimer/bp ratios. The linearity of the resulting products was confirmed by Exo III digestion (marked with ‘+/−’ signs). The 125-bp product was resolved on a 8% non-denaturing polyacrylamide gel, and the 2.8-kb product on a 1% TAE agarose gel. ( b ) Variations in intensity of DNA concatenation in the presence of HCc3 at different dimer/bp ratios. The marker bands indicate DNA sizes from 3 to 12 kb at 1-kb increments.
    Figure Legend Snippet: HCc3 promotes ligase-mediated DNA concatenation. All images are presented as negative images. ( a ) Concatenation of 125-bp (left panel) and 2.8-kb (right panel) DNA fragments to a higher level in the presence of HCc3 at respective dimer/bp ratios. The linearity of the resulting products was confirmed by Exo III digestion (marked with ‘+/−’ signs). The 125-bp product was resolved on a 8% non-denaturing polyacrylamide gel, and the 2.8-kb product on a 1% TAE agarose gel. ( b ) Variations in intensity of DNA concatenation in the presence of HCc3 at different dimer/bp ratios. The marker bands indicate DNA sizes from 3 to 12 kb at 1-kb increments.

    Techniques Used: Agarose Gel Electrophoresis, Marker

    4) Product Images from "Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity"

    Article Title: Molecular interactions of Escherichia coli ExoIX and identification of its associated 3?-5? exonuclease activity

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm396

    Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. ( A ) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/ xni , pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. ( B ) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. ( C ) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). ( D ) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). ( E ) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32 P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl 2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.
    Figure Legend Snippet: Chromatographic separation of 3′-5′ exodeoxyribonuclease activity associated with preparations of Exonuclease IX. ( A ) SDS–PAGE analysis of the purification of ExoIX from cell lysate of induced BL21 (pJONEX/ xni , pcI857). SPL, cleared cell lysate applied to SP/first Heparin column (28 µg); QL, Q load (6 µg); H2L, second Heparin column load (5 µg); IX, concentrated ExoIX eluate from second Heparin (7.5 µg). (B–D). Eluted fractions from first Heparin column were separated by SDS–PAGE. ( B ) Ethidium bromide stained substrate gel. High molecular weight DNA cast in the gel fluoresces with UV, while regions of DNA degradation appear as darker bands. Early fractions (lanes 1–4), contain detectable exonuclease activity. ( C ) The same gel counter-stained with Coomassie G250. Over-expressed ExoIX is eluted in later fractions (lanes 5 and 6). ( D ) Superimposition of images in panels B and C, demonstrating that exonuclease activity can be resolved from ExoIX. A fraction represented in lane 4 was used for subsequent enrichment and identification of the co-purifying nuclease. Lanes, 1–6, heparin fractions (2.5 µl); 7, loading sample (5 µl); 8, flow through (5 µl). ( E ) Highly purified ExoIX lacks activity on a single-stranded DNA substrate (34-mer). Protein samples taken during the purification of ExoIX were incubated with 15 fmol 32 P-labelled 34-mer at 37°C for 10 min in the presence of 10 mM MgCl 2 and the reaction products separated by denaturing PAGE. Reactions (10 µl) contained varying amounts of protein. SPL, 0.7 and 0.07 µg of protein from cell-free extract of induced cells expressing ExoIX; QL, 0.1 and 0.01 µg of protein loaded on to first anion exchange column; H2L, 3 and 0.3 µg of protein from sample loaded onto second heparin column; IX, contains samples from final purified fraction of ExoIX eluted from second heparin column, 5 and 0.5 µg; two positive controls are also shown, bacteriophage T5 D15 exonuclease (T5), 0.1 and 0.01 µg and exonuclease III (III), 0.03 and 0.003 µg.

    Techniques Used: Activity Assay, SDS Page, Purification, Staining, Molecular Weight, Flow Cytometry, Incubation, Polyacrylamide Gel Electrophoresis, Expressing

    5) Product Images from "Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations"

    Article Title: Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl559

    Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).
    Figure Legend Snippet: Probing of the EC translocation conformations. ( A ) Exo III footprints of EC14 and EC15. Tth EC14 (lanes 1 and 2) was assembled as described in Materials and Methods. EC15 (lanes 3 and 4) was obtained by incubation of EC14 with substrate ATP for 2 min at 60°C. Exo III (0.02 U/μl) was added for 5 (lanes 1 and 3) or 10 (lanes 2 and 4) min at 37°C. ( B ) Schematics of RNAP front edge oscillations in EC14 and EC15. ( C ) Photo cross-linking patterns of Tth EC14 and EC15, EC14 and EC15 containing 5′ 32 P-labeled RNA primers were prepared as illustrated (left). The photo cross-linking analog 4-thio UTP (50 μM) was incorporated into the transcript for 2 min at 60°C followed UV light irradiation for 5 min at RT. The cross-linked species were separated using gel electrophoresis (right).

    Techniques Used: Translocation Assay, Incubation, Labeling, Irradiation, Nucleic Acid Electrophoresis

    6) Product Images from "The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription"

    Article Title: The activation-induced cytidine deaminase (AID) efficiently targets DNA in nucleosomes but only during transcription

    Journal: The Journal of Experimental Medicine

    doi: 10.1084/jem.20082678

    Sequences of pKMP2 plasmids treated with AID during transcription and amplified in E. coli in the presence of ampicillin. The WT sequence is shown on the top lines. The two MP2 nucleosome positioning sequences are in green (BamHI sites underlined). The in-frame ACG initiation codons are in italics and underlined. The in-frame stop codons are underlined and not italicized. The AID-created Ts resulting in a potential TATA box present in all 35 clones are in bold font. The Shine-Dalgarno sequence (AGGAAG) is in blue. The transcription start site of unmutated pKMP2 plasmids in E. coli is in purple and underlined. The 5′ end of the Amp r coding region is in red. The ACG start codon for Amp r is at 3041–3043. Dashed lines represent agreement with the unmutated pKMP2 with mutations indicated by lowercase font. The sequences were obtained from one of three experiments.
    Figure Legend Snippet: Sequences of pKMP2 plasmids treated with AID during transcription and amplified in E. coli in the presence of ampicillin. The WT sequence is shown on the top lines. The two MP2 nucleosome positioning sequences are in green (BamHI sites underlined). The in-frame ACG initiation codons are in italics and underlined. The in-frame stop codons are underlined and not italicized. The AID-created Ts resulting in a potential TATA box present in all 35 clones are in bold font. The Shine-Dalgarno sequence (AGGAAG) is in blue. The transcription start site of unmutated pKMP2 plasmids in E. coli is in purple and underlined. The 5′ end of the Amp r coding region is in red. The ACG start codon for Amp r is at 3041–3043. Dashed lines represent agreement with the unmutated pKMP2 with mutations indicated by lowercase font. The sequences were obtained from one of three experiments.

    Techniques Used: Amplification, Sequencing, Clone Assay

    7) Product Images from "High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells"

    Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

    Journal: Theranostics

    doi: 10.7150/thno.23852

    Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.
    Figure Legend Snippet: Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.

    Techniques Used: Modification, Fluorescence

    8) Product Images from "Validation of DNA Sequences Using Mass Spectrometry Coupled with Nucleoside Mass Tagging"

    Article Title: Validation of DNA Sequences Using Mass Spectrometry Coupled with Nucleoside Mass Tagging

    Journal: Genome Research

    doi: 10.1101/gr.221402

    ( A ) Negative-ion MALDI-TOF-MS spectrum of unlabeled 10-bp PCR product after Hph I restriction digestion. The (−)-strand has an m/z ratio of 3068.2, and the (+)-strand has an m/z ratio of 3120.7. ( B ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dATP-labeled 10-bp product. Four different peaks represent the (−)-strand with m/z ratios of 3069.2, 3082.3, 3098.2, and 3112.1, reflecting the presence of three labeled adenines. Three different peaks with m/z ratios of 3120.6, 3135.8, and 3151.0 are detected for the (+)-strand. The peaks are separated by ∼15 Da each. ( C ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dTTP-labeled 10-bp product. The (−)-strand has three peaks with m/z ratios of 3068.4, 3079.9, and 3091.7. The (+)-strand was represented with four peaks with m/z ratios of 3118.5, 3132.8, 3144.5, and 3156.9. ( D ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dCTP-labeled 10-bp product. The (−)-strand had four peaks with m/z ratios of 3070.3, 3081.8, 3093.3, and 3105.7, whereas the (+)-strand had only two peaks with an m/z ratio of 3121.4 and 3133.6. ( E ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dGTP-labeled 10-bp product. The (−)-strand displayed two peaks with m/z values of 3067.0 and 3081.8. The (+)-strand had four peaks with a mass of 3120.4, 3135.1, 3150.6, and 3164.0. “+Na + ” indicates sodium adducts.
    Figure Legend Snippet: ( A ) Negative-ion MALDI-TOF-MS spectrum of unlabeled 10-bp PCR product after Hph I restriction digestion. The (−)-strand has an m/z ratio of 3068.2, and the (+)-strand has an m/z ratio of 3120.7. ( B ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dATP-labeled 10-bp product. Four different peaks represent the (−)-strand with m/z ratios of 3069.2, 3082.3, 3098.2, and 3112.1, reflecting the presence of three labeled adenines. Three different peaks with m/z ratios of 3120.6, 3135.8, and 3151.0 are detected for the (+)-strand. The peaks are separated by ∼15 Da each. ( C ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dTTP-labeled 10-bp product. The (−)-strand has three peaks with m/z ratios of 3068.4, 3079.9, and 3091.7. The (+)-strand was represented with four peaks with m/z ratios of 3118.5, 3132.8, 3144.5, and 3156.9. ( D ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dCTP-labeled 10-bp product. The (−)-strand had four peaks with m/z ratios of 3070.3, 3081.8, 3093.3, and 3105.7, whereas the (+)-strand had only two peaks with an m/z ratio of 3121.4 and 3133.6. ( E ) Negative-ion MALDI-TOF-MS spectrum of 50% 13 C/ 15 N-dGTP-labeled 10-bp product. The (−)-strand displayed two peaks with m/z values of 3067.0 and 3081.8. The (+)-strand had four peaks with a mass of 3120.4, 3135.1, 3150.6, and 3164.0. “+Na + ” indicates sodium adducts.

    Techniques Used: Mass Spectrometry, Polymerase Chain Reaction, Labeling

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa473

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

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

    10) Product Images from "Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc01652g"

    Article Title: Rolling circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level circle amplification-driven encoding of different fluorescent molecules for simultaneous detection of multiple DNA repair enzymes at the single-molecule level †Electronic supplementary information (ESI) available. See DOI: 10.1039/d0sc01652g

    Journal: Chemical Science

    doi: 10.1039/d0sc01652g

    (A and B) Measurement of Cy3 counts (A) and Cy5 counts (B) in the presence of A549 cells, respectively. (C and D) Measurement of Cy3 counts (C) and Cy5 counts (D) in the presence of HeLa cells, respectively. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars represent standard deviations of three experiments.
    Figure Legend Snippet: (A and B) Measurement of Cy3 counts (A) and Cy5 counts (B) in the presence of A549 cells, respectively. (C and D) Measurement of Cy3 counts (C) and Cy5 counts (D) in the presence of HeLa cells, respectively. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars represent standard deviations of three experiments.

    Techniques Used:

    Variance of Cy3 counts (green color) and Cy5 counts (red color) in response to 0.1 U μL –1 hAAG + 0.1 U μL –1 UDG, 0.1 U μL –1 hAAG, 0.1 U μL –1 UDG, 0.1 U μL –1 hOGG1, 0.1 U μL –1 TDG, 0.1 μg μL –1 BSA, 0.2 U μL –1 FPG, and the control group with only reaction buffer, respectively. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: Variance of Cy3 counts (green color) and Cy5 counts (red color) in response to 0.1 U μL –1 hAAG + 0.1 U μL –1 UDG, 0.1 U μL –1 hAAG, 0.1 U μL –1 UDG, 0.1 U μL –1 hOGG1, 0.1 U μL –1 TDG, 0.1 μg μL –1 BSA, 0.2 U μL –1 FPG, and the control group with only reaction buffer, respectively. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars show the standard deviation of three experiments.

    Techniques Used: Standard Deviation

    (A) Variance of initial velocity ( V ) with the concentration of DNA substrates in response to 0.1 U μL –1 hAAG. (B) Variance of initial velocity ( V ) with the concentration of DNA substrates in response to 0.1 U μL –1 UDG. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: (A) Variance of initial velocity ( V ) with the concentration of DNA substrates in response to 0.1 U μL –1 hAAG. (B) Variance of initial velocity ( V ) with the concentration of DNA substrates in response to 0.1 U μL –1 UDG. Error bars show the standard deviation of three experiments.

    Techniques Used: Concentration Assay, Standard Deviation

    Schematic illustration of the simultaneous detection of multiple DNA repair enzymes by the integration of RCA with single-molecule detection. This strategy involves four steps: (1) specific excision of dsDNA substrate by hAAG and UDG, (2) the hybridization of primers with circular templates and the subsequent RCA reaction, (3) magnetic separation and the cleavage of amplified products by Exonucleases I and III to release fluorescent molecules, and (4) single-molecule detection of the released fluorescent molecules.
    Figure Legend Snippet: Schematic illustration of the simultaneous detection of multiple DNA repair enzymes by the integration of RCA with single-molecule detection. This strategy involves four steps: (1) specific excision of dsDNA substrate by hAAG and UDG, (2) the hybridization of primers with circular templates and the subsequent RCA reaction, (3) magnetic separation and the cleavage of amplified products by Exonucleases I and III to release fluorescent molecules, and (4) single-molecule detection of the released fluorescent molecules.

    Techniques Used: Hybridization, Amplification

    (A) Variance of the relative activity of hAAG in response to different-concentration Cd 2+ . (B) Variance of the relative activity of UDG in response to different-concentration Cd 2+ . The 100 nM bifunctional dsDNA substrates, 2 U of APE1, 0.1 U μL –1 hAAG, and 0.1 U μL –1 UDG were used in this research. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: (A) Variance of the relative activity of hAAG in response to different-concentration Cd 2+ . (B) Variance of the relative activity of UDG in response to different-concentration Cd 2+ . The 100 nM bifunctional dsDNA substrates, 2 U of APE1, 0.1 U μL –1 hAAG, and 0.1 U μL –1 UDG were used in this research. Error bars show the standard deviation of three experiments.

    Techniques Used: Activity Assay, Concentration Assay, Standard Deviation

    (A) Fluorescence spectra in response to different concentrations of hAAG. (B) Fluorescence spectra in response to different concentrations of UDG. (C) The log–linear correlation between the fluorescence intensity at 568 nm and the concentration of hAAG. (D) The log–linear correlation between the fluorescence intensity at 670 nm and the concentration of UDG. Error bars show the standard deviations of three experiments. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research.
    Figure Legend Snippet: (A) Fluorescence spectra in response to different concentrations of hAAG. (B) Fluorescence spectra in response to different concentrations of UDG. (C) The log–linear correlation between the fluorescence intensity at 568 nm and the concentration of hAAG. (D) The log–linear correlation between the fluorescence intensity at 670 nm and the concentration of UDG. Error bars show the standard deviations of three experiments. The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research.

    Techniques Used: Fluorescence, Concentration Assay

    (A) Variance of Cy3 counts with the hAAG concentration. The inset shows the linear relationship between Cy3 counts and the logarithm of hAAG concentration in the range from 1 × 10 –11 to 1 × 10 –3 U μL –1 . (B) Variance of Cy5 counts with the UDG concentration. The inset shows the linear relationship between Cy5 counts and the logarithm of UDG concentration in the range from 1 × 10 –11 to 1 × 10 –3 U μL –1 . The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars show the standard deviation of three experiments.
    Figure Legend Snippet: (A) Variance of Cy3 counts with the hAAG concentration. The inset shows the linear relationship between Cy3 counts and the logarithm of hAAG concentration in the range from 1 × 10 –11 to 1 × 10 –3 U μL –1 . (B) Variance of Cy5 counts with the UDG concentration. The inset shows the linear relationship between Cy5 counts and the logarithm of UDG concentration in the range from 1 × 10 –11 to 1 × 10 –3 U μL –1 . The 100 nM bifunctional dsDNA substrates and 2 U of APE1 were used in this research. Error bars show the standard deviation of three experiments.

    Techniques Used: Concentration Assay, Standard Deviation

    11) Product Images from "In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium"

    Article Title: In vivo and in vitro characterization of DdrC, a DNA damage response protein in Deinococcus radiodurans bacterium

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0177751

    DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.
    Figure Legend Snippet: DdrC protects DNA against degradation by nucleases. Protection of supercoiled pBR322 plasmid (3.5 nM) from DNase I activity (0.1 U) (panel a), linear pBR322 (3.5 nM) from Exonuclease III activity (200 U) (panel b) and phiX174 ssDNA (5.9 nM) from Mung Bean Nuclease activity (1 U) (panel c) by 7 μM, 7 μM, and 2 μM DdrC, respectively. Lanes C: DNA controls without protein. Lanes 1: DNA incubation with nuclease alone. Lanes 2: DNA incubation with DdrC alone. Lanes 3: DNA pre-incubated with DdrC 15 min at 4°C before addition of nuclease. Lanes 4: Reaction products corresponding to lane 3 were further treated with Proteinase K/SDS. Panel a, lane 5: DdrC and DNase I were simultaneously incubated with supercoiled DNA before treatment with Proteinase K/SDS.

    Techniques Used: Plasmid Preparation, Activity Assay, Incubation

    12) Product Images from "Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification"

    Article Title: Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification

    Journal: ACS Omega

    doi: 10.1021/acsomega.8b03376

    Inhibition effect of different concentrations of UGI on the UDG activity. The concentration of UDG was 5 × 10 –4 U/mL. Error bars were obtained from three repetitive measurements.
    Figure Legend Snippet: Inhibition effect of different concentrations of UGI on the UDG activity. The concentration of UDG was 5 × 10 –4 U/mL. Error bars were obtained from three repetitive measurements.

    Techniques Used: Inhibition, Activity Assay, Concentration Assay

    13) Product Images from "The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA"

    Article Title: The Binding Site of Transcription Factor YY1 Is Required for Intramolecular Recombination between Terminally Repeated Sequences of Linear Replicative Hepatitis B Virus DNA

    Journal: Journal of Virology

    doi:

    Southern blot analysis of HBV DNA in viral or core particles. (A) HBV DNA in viral particles. HBV particles secreted into the culture medium of the cells transfected with pBS-HBV3, WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (lanes 1 to 5) were treated with 1 mg of proteinase K per ml and 1% SDS and then directly subjected to 1% agarose gel electrophoresis. The resultant DNA was blotted to the filter paper and hybridized with an HBV DNA probe. Arrowheads indicate the positions corresponding to three different forms of HBV DNA (RC, L, and SS) and the bracket shows the position of transfected plasmid DNA. (B) HBV DNA in core particles was treated as described for panel A. Lanes 1 to 5 contain the samples from pBS-HBV3-, WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The arrowhead indicates the position of the SS form of HBV DNA. The positions of the transfected plasmid and linear DNA are also indicated.
    Figure Legend Snippet: Southern blot analysis of HBV DNA in viral or core particles. (A) HBV DNA in viral particles. HBV particles secreted into the culture medium of the cells transfected with pBS-HBV3, WT HBV, HBV ΔDR, HBV Δr, or HBV ΔYY (lanes 1 to 5) were treated with 1 mg of proteinase K per ml and 1% SDS and then directly subjected to 1% agarose gel electrophoresis. The resultant DNA was blotted to the filter paper and hybridized with an HBV DNA probe. Arrowheads indicate the positions corresponding to three different forms of HBV DNA (RC, L, and SS) and the bracket shows the position of transfected plasmid DNA. (B) HBV DNA in core particles was treated as described for panel A. Lanes 1 to 5 contain the samples from pBS-HBV3-, WT-HBV-, HBV ΔDR-, HBV Δr-, and HBV ΔYY-transfected cells, respectively. The arrowhead indicates the position of the SS form of HBV DNA. The positions of the transfected plasmid and linear DNA are also indicated.

    Techniques Used: Southern Blot, Transfection, Agarose Gel Electrophoresis, Plasmid Preparation

    14) Product Images from "Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase"

    Article Title: Expression and the Peculiar Enzymatic Behavior of the Trypanosoma cruzi NTH1 DNA Glycosylase

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0157270

    TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.
    Figure Legend Snippet: TcNTH1 does not present mono nor bifunctional DNA glycosylase activities but an AP endonuclease activity. A, B and C: A [γ-32P]ATP labeled 32 mer oligonucleotide containing a thymine glycol residue at position 18 incubated without enzyme (negative control, lane 1) or with E . coli Endo III (bacterial NTH1, positive control, lane 2). A: Lanes 3 and 4, same oligo incubated with native TcNTH1 purified from transformed bacteria or purified from transfected epimastigotes, respectively. B: Lane 3 same oligo co-incubated with native TcNTH1 purified from transformed bacteria and with native TcAP1 endonuclease. Lanes 4 and 5 same oligo incubated with native TcNTH1 purified from transformed bacteria or incubated with native TcAP1, respectively. C: Lanes 3 and 4 same oligo incubated with epimastigote or trypomastigote homogenates, respectively. D: A [γ- 32 P]ATP labeled 25-mer oligonucleotide with an AP site at position 8, was incubated with E . coli Endo III (AP lyase, positive control, lane 3), with native TcNTH1 purified from transformed bacteria (lane 4) and with native TcNTH1 purified from transfected epimastigotes (lane 5). Lane 1 same oligo incubated without enzyme (negative control). Lanes 2 and 6 same oligo incubated with E . coli Exo III (canonic AP endonuclease, positive control) or with TcAP1 AP endonuclease, respectively. A densitometric analysis of bands was performed using the Quantity One version 4.6.3 program (Bio Rad). S: substrate, P: product.

    Techniques Used: Activity Assay, Labeling, Incubation, Negative Control, Positive Control, Purification, Transformation Assay, Transfection

    15) Product Images from "Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification"

    Article Title: Sensitive RNA detection by combining three-way junction formation and primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr909

    RNA detection mechanism by three-way junction probe and primer generation-rolling circle amplification. ( A ) Three-way junction (3WJ) probes (primer and template) are designed to form a 3WJ structure on target RNA, however they do not interact each other without target RNA because their complementary sequence is only 6–8 bases. ( B ) Addition of DNA polymerase and nicking enzyme initiates a reaction cycle of primer extension, nicking reaction and signal primer generation under an isothermal condition to generate signal primers. ( C ) The generated signal primers can be detected by primer generation-rolling circle amplification.
    Figure Legend Snippet: RNA detection mechanism by three-way junction probe and primer generation-rolling circle amplification. ( A ) Three-way junction (3WJ) probes (primer and template) are designed to form a 3WJ structure on target RNA, however they do not interact each other without target RNA because their complementary sequence is only 6–8 bases. ( B ) Addition of DNA polymerase and nicking enzyme initiates a reaction cycle of primer extension, nicking reaction and signal primer generation under an isothermal condition to generate signal primers. ( C ) The generated signal primers can be detected by primer generation-rolling circle amplification.

    Techniques Used: RNA Detection, Amplification, Sequencing, Generated

    16) Product Images from "Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2"

    Article Title: Molecular differences between two Jeryl Lynn mumps virus vaccine component strains, JL5 and JL2

    Journal: The Journal of General Virology

    doi: 10.1099/vir.0.013946-0

    Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.
    Figure Legend Snippet: Molecular clone of MuV JL2 , indicating gene boundaries and restriction sites in pMuV JL2 . The bar shows the antigenome of pMuV JL2 and the locations of viral genes (not to scale). Arrows beneath the bar indicate the location of unique restriction sites suitable for ligation-independent cloning using exonuclease III in pMuV JL2 . The vector sequence flanking the antigenome contains a Not I site upstream of a T7 RNA polymerase promoter located 5′ to the antigenome (i.e. to the left of N) and a Kas I site downstream of the antigenome 3′ terminus (i.e. to the right of L) which is internal to the hepatitis delta ribozyme (these restriction sites are shown in bold). (a) Restriction sites present in the consensus MuV JL2 sequence – these were either already unique in the consensus MuV JL2 sequence or made unique by mutagenesis of sites at other locations in the MuV genome or the plasmid vector. (b) Restriction sites introduced into the final clone by in vitro mutagenesis. Additional Sma I, Avr II, Bsr GI and Xho I restriction sites in the MuV JL2 sequence (c) were removed by in vitro mutagenesis. A Sap I site and two Fsp I sites were removed from the vector sequence by in vitro mutagenesis or deletion to render sites in the MuV JL2 sequence unique in the final clone. Restriction-enzyme names are abbreviated for clarity. Details of their position in the MuV JL2 sequence are available on request. The asterisks indicate that these sites are unique in the plasmid DNA which is methylated, as there are two sites at 11408–11413 and 11608–11613 that are also cleavable with Stu I and Nru I, respectively, in unmethylated plasmid DNA.

    Techniques Used: Ligation, Clone Assay, Plasmid Preparation, Sequencing, Mutagenesis, In Vitro, Methylation

    17) Product Images from "Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification"

    Article Title: Sensitive isothermal detection of nucleic-acid sequence by primer generation-rolling circle amplification

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn1014

    Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.
    Figure Legend Snippet: Real-time quantification of hly gene in L. monocytogenes genomic DNA by PG–RCA. ( A ) Circular probe LM for detection of pathogenic L. monocytogenes genomic DNA. The probe targets the complementary strand of virulence gene, hly (GeneBank GeneID 2797098), encoding a cholesterol-dependent cytolysin, listeriolysin O (LLO). Circular probe LM contains three repeats of a 26-base sequence complementary to the gene including a nicking site for Nb.BsmI. Since these repeat sequences have 5-base overlaps each other, the circular probe comprises three repeats of a 21-base sequence (red, blue and green). ( B ) Genomic DNA from L. monocytogenes (0.1–100 pg) was analyzed by real-time PG–RCA with circular probe LM. Threshold time ( T T ) was plotted against the L. monocytogenes genomic DNA concentration (S) of the reaction. Solid line indicates linear least squares fitting between 0.1 and 100 pg L. monocytogenes genomic DNA and its formulation is T T = −19.1 log 10 (S) + 233 ( R 2 =0.964). Perforated line indicates average T T value of the negative controls ( n =2). Limit of detection is 0.163 pg (∼60 molecules) of L. monocytogenes genomic DNA by calculation from the intersection of both lines. ( C ) Genomic DNA (100 pg) from L. monocytogenes , L. innocua , E. coli and S. enterica were analyzed by real-time PG–RCA with circular probe LM and their threshold times were compared with the values for L. monocytogenes (100 pg). ‘No DNA’ indicates the negative controls.

    Techniques Used: Sequencing, Concentration Assay

    18) Product Images from "Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination"

    Article Title: Strand break-induced replication fork collapse leads to C-circles, C-overhangs and telomeric recombination

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1007925

    C-circles and 5' C-overhangs are linked to DNA damage-induced replication fork collapse. (A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t- test was used to calculate P-values. *P
    Figure Legend Snippet: C-circles and 5' C-overhangs are linked to DNA damage-induced replication fork collapse. (A) Replication fork stalling induced by HU or aphidicolin decreases abundance of C-circles in U2OS cells. U2OS cells were treated with HU (hydroxyurea, 2mM) or aphidicolin (Aphi, 1μg/mL) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t- test was used to calculate P-values. *P

    Techniques Used: Purification, Two Tailed Test

    Endogenous ssDNA break/gap or induced ssDNA break in C-rich strand stimulates formation of C-circles and 5' C-overhangs. (A) Experimental protocol to study strand specific (G-rich or C-rich) breaks/gaps on telomere is shown schematically. HinfI and RsaI digested genomic DNA was purified and further digested with Exo III to examine potential breaks/gaps on G-strand or C-strand of telomeres. If breaks/gaps occur on C-strand, Exo III would degrade all C-strand, leaving single-stranded G-strand that can be detected by hybridization with C-rich probe under native or denatured condition. Contrariwise, only C-strand can be detected if breaks/gaps occur on G-strand. (B) Breaks/gaps occur more frequently on C-rich strand of telomere. Exo III digestion produces single-stranded DNA that is less in molecular weight than corresponding double-stranded DNA, thereby migrating faster during electrophoresis. (C) Methyl-methane sulfonate (MMS) stimulates formation of C-circle DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t -test was used to calculate P-values. ***P
    Figure Legend Snippet: Endogenous ssDNA break/gap or induced ssDNA break in C-rich strand stimulates formation of C-circles and 5' C-overhangs. (A) Experimental protocol to study strand specific (G-rich or C-rich) breaks/gaps on telomere is shown schematically. HinfI and RsaI digested genomic DNA was purified and further digested with Exo III to examine potential breaks/gaps on G-strand or C-strand of telomeres. If breaks/gaps occur on C-strand, Exo III would degrade all C-strand, leaving single-stranded G-strand that can be detected by hybridization with C-rich probe under native or denatured condition. Contrariwise, only C-strand can be detected if breaks/gaps occur on G-strand. (B) Breaks/gaps occur more frequently on C-rich strand of telomere. Exo III digestion produces single-stranded DNA that is less in molecular weight than corresponding double-stranded DNA, thereby migrating faster during electrophoresis. (C) Methyl-methane sulfonate (MMS) stimulates formation of C-circle DNA in U2OS cells. U2OS cells were treated with MMS (0.25mM) for 24h and genomic DNA was purified for C-circle assay. Error bars represent the mean ± SEM of three independent experiments. Two-tailed unpaired student’s t -test was used to calculate P-values. ***P

    Techniques Used: Purification, Hybridization, Molecular Weight, Electrophoresis, Two Tailed Test

    Nascent C-circles and 5' C-overhangs are generated during telomere replication. (A) FACS analysis of G1/S synchronized U2OS cells. Cells were synchronized by double thymidine block, then released and harvested at the indicated time. (B) C-circle assay was performed at the indicated time after release from G1/S. (C) Representative image and statistical analysis showing that RPA2 foci colocalize with telomere at each time point. Cells with more than 5 colocalized foci/cell were scored positively, > 100 cells were counted per time point. Error bars represent the mean ± SEM of three independent experiments.(D) BrdU pulse-labeling strategy. U2OS cells were synchronized at G1/S, released in presence of BrdU for 12h. (E) Leading, lagging and unreplicated telomeric fractions were resolved by CsCl gradient ultracentrifugation and hybridized with telomeric probe. Non-BrdU labeled U2OS was used as a negative control (upper figure). “Area under peak” for leading, lagging and unreplicated telomeres was analyzed by Graphpad Prism and the relative amount of telomeres was indicated above individual peak. (F) Nascent C-circle is predominantly associated with lagging strand DNA synthesis. C-circle assay analysis of CsCl gradient fractions in (E). The amount of C-circle in leading, lagging and unreplicated telomeres was calculated by determining
    Figure Legend Snippet: Nascent C-circles and 5' C-overhangs are generated during telomere replication. (A) FACS analysis of G1/S synchronized U2OS cells. Cells were synchronized by double thymidine block, then released and harvested at the indicated time. (B) C-circle assay was performed at the indicated time after release from G1/S. (C) Representative image and statistical analysis showing that RPA2 foci colocalize with telomere at each time point. Cells with more than 5 colocalized foci/cell were scored positively, > 100 cells were counted per time point. Error bars represent the mean ± SEM of three independent experiments.(D) BrdU pulse-labeling strategy. U2OS cells were synchronized at G1/S, released in presence of BrdU for 12h. (E) Leading, lagging and unreplicated telomeric fractions were resolved by CsCl gradient ultracentrifugation and hybridized with telomeric probe. Non-BrdU labeled U2OS was used as a negative control (upper figure). “Area under peak” for leading, lagging and unreplicated telomeres was analyzed by Graphpad Prism and the relative amount of telomeres was indicated above individual peak. (F) Nascent C-circle is predominantly associated with lagging strand DNA synthesis. C-circle assay analysis of CsCl gradient fractions in (E). The amount of C-circle in leading, lagging and unreplicated telomeres was calculated by determining "area under peak" using Graphpad Prism. The relative amount of C-circles was indicated above individual peak. (G) Schematic of the migration of linear dsDNA, ssDNA (C-overhangs) and telomeric open circles (T-circle) during 2D agarose gel electrophoresis and hybridization to a telomere-specific G-rich probe under native or denatured condition. (H) 5' C-overhang DNA is predominantly associated with leading strand DNA synthesis. The fractions corresponding to leading, lagging or non-replication telomeres from 12h BrdU labeled sample in (E) were pooled. DNA was incubated with or without RecJf, analyzed by 2D agarose gel electrophoresis and hybridized with G-rich telomeric probe under native and denaturing conditions. C-overhangs were indicated by red arrows. C-overhangs abundance was expressed as a ratio between the native and denatured signals. Values were then normalized with leading C-overhangs to obtain relative abundance.

    Techniques Used: Generated, FACS, Blocking Assay, Labeling, Negative Control, DNA Synthesis, Migration, Agarose Gel Electrophoresis, Hybridization, Incubation

    19) Product Images from "Fluorescence spectroscopic detection and measurement of single telomere molecules"

    Article Title: Fluorescence spectroscopic detection and measurement of single telomere molecules

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky627

    Principle of telomere measurement by PHAST. ( A ) The biological effects of telomeres are mediated primarily by the proportion of telomeres below a critical length. ( i ) This most commonly happens in ageing, where the telomeres in a cell population shorten with doubling. However, the proportion of short telomeres can also reach critical levels when the average telomere length is normal. ( ii ) This can happen due to the naturally occurring diversity in the shape of the distribution between individuals, or ( iii ) if the telomere distribution is highly heterogeneous. ( B ) To perform our assay, cells are lyzed to release the DNA from the nucleus, and incubated with biotinylated PNA probes. Telomeric DNA is separated from genomic DNA using magnetic beads, and released after washing, whereupon fluorescent probes are hybridized to the telomeric sequences. ( C ) The labelled DNA is then flowed through a microchannel, and excited by a laser through an objective as it transits the observation volume (OV). These peaks can then be processed to yield the telomere distribution.
    Figure Legend Snippet: Principle of telomere measurement by PHAST. ( A ) The biological effects of telomeres are mediated primarily by the proportion of telomeres below a critical length. ( i ) This most commonly happens in ageing, where the telomeres in a cell population shorten with doubling. However, the proportion of short telomeres can also reach critical levels when the average telomere length is normal. ( ii ) This can happen due to the naturally occurring diversity in the shape of the distribution between individuals, or ( iii ) if the telomere distribution is highly heterogeneous. ( B ) To perform our assay, cells are lyzed to release the DNA from the nucleus, and incubated with biotinylated PNA probes. Telomeric DNA is separated from genomic DNA using magnetic beads, and released after washing, whereupon fluorescent probes are hybridized to the telomeric sequences. ( C ) The labelled DNA is then flowed through a microchannel, and excited by a laser through an objective as it transits the observation volume (OV). These peaks can then be processed to yield the telomere distribution.

    Techniques Used: Incubation, Magnetic Beads

    Telomere length (TL) determination using peak height. ( A ) Peak height is converted to TL using a simple linear equation derived from telomere standards of known lengths. The resulting three sets of TL estimates are nearly identical despite having been performed at 6-month intervals, with high linearity ( R 2 > 0.99). This reproducibility demonstrates the ability of PHAST to provide absolute telomere lengths with minimal calibration. ( B ) The six standards can be distinguished from each other by PHAST even when pre-mixed prior to detection, and have a smaller CV as the TL increases, as evidenced by the narrowing of the distributions with longer TL. ( C ) We have demonstrated the robustness of PHAST, by detecting a 2.4 kb telomere standard with and without a large excess of genomic DNA. Because the spurious peaks far outnumber the real peaks (by a factor of 10 to 1), the traces are normalized for clarity. After gating, the sample with genomic DNA (red solid trace) was essentially the same as that without any genomic DNA (black dashed trace). ( D ) To determine the ability to detect short telomeres using PHAST, 200-bp telomere standard is mixed at varying proportions with the 900-bp standard (0–50%). ( E ) After gating and counting the two sub-populations, the measured proportion was plotted against the nominal proportion. The two numbers were very consistent, with a slightly higher proportion measured than expected. This is attributed to incomplete labeling of some of the longer telomeres, which are misidentified as short telomeres. ( F ) Q – Q plots for the telomere estimates determined by PHAST and q-FISH are presented with three different cell lines (WI-83, U2OS and R83). This allows the distributions between two measurement methods to be compared directly even in the absence of common units and when the number of data points differs. Although PHAST and q-FISH are different methods, they yielded similar distribution shapes, as evidenced by the largely linear relationship between the corresponding quantiles. The red dashed line in each plot is the extrapolation of the interquartile range.
    Figure Legend Snippet: Telomere length (TL) determination using peak height. ( A ) Peak height is converted to TL using a simple linear equation derived from telomere standards of known lengths. The resulting three sets of TL estimates are nearly identical despite having been performed at 6-month intervals, with high linearity ( R 2 > 0.99). This reproducibility demonstrates the ability of PHAST to provide absolute telomere lengths with minimal calibration. ( B ) The six standards can be distinguished from each other by PHAST even when pre-mixed prior to detection, and have a smaller CV as the TL increases, as evidenced by the narrowing of the distributions with longer TL. ( C ) We have demonstrated the robustness of PHAST, by detecting a 2.4 kb telomere standard with and without a large excess of genomic DNA. Because the spurious peaks far outnumber the real peaks (by a factor of 10 to 1), the traces are normalized for clarity. After gating, the sample with genomic DNA (red solid trace) was essentially the same as that without any genomic DNA (black dashed trace). ( D ) To determine the ability to detect short telomeres using PHAST, 200-bp telomere standard is mixed at varying proportions with the 900-bp standard (0–50%). ( E ) After gating and counting the two sub-populations, the measured proportion was plotted against the nominal proportion. The two numbers were very consistent, with a slightly higher proportion measured than expected. This is attributed to incomplete labeling of some of the longer telomeres, which are misidentified as short telomeres. ( F ) Q – Q plots for the telomere estimates determined by PHAST and q-FISH are presented with three different cell lines (WI-83, U2OS and R83). This allows the distributions between two measurement methods to be compared directly even in the absence of common units and when the number of data points differs. Although PHAST and q-FISH are different methods, they yielded similar distribution shapes, as evidenced by the largely linear relationship between the corresponding quantiles. The red dashed line in each plot is the extrapolation of the interquartile range.

    Techniques Used: Derivative Assay, Labeling, Fluorescence In Situ Hybridization

    20) Product Images from "Exonuclease III-Regulated Target Cyclic Amplification-Based Single Nucleotide Polymorphism Detection Using Ultrathin Ternary Chalcogenide Nanosheets"

    Article Title: Exonuclease III-Regulated Target Cyclic Amplification-Based Single Nucleotide Polymorphism Detection Using Ultrathin Ternary Chalcogenide Nanosheets

    Journal: Frontiers in Chemistry

    doi: 10.3389/fchem.2019.00844

    (A) Fluorescence spectra of P/MT + Exo III + Ta 2 NiS 5 (black), P/WT + Exo III + Ta 2 NiS 5 (red), P/MT + Ta 2 NiS 5 (pink), and P/WT + Ta 2 NiS 5 (green). (B) The fluorescence intensity ratio (FP/MT/FP/WT) at 610 nm for P/MT + Exo III and P/WT + Exo III in the absence (black) and presence (red) of Ta 2 NiS 5 nanosheets. (C) Fluorescence intensity of P/MT + Exo III (black) and P/WT + Exo III (red) in the presence of Ta 2 NiS 5 nanosheets with different final concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 μg ml −1 (P = 1 μM; MT = 100 nM; WT = 100 nM; Exo III = 0.25 U μl −1 ). (D) The fluorescence intensity ratio (FP/MT/FP/WT) at 610 nm in the presence of Ta2NiS5 nanosheets with different final concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 μg ml −1 (P = 1 μM; MT = 100 nM; WT = 100 nM; Exo III = 0.25 U μl −1) . The excitation wavelength is 590 nm.
    Figure Legend Snippet: (A) Fluorescence spectra of P/MT + Exo III + Ta 2 NiS 5 (black), P/WT + Exo III + Ta 2 NiS 5 (red), P/MT + Ta 2 NiS 5 (pink), and P/WT + Ta 2 NiS 5 (green). (B) The fluorescence intensity ratio (FP/MT/FP/WT) at 610 nm for P/MT + Exo III and P/WT + Exo III in the absence (black) and presence (red) of Ta 2 NiS 5 nanosheets. (C) Fluorescence intensity of P/MT + Exo III (black) and P/WT + Exo III (red) in the presence of Ta 2 NiS 5 nanosheets with different final concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 μg ml −1 (P = 1 μM; MT = 100 nM; WT = 100 nM; Exo III = 0.25 U μl −1 ). (D) The fluorescence intensity ratio (FP/MT/FP/WT) at 610 nm in the presence of Ta2NiS5 nanosheets with different final concentrations of 2.5, 5.0, 7.5, 10.0, and 12.5 μg ml −1 (P = 1 μM; MT = 100 nM; WT = 100 nM; Exo III = 0.25 U μl −1) . The excitation wavelength is 590 nm.

    Techniques Used: Fluorescence

    (A) The fluorescence spectra of P (1 μM) in the presence of different concentrations of mutant-type target (0, 0.001, 0.01, 0.1, 1, 10, and 100 nM) and Exo III (0.25 U μl −1 ) with addition of Ta 2 NiS 5 nanosheets (5.0 μg ml −1 ). (B) Relationship between fluorescence intensity at 610 nm and the concentrations of mutant-type target. Inset: Calibration curve for detection of mutant-type target. (C) Fluorescence spectra of different percentage of mutant-type target in mixed DNA samples (MT/(MT + WT) was 0, 5, 10, 20, 40, 60, 80, and 100%). (D) Fluorescence intensity at 610 nm as a function of allele frequency. The total concentration of the mutant and wild-type target is 100 pM. The excitation wavelength is 590 nm.
    Figure Legend Snippet: (A) The fluorescence spectra of P (1 μM) in the presence of different concentrations of mutant-type target (0, 0.001, 0.01, 0.1, 1, 10, and 100 nM) and Exo III (0.25 U μl −1 ) with addition of Ta 2 NiS 5 nanosheets (5.0 μg ml −1 ). (B) Relationship between fluorescence intensity at 610 nm and the concentrations of mutant-type target. Inset: Calibration curve for detection of mutant-type target. (C) Fluorescence spectra of different percentage of mutant-type target in mixed DNA samples (MT/(MT + WT) was 0, 5, 10, 20, 40, 60, 80, and 100%). (D) Fluorescence intensity at 610 nm as a function of allele frequency. The total concentration of the mutant and wild-type target is 100 pM. The excitation wavelength is 590 nm.

    Techniques Used: Fluorescence, Mutagenesis, Concentration Assay

    21) Product Images from "Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes"

    Article Title: Inhibition of nuclear factor kappaB proteins-platinated DNA interactions correlates with cytotoxic effectiveness of the platinum complexes

    Journal: Scientific Reports

    doi: 10.1038/srep28474

    Differential inhibition of dumbbell decoy activity by DNA adducts of BBR 3464 as compared with cisplatin and transplatin in HEK293-NF-кB-luciferase reporter cell line. The percentage of inhibition of the specific decoy activity was calculated by measuring, for each experiment, the difference between luciferase activities obtained with cells transfected with platinated DUMBBELL-кB and nonplatinated DUMBBELL-kB divided by the difference between luciferase activities obtained with cells transfected with nonplatinated scrambled and specific (κB-site containing) DUMBBELL decoy oligonucleotides. Data represent the mean ± SD obtained from triplicate wells and are representative of at least three independent experiments. Data for ciplatin and transplatin were taken from ref. 10 .
    Figure Legend Snippet: Differential inhibition of dumbbell decoy activity by DNA adducts of BBR 3464 as compared with cisplatin and transplatin in HEK293-NF-кB-luciferase reporter cell line. The percentage of inhibition of the specific decoy activity was calculated by measuring, for each experiment, the difference between luciferase activities obtained with cells transfected with platinated DUMBBELL-кB and nonplatinated DUMBBELL-kB divided by the difference between luciferase activities obtained with cells transfected with nonplatinated scrambled and specific (κB-site containing) DUMBBELL decoy oligonucleotides. Data represent the mean ± SD obtained from triplicate wells and are representative of at least three independent experiments. Data for ciplatin and transplatin were taken from ref. 10 .

    Techniques Used: Inhibition, Activity Assay, Luciferase, Transfection

    Binding of NF-кB proteins to the DNA duplex containing the кB site. ( A ) The nucleotide sequence of the 22-bp oligodeoxyribonucleotide duplex containing κB site (DUPLEX-κB). The bold letters in the sequence indicate the κB recognition sequence. Left panels in Fig. 2B–D. Binding of p50/p65 heterodimer and p50/p50 and p65/p65 homodimers to the DUPLEX-κB containing the кB site (( B–D ), respectively). The panels show autoradiograms of the EMSA experiments showing the binding of p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 (Fig. 2D) homodimers to the DUPLEX-κB. Lanes 1 and 2, non-modified duplex; lanes 3–5, duplex globally modified by BBR3464 at r b = 0.023, 0.045, or 0.091, respectively. The gel mobility shift assay was performed as described in the section Materials and Methods; concentration of the oligonucleotide duplex was 1 nM and the concentrations of p50/p50, p65/p65 and p50/p65 were 10, 15 and 15 nM, respectively. Right panels in Fig. 2B–D. Plots of the amount of the DUPLEX-κB modified by BBR3464 (full line), cisplatin (dashed line) or transplatin (dotted line) in complex with p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 homodimers (Fig. 2D) on r b ; the data for cisplatin and transplatin were taken from ref. 10 . Data are the mean ± SD obtained from three different experiments.
    Figure Legend Snippet: Binding of NF-кB proteins to the DNA duplex containing the кB site. ( A ) The nucleotide sequence of the 22-bp oligodeoxyribonucleotide duplex containing κB site (DUPLEX-κB). The bold letters in the sequence indicate the κB recognition sequence. Left panels in Fig. 2B–D. Binding of p50/p65 heterodimer and p50/p50 and p65/p65 homodimers to the DUPLEX-κB containing the кB site (( B–D ), respectively). The panels show autoradiograms of the EMSA experiments showing the binding of p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 (Fig. 2D) homodimers to the DUPLEX-κB. Lanes 1 and 2, non-modified duplex; lanes 3–5, duplex globally modified by BBR3464 at r b = 0.023, 0.045, or 0.091, respectively. The gel mobility shift assay was performed as described in the section Materials and Methods; concentration of the oligonucleotide duplex was 1 nM and the concentrations of p50/p50, p65/p65 and p50/p65 were 10, 15 and 15 nM, respectively. Right panels in Fig. 2B–D. Plots of the amount of the DUPLEX-κB modified by BBR3464 (full line), cisplatin (dashed line) or transplatin (dotted line) in complex with p50/p65 heterodimer (Fig. 2B), p50/p50 (Fig. 2C) and p65/p65 homodimers (Fig. 2D) on r b ; the data for cisplatin and transplatin were taken from ref. 10 . Data are the mean ± SD obtained from three different experiments.

    Techniques Used: Binding Assay, Sequencing, Modification, Mobility Shift, Concentration Assay

    22) Product Images from "Detection of 5-Hydroxymethylcytosine in DNA by Transferring a Keto-Glucose Using T4 phage β-Glucosyltransferase**"

    Article Title: Detection of 5-Hydroxymethylcytosine in DNA by Transferring a Keto-Glucose Using T4 phage β-Glucosyltransferase**

    Journal: Chembiochem : a European journal of chemical biology

    doi: 10.1002/cbic.201100278

    Exonuclease III digestion assay of a 11-mer biotin-keto-5-gmC-contaning DNA in the presence of streptavidin, showing MALDI spectra after the digestion. Exonuclease III digestion can be blocked mainly at one base before the modification, but also right at the modification position. Black is theoretical MS; grey is the observed MS.
    Figure Legend Snippet: Exonuclease III digestion assay of a 11-mer biotin-keto-5-gmC-contaning DNA in the presence of streptavidin, showing MALDI spectra after the digestion. Exonuclease III digestion can be blocked mainly at one base before the modification, but also right at the modification position. Black is theoretical MS; grey is the observed MS.

    Techniques Used: Modification, Mass Spectrometry

    23) Product Images from "The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini"

    Article Title: The mitochondrial genome of the pathogenic yeast Candida subhashii: GC-rich linear DNA with a protein covalently attached to the 5? termini

    Journal: Microbiology

    doi: 10.1099/mic.0.038646-0

    The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.
    Figure Legend Snippet: The termini of the linear mtDNA are bound by a protein. (a) DNA samples were prepared from C. subhashii (see Methods) and separated by PFGE in a 1.5 % agarose gel. The gel was stained with 0.5 μg ml −1 ethidium bromide (EtBr) and transferred onto a nylon membrane. The blot was hybridized with radioactively labelled mtDNA from C. subhashii . Lane 1, isolated mtDNA; lanes 2 and 3, total cellular DNA prepared in agarose blocks treated or untreated with proteinase K, respectively. (b) Approximately 1 μg of isolated mtDNA was treated with exonuclease III (ExoIII) (left panel) or BAL-31 nuclease (right panel), as indicated. The mtDNA was then extracted from reactions, digested with Xba I endonuclease and electrophoretically separated. Note that the terminal fragments are sensitive to ExoIII but apparently not to BAL-31, indicating the possibility that the linear molecules have their 5′ termini blocked. L and R, positions of the 1527 and 2803 bp terminal restriction enzyme fragments, respectively; C, position of the internal control (a 1040 bp long linear blunt-ended DNA fragment) mixed with mtDNA prior to digestion with BAL-31 nuclease. (c) The mtDNA–protein complexes were isolated as described in Methods, digested with restriction endonucleases Cla I or Pvu I, and treated or not treated with proteinase K. The positions of terminal restriction enzyme fragments generated by Pvu I (833 and 2339 bp) and Cla I (547 bp) are indicated as L, R and L+R, respectively. Note that both terminal Cla I fragments have identical sizes, as this enzyme digests the C. subhashii mtDNA within TIRs.

    Techniques Used: Agarose Gel Electrophoresis, Staining, Isolation, Generated

    24) Product Images from "First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid"

    Article Title: First Description of Natural and Experimental Conjugation between Mycobacteria Mediated by a Linear Plasmid

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029884

    PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.
    Figure Legend Snippet: PFGE of DNA genomic preparations. (A) PFGE with undigested DNAs from M. avium 88.3 (1) and M. kansasii 88.8 (2) under different switch times, indicated below each figure; (B) pMA100 extracted from PFGE gels and treated with exonuclease III (3) or exonuclease lambda (4); (C) pMA100 extracted from PFGE gels and treated (+) or not (-) with topoisomerase I; (D) DNA prepared with (+) or without (-) adding proteinase K to the lysis buffer; (E) same as in (D) in PFGE gels and running buffer prepared with 0.2% SDS. λ: DNA concatemers of the bacteriophage λ genome.

    Techniques Used: Lysis

    25) Product Images from "Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates"

    Article Title: Escherichia coli exonuclease III enhances long PCR amplification of damaged DNA templates

    Journal: Nucleic Acids Research

    doi:

    PCR product ratio (with/without exonuclease III) as a function of the percentage of heat-induced mtDNA loss. Four Qiagen-extracted mouse liver DNA samples were heated at 99°C for 0, 30, 60 and 90 s. Residual mtDNA was quantitated by Southern blot with an mtDNA probe while a 8636 bp mtDNA fragment was amplified with Protocol 1b, with or without 25 U of exonuclease III.
    Figure Legend Snippet: PCR product ratio (with/without exonuclease III) as a function of the percentage of heat-induced mtDNA loss. Four Qiagen-extracted mouse liver DNA samples were heated at 99°C for 0, 30, 60 and 90 s. Residual mtDNA was quantitated by Southern blot with an mtDNA probe while a 8636 bp mtDNA fragment was amplified with Protocol 1b, with or without 25 U of exonuclease III.

    Techniques Used: Polymerase Chain Reaction, Southern Blot, Amplification

    Exonuclease III enhances long PCR amplification from phenol-extracted DNA samples. DNA samples were extracted with phenol/chloroform and either stored at –20 or –80°C for several years (mouse and human DNA, respectively) or used immediately (rat DNA). After PCR, agarose gels (0.7–1.2%) were loaded with 22 µl of the PCR products together with Hin dIII-digested phage λ DNA (M). ( A ) Five mouse liver DNA samples (ML1–ML5) were used for PCR co-amplification of the 316 and 8636 bp mtDNA fragments, using Protocol 1a without (exo 0) or with 25 U of exonuclease III (exo +). ( B ) Four rat liver DNA samples (RL1–RL4) were used for long PCR amplification of a 15.4 kb mtDNA fragment, using Protocol 2 without (exo 0) or with 25 U of exonuclease III (exo +). ( C ) Five human blood DNA samples (HB1–HB5) were used for long PCR amplification of a 5 kb fragment from the human CYP2D6 nuclear gene, using Protocol 3 without (exo 0) or with 50 U of exonuclease III (exo +).
    Figure Legend Snippet: Exonuclease III enhances long PCR amplification from phenol-extracted DNA samples. DNA samples were extracted with phenol/chloroform and either stored at –20 or –80°C for several years (mouse and human DNA, respectively) or used immediately (rat DNA). After PCR, agarose gels (0.7–1.2%) were loaded with 22 µl of the PCR products together with Hin dIII-digested phage λ DNA (M). ( A ) Five mouse liver DNA samples (ML1–ML5) were used for PCR co-amplification of the 316 and 8636 bp mtDNA fragments, using Protocol 1a without (exo 0) or with 25 U of exonuclease III (exo +). ( B ) Four rat liver DNA samples (RL1–RL4) were used for long PCR amplification of a 15.4 kb mtDNA fragment, using Protocol 2 without (exo 0) or with 25 U of exonuclease III (exo +). ( C ) Five human blood DNA samples (HB1–HB5) were used for long PCR amplification of a 5 kb fragment from the human CYP2D6 nuclear gene, using Protocol 3 without (exo 0) or with 50 U of exonuclease III (exo +).

    Techniques Used: Polymerase Chain Reaction, Amplification

    Exonuclease III enhances long PCR amplification of the 8636 bp mtDNA fragment from depurinated mouse liver DNA samples. Aliquots of the same Qiagen-extracted mouse liver DNA preparation were treated in depurination buffer at 70°C for 0, 20, 40 or 60 min (AP0, AP20, AP40 and AP60, respectively) and the 8636 bp mtDNA fragment was amplified with Protocol 1b without (exo 0) or with 25 U of exonuclease III (exo +). The agarose gel (0.8%) was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.
    Figure Legend Snippet: Exonuclease III enhances long PCR amplification of the 8636 bp mtDNA fragment from depurinated mouse liver DNA samples. Aliquots of the same Qiagen-extracted mouse liver DNA preparation were treated in depurination buffer at 70°C for 0, 20, 40 or 60 min (AP0, AP20, AP40 and AP60, respectively) and the 8636 bp mtDNA fragment was amplified with Protocol 1b without (exo 0) or with 25 U of exonuclease III (exo +). The agarose gel (0.8%) was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    Effect of exonuclease III on long PCR amplification performed with either rTth DNA polymerase alone (rTth) or in combination with Vent DNA polymerase (Vent). Aliquots of two phenol-extracted mouse liver DNA samples were used for long PCR amplification of the 8636 bp mtDNA fragment in the absence (exo 0) or presence of 25 U of exonuclease III (exo +). The 0.8% agarose gel was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.
    Figure Legend Snippet: Effect of exonuclease III on long PCR amplification performed with either rTth DNA polymerase alone (rTth) or in combination with Vent DNA polymerase (Vent). Aliquots of two phenol-extracted mouse liver DNA samples were used for long PCR amplification of the 8636 bp mtDNA fragment in the absence (exo 0) or presence of 25 U of exonuclease III (exo +). The 0.8% agarose gel was loaded with 22 µl of the PCR products. M, Hin dIII-digested phage λ DNA.

    Techniques Used: Polymerase Chain Reaction, Amplification, Agarose Gel Electrophoresis

    Escherichia coli exonuclease III enhances long PCR amplification of mtDNA from heat-damaged mouse liver DNA templates. Qiagen-extracted mouse liver DNA was heated at 99°C for 30–120 s and two distinct regions of the mtDNA were co-amplified with Protocol 1a using 14 pmol of primers for the 316 bp PCR product and 40 pmol for the 8636 bp PCR product. Lanes 1–4 correspond to aliquots of the same mouse liver DNA sample heated for 30, 60, 90 and 120 s, respectively. Agarose gels (1.2%) were loaded with 22 µl of the products. M is Hin dIII-digested phage λ DNA (fragment sizes 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.56 kb). ( A ) PCR reactions were performed without exonuclease III (exo 0) or with 25 U of exonuclease III (exo 25 U). ( B ) PCR reactions were performed with either 5 or 1 U of exonuclease III (exo 5 U and exo 1 U) or with 25 U of exonuclease III preheated at 99°C for 10 min (preheated exo).
    Figure Legend Snippet: Escherichia coli exonuclease III enhances long PCR amplification of mtDNA from heat-damaged mouse liver DNA templates. Qiagen-extracted mouse liver DNA was heated at 99°C for 30–120 s and two distinct regions of the mtDNA were co-amplified with Protocol 1a using 14 pmol of primers for the 316 bp PCR product and 40 pmol for the 8636 bp PCR product. Lanes 1–4 correspond to aliquots of the same mouse liver DNA sample heated for 30, 60, 90 and 120 s, respectively. Agarose gels (1.2%) were loaded with 22 µl of the products. M is Hin dIII-digested phage λ DNA (fragment sizes 23.1, 9.4, 6.6, 4.4, 2.3, 2.0 and 0.56 kb). ( A ) PCR reactions were performed without exonuclease III (exo 0) or with 25 U of exonuclease III (exo 25 U). ( B ) PCR reactions were performed with either 5 or 1 U of exonuclease III (exo 5 U and exo 1 U) or with 25 U of exonuclease III preheated at 99°C for 10 min (preheated exo).

    Techniques Used: Polymerase Chain Reaction, Amplification

    26) Product Images from "Evolution of linear chromosomes and multipartite genomes in yeast mitochondria"

    Article Title: Evolution of linear chromosomes and multipartite genomes in yeast mitochondria

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq1345

    PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region
    Figure Legend Snippet: PFGE analysis of the yeast mtDNAs. The whole-cell DNA samples were separated by PFGE using a CHEF Mapper XA Chiller System (Biorad), blotted onto a nylon membrane and hybridized with mtDNA-derived probes as described in ‘Material and Methods’ section. Lane 1— C. viswanathii CBS 4024; lane 2— C. sojae CBS 7871; lane 3— C. maltosa CBS 5611; lane 4— C. neerlandica NRRL Y-27057; lane 5— C. alai NRRL Y-27739; lane 6— C. labiduridarum NRRL Y-27940; lane 7— C. frijolesensis NRRL Y-48060; lane 8— C. subhashii CBS 10753; lane 9— C. jiufengensis CBS 10846; lane 10— C. albicans CBS 562. Note that three discrete bands migrating in the region

    Techniques Used: Derivative Assay

    Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.
    Figure Legend Snippet: Multipartite linear-mapping genomes in C. labiduridarum and C. frijolesensis ( A ) PFGE separated samples of C. labiduridarum NRRL Y-27940 (lane 1) and C. frijolesensis NRRL Y-48060 (lane 2) were blotted onto a nylon membrane and hybridized with the radioactively labeled probes P-668 and H-1030 (regions hybridizing with both probes are shown as dashed lines). Presumed master (I) and two smaller chromosomes (II and III) are indicated. Note that the master chromosome occurs in four isomers (i.e. L III − R III − L II − R II (shown in the scheme), L III − R III − R II − L II , R III − L III − L II − R II and R III − L III − R II − L II . ‘L’ and ‘R’ indicate the left and the right telomere, respectively). The C. frijolesensis mtDNA (∼1 µg) was digested with BAL-31 nuclease ( B ) or exonuclease III (ExoIII) ( C ) as indicated. After nuclease inactivation, the DNA was digested with EcoRV, separated in 0.9% (w/v) agarose gel. The Southern blots were hybridized with the P-668 and EH-1350 probes specific for the left and the right arm of the master chromosome, respectively (see ‘Materials and Methods’ section). Arrows show the positions of the left (L) and right (R) terminal fragments and their fusions (R + R, R + L and L + L). Note that after ExoIII treatment the telomeric fragments form two subpopulations that differ in their sensitivity to the ExoIII treatment. This indicates that the linear mtDNA molecules possess an open structure with 5′ overhang or blunt end or covalently closed t-hairpin. ( D ) The C. frijolesensis mtDNA was treated with antarctic phosphatase and labeled with [γ 32 P]ATP and T4 polynucleotide kinase. The mtDNA was then digested with restriction endonuclease EcoRV (lane 1) or BglII (lane 2) and separated in 0.8% (w/v) agarose gel (left panel). The gel was fixed in 10% (v/v) methanol/10% (v/v) acetic acid for 30 min, dried overnight and autoradiographed (right panel). Arrows indicate the position of telomeric fragments containing the open structures accessible to terminal labeling.

    Techniques Used: Labeling, Agarose Gel Electrophoresis

    27) Product Images from "Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta"

    Article Title: Robust physical methods that enrich genomic regions identical by descent for linkage studies: confirmation of a locus for osteogenesis imperfecta

    Journal: BMC Genetics

    doi: 10.1186/1471-2156-10-16

    Physical IBD enrichment process . Genomic DNAs (gDNA) are isolated from two related, afflicted individuals and digested with a restriction enzyme that leaves Exonuclease III-resistant ends and generates fragments of about 4 Kb. DNA fragments are modified (diamonds or ovals) to permit discrimination between the individuals, for example by presence or absence of methylation at GATC sequences. The fragments are mixed, denatured and renatured under conditions to favour unique copy reannealing. Non-annealed strands and hybrids of strands from the same individual are eliminated. Reannealed DNA fragments with one strand from each individual are called heterohybrids, which may be perfectly paired due to inheritance of both strands from the same ancestral sequence or mismatched due to variation between different ancestral sequences. Mismatched heterohybrid fragments are removed by LSHase, a nucleolytic cocktail of MutL, MutS and MutH, and subsequent digestion by Exonuclease III. The resulting IBD-enriched DNA is generically amplified, labelled and mapped by two-colour hybridization to genomic topographic arrays, using the reannealed DNA as reference. The process is repeated for other afflicted pairs in the same family and in additional families. Variations include use of oligonucleotides as discrimination tags, reducing the number of steps by combining similar intermediate purification procedures, and eventually mapping IBD regions by high throughput redundant sequencing, with or without amplification.
    Figure Legend Snippet: Physical IBD enrichment process . Genomic DNAs (gDNA) are isolated from two related, afflicted individuals and digested with a restriction enzyme that leaves Exonuclease III-resistant ends and generates fragments of about 4 Kb. DNA fragments are modified (diamonds or ovals) to permit discrimination between the individuals, for example by presence or absence of methylation at GATC sequences. The fragments are mixed, denatured and renatured under conditions to favour unique copy reannealing. Non-annealed strands and hybrids of strands from the same individual are eliminated. Reannealed DNA fragments with one strand from each individual are called heterohybrids, which may be perfectly paired due to inheritance of both strands from the same ancestral sequence or mismatched due to variation between different ancestral sequences. Mismatched heterohybrid fragments are removed by LSHase, a nucleolytic cocktail of MutL, MutS and MutH, and subsequent digestion by Exonuclease III. The resulting IBD-enriched DNA is generically amplified, labelled and mapped by two-colour hybridization to genomic topographic arrays, using the reannealed DNA as reference. The process is repeated for other afflicted pairs in the same family and in additional families. Variations include use of oligonucleotides as discrimination tags, reducing the number of steps by combining similar intermediate purification procedures, and eventually mapping IBD regions by high throughput redundant sequencing, with or without amplification.

    Techniques Used: Isolation, Modification, Methylation, Sequencing, Amplification, Hybridization, Purification, High Throughput Screening Assay

    28) Product Images from "A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells"

    Article Title: A specific DNA-nanoprobe for tracking the activities of human apurinic/apyrimidinic endonuclease 1 in living cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1205

    ( A ) Comparison of the responses of four different Nanoprobes to APE1 and DNase I. ( B ) Zeta potential values of the four different Nanoprobes. ( C ) Influences of free avidin or streptavidin molecules on the reactions between APE1 and AP-DNA in homogeneous solutions. Probe 1 (BHQ1-46-biotin/18-FAM duplex, Supplementary Table S1 ) and Probe 1΄ (BHQ1-46/18-FAM duplex) were used as the Biotin-labeled and Non-biotin-labeled AP-DNA, respectively. All experiments were repeated at least three times.
    Figure Legend Snippet: ( A ) Comparison of the responses of four different Nanoprobes to APE1 and DNase I. ( B ) Zeta potential values of the four different Nanoprobes. ( C ) Influences of free avidin or streptavidin molecules on the reactions between APE1 and AP-DNA in homogeneous solutions. Probe 1 (BHQ1-46-biotin/18-FAM duplex, Supplementary Table S1 ) and Probe 1΄ (BHQ1-46/18-FAM duplex) were used as the Biotin-labeled and Non-biotin-labeled AP-DNA, respectively. All experiments were repeated at least three times.

    Techniques Used: Avidin-Biotin Assay, Labeling

    ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.
    Figure Legend Snippet: ( A ) Fluorescence responses of Nanoprobe A (0.1 mg/ml) to APE1 at different concentrations. ( B ) Linear calibration curve for detection of the activity of APE1. The linear regression equation is F = 0.20 c (U/ml) – 2.1 × 10 −4 , and the detection limit is 0.01 U/ml. ( C ) Selectivity of Nanoprobe A toward APE1 (2.0 U/ml) over other nucleases. (DNase I: 5.0 U/ml; Exo III: 4.0 U/ml; lambda exo: 66.7 U/ml; Exo I: 12.5 U/ml; T5: 5.0 U/ml; T7: 50 U/ml). All experiments were repeated at least three times.

    Techniques Used: Fluorescence, Activity Assay

    ( A ) SPR measurement results of the interactions between immobilized APE1 and avidin. ( B ) Selective binding of APE1 by immobilized avidin. For comparison, RNase A which has the same pI as APE1 and Exo III which has the same molecular weight as APE1 were also tested. All experiments were repeated at least three times.
    Figure Legend Snippet: ( A ) SPR measurement results of the interactions between immobilized APE1 and avidin. ( B ) Selective binding of APE1 by immobilized avidin. For comparison, RNase A which has the same pI as APE1 and Exo III which has the same molecular weight as APE1 were also tested. All experiments were repeated at least three times.

    Techniques Used: SPR Assay, Avidin-Biotin Assay, Binding Assay, Molecular Weight

    29) Product Images from "A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification"

    Article Title: A Label-Free and Sensitive Fluorescent Qualitative Assay for Bisphenol A Based on Rolling Circle Amplification/Exonuclease III-Combined Cascade Amplification

    Journal: Nanomaterials

    doi: 10.3390/nano6100190

    Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.
    Figure Legend Snippet: Fluorescence-emission spectra of zinc(II)-protoporphyrin IX (ZnPPIX)/G-quadruplex supramolecular fluorescent labels under different conditions: ( a ) Buffer; ( b ) Buffer + ZnPPIX; ( c ) RP (DNA duplex probe) + Circle DNA + hairpin probes (GHP) + Exonuclease III (Exo III) + ZnPPIX; ( d ) BPA + RP + Circle DNA + GHP + Exo III + ZnPPIX; C BPA = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, C ZnPPIX = 20 μM, RCA reaction time 1.5 h.

    Techniques Used: Fluorescence

    Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.
    Figure Legend Snippet: Schematic illustration the principle of the fluorescent assay of Bisphenol A (BPA) based on the rolling circle amplification (RCA)/Exo III-combined cascade signal amplification strategy.

    Techniques Used: Fluorescence, Amplification

    ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.
    Figure Legend Snippet: ( a ) Agarose gel (0.7%) electrophoresis: (1) DNA 1 alone; (2) P 1 alone; (3) Circle DNA alone; (4) GHP alone; (5) RP + Circle DNA; (6) BPA + RP + Circle DNA; (7) RP + Circle DNA + GHP; (8) BPA + RP + Circle DNA + GHP; ( b ) Atomic force microscope (AFM) images of amplification products of RCA/Exo III-combined cascade signal amplification reaction. C DNA1 = 1.0 μM, C P1 = 1.0 μM, C RP = 1.0 μM, C Circle DNA = 100 nM, C GHP = 25 μM, C Exo III = 100 U, RCA reaction time 1.5 h.

    Techniques Used: Agarose Gel Electrophoresis, Electrophoresis, Microscopy, Amplification

    30) Product Images from "Platinum anticancer drug damage enforces a particular rotational setting of DNA in nucleosomes"

    Article Title: Platinum anticancer drug damage enforces a particular rotational setting of DNA in nucleosomes

    Journal:

    doi: 10.1073/pnas.0506025102

    Exonuclease III analysis of native and platinated nucleosomes. All four nucleosome samples, and the corresponding free DNA, were treated with exonuclease III to assess the positioning of the DNA on the histone core. ( A ) Denaturing PAGE analysis (8% polyacrylamide)
    Figure Legend Snippet: Exonuclease III analysis of native and platinated nucleosomes. All four nucleosome samples, and the corresponding free DNA, were treated with exonuclease III to assess the positioning of the DNA on the histone core. ( A ) Denaturing PAGE analysis (8% polyacrylamide)

    Techniques Used: Polyacrylamide Gel Electrophoresis

    31) Product Images from "T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR"

    Article Title: T5 Exonuclease Hydrolysis of Hepatitis B Virus Replicative Intermediates Allows Reliable Quantification and Fast Drug Efficacy Testing of Covalently Closed Circular DNA by PCR

    Journal: Journal of Virology

    doi: 10.1128/JVI.01117-18

    T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).
    Figure Legend Snippet: T5 Exo and Exo III remove HBV replicative intermediates without affecting cccDNA. HepG2 hNTCP cells were seeded in a 6-well plate and infected at an mge/cell of 3,000. To block entry, Myrcludex B (2 μM) was used as a control. (A) On day 7 p.i., cytosolic DNA samples were extracted as described in Materials and Methods and hydrolyzed by Exo I (5 U, 60 min), Exo III (25 U, 60 min), Exo I and III (5 U plus 25 U, 60 min), T5 Exo (5 U, 60 min), PSD (10 U, 60 min), and EcoRI (10 U, 60 min) at 37°C for 1 h, and later on, all enzymes were heat denatured at 70°C. Samples were analyzed by Southern blotting (left) and PCR with pp466-541 (right). (B) HepG2 hNTCP cells were infected in a 6-well plate format for 7 days, and the DNA samples were Hirt extracted and hydrolyzed by the respective enzymes prior to Southern blotting (left) and cccDNA-specific PCR using pp1040-1996 (right).

    Techniques Used: Infection, Blocking Assay, Southern Blot, Polymerase Chain Reaction

    32) Product Images from "Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed"

    Article Title: Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed

    Journal: Mobile DNA

    doi: 10.1186/1759-8753-2-1

    Model for protein-primed reverse transcription by the pFOXC-reverse transcriptase (RT) . Transcription of the pFOXC plasmid DNA molecules produces full-length RNAs that appear to function as both mRNAs for the synthesis of the RT and as templates for (-) strand cDNA synthesis [ 6 ]. Transcripts of pFOXC3 terminate in approximately three pentameric repeats, whereas transcripts of pFOXC1 terminate in approximately four copies of a 3 bp sequence (the 3' terminus of in vitro RNA used in this study is shown). Following production of the plasmid-encoded RT, deoxynucleotidylation occurs with the covalent addition of dAMP to a tyrosine residue of the 60 kDa pFOXC3-RT, followed by incorporation of deoxyguanosine monophosphate (dGMP) and a third nucleotide. Deoxynucleotidylation of the pFOXC1-RT results in the addition of thymidine monophosphate (TMP) to the RT, followed by one or more deoxynucleotide monophosphates (dNMPs) (a second TMP is shown). The resulting RT-(dNMP) n complex would have complementarity to the corresponding terminal repeat. Based on studies of protein-primed DNA elements, the model predicts that the complex anneals to the penultimate 3' repeat of the template (shown for pFOXC1 only). Following the synthesis of a unit-length repeat, the RT-(dNMP) n complex undergoes a slideback and is repositioned opposite the terminal repeat. The nascent cDNA is elongated via reverse transcription of the template by the 5'-linked RT or by a separate RT recruited to the complex. The model could also accommodate an increase in the number of repeats, depending on the number of slideback events that occur.
    Figure Legend Snippet: Model for protein-primed reverse transcription by the pFOXC-reverse transcriptase (RT) . Transcription of the pFOXC plasmid DNA molecules produces full-length RNAs that appear to function as both mRNAs for the synthesis of the RT and as templates for (-) strand cDNA synthesis [ 6 ]. Transcripts of pFOXC3 terminate in approximately three pentameric repeats, whereas transcripts of pFOXC1 terminate in approximately four copies of a 3 bp sequence (the 3' terminus of in vitro RNA used in this study is shown). Following production of the plasmid-encoded RT, deoxynucleotidylation occurs with the covalent addition of dAMP to a tyrosine residue of the 60 kDa pFOXC3-RT, followed by incorporation of deoxyguanosine monophosphate (dGMP) and a third nucleotide. Deoxynucleotidylation of the pFOXC1-RT results in the addition of thymidine monophosphate (TMP) to the RT, followed by one or more deoxynucleotide monophosphates (dNMPs) (a second TMP is shown). The resulting RT-(dNMP) n complex would have complementarity to the corresponding terminal repeat. Based on studies of protein-primed DNA elements, the model predicts that the complex anneals to the penultimate 3' repeat of the template (shown for pFOXC1 only). Following the synthesis of a unit-length repeat, the RT-(dNMP) n complex undergoes a slideback and is repositioned opposite the terminal repeat. The nascent cDNA is elongated via reverse transcription of the template by the 5'-linked RT or by a separate RT recruited to the complex. The model could also accommodate an increase in the number of repeats, depending on the number of slideback events that occur.

    Techniques Used: Plasmid Preparation, Sequencing, In Vitro

    33) Product Images from "Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition"

    Article Title: Multiple serine transposase dimers assemble the transposon-end synaptic complex during IS607-family transposition

    Journal: eLife

    doi: 10.7554/eLife.39611

    Footprint analysis of IS 1535 deletion substrate LE(v54-20v) containing the minimal transposon sequences required for efficient PEC assembly. ( A ) DNase I footprints of PEC assembly reactions on 5′- 32 P-labeled bottom and top strands of LE(v54-20v). TnpA concentrations were from 4 to 128 nM in 2-fold increasing amounts. Shaded rectangles on the left of the gels denote the positions of transposon sequences; coordinates labeled with v are vector sequences with vH being the equivalent locations of host DNA. The bars on the right of the gels denote regions of significant changes in DNase I reactivity by TnpA with dashes indicating weakly protected regions. ( B ) Exonuclease III delineated boundaries of TnpA binding. TnpA concentrations are the same as in panel A. ( C ) Summary of DNase I (strongly protected regions, blue) and Exo III digestion boundaries on the LE(v54-20v) sequence. Small letters denote vector sequence.
    Figure Legend Snippet: Footprint analysis of IS 1535 deletion substrate LE(v54-20v) containing the minimal transposon sequences required for efficient PEC assembly. ( A ) DNase I footprints of PEC assembly reactions on 5′- 32 P-labeled bottom and top strands of LE(v54-20v). TnpA concentrations were from 4 to 128 nM in 2-fold increasing amounts. Shaded rectangles on the left of the gels denote the positions of transposon sequences; coordinates labeled with v are vector sequences with vH being the equivalent locations of host DNA. The bars on the right of the gels denote regions of significant changes in DNase I reactivity by TnpA with dashes indicating weakly protected regions. ( B ) Exonuclease III delineated boundaries of TnpA binding. TnpA concentrations are the same as in panel A. ( C ) Summary of DNase I (strongly protected regions, blue) and Exo III digestion boundaries on the LE(v54-20v) sequence. Small letters denote vector sequence.

    Techniques Used: Labeling, Plasmid Preparation, Binding Assay, Sequencing

    34) Product Images from "Analysis of variants in Chinese individuals with primary open-angle glaucoma using molecular inversion probe (MIP)-based panel sequencing"

    Article Title: Analysis of variants in Chinese individuals with primary open-angle glaucoma using molecular inversion probe (MIP)-based panel sequencing

    Journal: Molecular Vision

    doi:

    Design of molecular inversion probe and MIP-based target-sequencing workflow. A : Molecular inversion probe (MIP) structure and design for regions of interest. The general structure for the MIP is a common 41-bp linker flanked by a target-specific extension and a ligation arm of 16 to 28 bp. B : MIP-based target-sequencing workflow. (i) Genomic DNA was sheared into 300–500 bp fragments. (ii) Pooled probes are added to the genomic DNA, and each probe is hybridized to the target DNA sequences through the target-specific sequences on both ends. (iii) The gaps are filled and circularized with DNA polymerase and ligase. (iv) The linear genomic DNA and uncircularized padlock probes are removed with exonuclease. (v) The circularized probes are amplified with a universal sequencing primer and a sample barcode primer.
    Figure Legend Snippet: Design of molecular inversion probe and MIP-based target-sequencing workflow. A : Molecular inversion probe (MIP) structure and design for regions of interest. The general structure for the MIP is a common 41-bp linker flanked by a target-specific extension and a ligation arm of 16 to 28 bp. B : MIP-based target-sequencing workflow. (i) Genomic DNA was sheared into 300–500 bp fragments. (ii) Pooled probes are added to the genomic DNA, and each probe is hybridized to the target DNA sequences through the target-specific sequences on both ends. (iii) The gaps are filled and circularized with DNA polymerase and ligase. (iv) The linear genomic DNA and uncircularized padlock probes are removed with exonuclease. (v) The circularized probes are amplified with a universal sequencing primer and a sample barcode primer.

    Techniques Used: Sequencing, Ligation, Amplification

    35) Product Images from "hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism"

    Article Title: hSWI/SNF-Catalyzed Nucleosome Sliding Does Not Occur Solely via a Twist-Diffusion Mechanism

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.21.7484-7490.2002

    Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.
    Figure Legend Snippet: Exo III analysis of nucleosome positioning before and after remodeling by hSWI/SNF. Glycerol-gradient-purified nucleosomes reconstituted with native (A), flap (B), hairpin (C), or nick (D) templates were incubated in the absence or presence of hSWI/SNF+ATP and then subjected to Exo III digestion for 5 min. Each panel shows, from left to right, G-specific cleavage of native 5S DNA as a marker, Exo III digestion pattern of naked template DNA, undigested nucleosomes, and Exo III digestion of nucleosomes before and after hSWI/SNF remodeling. The positions of the downstream edge of the nucleosomes before and after SWI/SNF remodeling are schematically represented on the left and right sides of the gel, respectively. The dark half ovals and the light ovals represent the major and minor nucleosome positions as determined from the Exo III data.

    Techniques Used: Purification, Incubation, Marker

    36) Product Images from "Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus"

    Article Title: Biochemical analysis of components of the pre-replication complex of Archaeoglobus fulgidus

    Journal: Nucleic Acids Research

    doi:

    Exonuclease III digestion of a bubble and a flayed duplex DNA substrate, on either naked DNA or DNA bound by wt Mcm (400 nM) or AF0244 (200 nM). The bubble used here consisted of dT 10 region on each strand, flanked by 18 bp duplex on either side. In the absence of protein, digestion proceeds up to the end of the duplex DNA and no further. In the presence of either AF0244 or Mcm there is protection of the final 2–3 bp of duplex DNA before the bubble region. A similar pattern is seen with the flayed duplex where digestion can proceed up to the end of the duplex DNA but is halted 3 bases earlier in the presence of Mcm or AF0244.
    Figure Legend Snippet: Exonuclease III digestion of a bubble and a flayed duplex DNA substrate, on either naked DNA or DNA bound by wt Mcm (400 nM) or AF0244 (200 nM). The bubble used here consisted of dT 10 region on each strand, flanked by 18 bp duplex on either side. In the absence of protein, digestion proceeds up to the end of the duplex DNA and no further. In the presence of either AF0244 or Mcm there is protection of the final 2–3 bp of duplex DNA before the bubble region. A similar pattern is seen with the flayed duplex where digestion can proceed up to the end of the duplex DNA but is halted 3 bases earlier in the presence of Mcm or AF0244.

    Techniques Used:

    Autoradiogram of a denaturing polyacrylamide gel showing exonuclease III digestion of a bubble substrate with and without Mcm present. The bubble in this substrate was made by repeats of d(GACT) on each strand. Digestion without Mcm present proceeds within the bubble region indicating that it is not fully single stranded in the reaction conditions. The frequent pause sites do indicate that it is not stable duplex DNA either. In the presence of Mcm digestion is halted 2–3 bases within the duplex region, indicating protection of this region by the bound protein.
    Figure Legend Snippet: Autoradiogram of a denaturing polyacrylamide gel showing exonuclease III digestion of a bubble substrate with and without Mcm present. The bubble in this substrate was made by repeats of d(GACT) on each strand. Digestion without Mcm present proceeds within the bubble region indicating that it is not fully single stranded in the reaction conditions. The frequent pause sites do indicate that it is not stable duplex DNA either. In the presence of Mcm digestion is halted 2–3 bases within the duplex region, indicating protection of this region by the bound protein.

    Techniques Used:

    37) Product Images from "DNA microarrays with stem-loop DNA probes: preparation and applications"

    Article Title: DNA microarrays with stem-loop DNA probes: preparation and applications

    Journal: Nucleic Acids Research

    doi:

    Exonuclease III digests of Sanger sequencing ladders. ( A ) Outline of the experiments. ( B ) Image of the sequencing gel generated by software provided with the ALF sequencing instrument (Pharmacia Biotech, Uppsala, Sweden). Sanger sequencing ladders without and with exo III treatment. Arrows mark matching fragments. ( C ) Sequencing reads obtained before and after exo III digestion.
    Figure Legend Snippet: Exonuclease III digests of Sanger sequencing ladders. ( A ) Outline of the experiments. ( B ) Image of the sequencing gel generated by software provided with the ALF sequencing instrument (Pharmacia Biotech, Uppsala, Sweden). Sanger sequencing ladders without and with exo III treatment. Arrows mark matching fragments. ( C ) Sequencing reads obtained before and after exo III digestion.

    Techniques Used: Sequencing, Generated, Software

    38) Product Images from "High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells"

    Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells

    Journal: Theranostics

    doi: 10.7150/thno.23852

    Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.
    Figure Legend Snippet: Enzymatic resistance of phosphorothioate-modified Au-TDNNs. (A, B) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by DNase I. (C, D) Fluorescence time graph depicting terminal-modified Au-TDNN and overall-modified Au-TDNN degradation by Exo III.

    Techniques Used: Modification, Fluorescence

    39) Product Images from "Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening"

    Article Title: Quantum Dot Doping-Induced Photoluminescence for Facile, Label-Free, and Sensitive Pyrophosphatase Activity Assay and Inhibitor Screening

    Journal: Nanomaterials

    doi: 10.3390/nano9010111

    ( A ) Fluorescence spectra of QD upon incubation of different concentrations of PPase from 0 to 20 mU/mL; ( B ) Relationship of the fluorescence intensity of QD at 510 nm, with the PPase concentration. Inset shows the corresponding linear range. ( C ) The specificity of the proposed sensing strategy toward PPase, against Exo I, Exo III, GOx, and lysozyme. The concentration of PPase was 10 mU/mL, and the concentrations for all other proteins were 0.1 U/mL. ( D ) The fluorescence intensities of QD, into the mixture of Cu 2+ (10 µM), Cu 2+ (10 µM) + PPi (20 µM), and Cu 2+ (10 µM) + PPi (20 µM) + PPase (1, 10 mU/mL), respectively, in the buffer solution and 5% diluted fetal bovine serum (FBS). Error bars in ( B – D ), for each data point, indicate the standard deviations, which were calculated on the basis of three repetitive experiments.
    Figure Legend Snippet: ( A ) Fluorescence spectra of QD upon incubation of different concentrations of PPase from 0 to 20 mU/mL; ( B ) Relationship of the fluorescence intensity of QD at 510 nm, with the PPase concentration. Inset shows the corresponding linear range. ( C ) The specificity of the proposed sensing strategy toward PPase, against Exo I, Exo III, GOx, and lysozyme. The concentration of PPase was 10 mU/mL, and the concentrations for all other proteins were 0.1 U/mL. ( D ) The fluorescence intensities of QD, into the mixture of Cu 2+ (10 µM), Cu 2+ (10 µM) + PPi (20 µM), and Cu 2+ (10 µM) + PPi (20 µM) + PPase (1, 10 mU/mL), respectively, in the buffer solution and 5% diluted fetal bovine serum (FBS). Error bars in ( B – D ), for each data point, indicate the standard deviations, which were calculated on the basis of three repetitive experiments.

    Techniques Used: Fluorescence, Incubation, Concentration Assay

    40) Product Images from "A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta"

    Article Title: A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta

    Journal: The Plant Cell

    doi:

    Identification and Mapping of the AVR-Pita Telomere by Using Virulent Mutants. Genomic DNAs were digested with EcoRI (A) or NcoI or SacI (B) , electrophoresed on 0.7% agarose gels, blotted to a Hybond-N membrane, and hybridized with the 32 P-labeled telomere repeat oligonucleotide 5′-(AACCCT) 4 -3′. (A) Lanes 1 and 2 contain the DNAs of 6043 (virulent on Yashiro-mochi) and 4224-7-8 ( AVR-Pita ). DNAs loaded in the remaining lanes are from three pairs of avirulent strains and virulent mutants derived from them: 4360-17-1/CP917 (lanes 3 and 4); 4375-R-6/CP983 (lanes 5 and 6); and 4375-R-26/CP984 (lanes 7 and 8). The arrow marks the band corresponding to Tel 5. Bars at left indicate positions of λ HindIII DNA fragments used as length standards; from the top, they are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. (B) Distal restriction fragments of the AVR-Pita telomere were identified by restriction digestion and hybridization of genomic DNAs from two avirulent strain/mutant pairs (4375-R-26/CP984 in lanes 1 and 2, and 4360-17-1/CP917 in lanes 3 and 4). No changes were observed in telomere fragments > 4.4 kb, so these fragments are not shown. The telomeric 2.0-kb NcoI fragment and the telomeric 0.8-kb SacI fragment (arrows) were altered in each of the two independent mutants. Not shown are data identifying the distal 9-kb SalI fragment, 6.9-kb BamHI fragment, 6.5-kb BglII fragment, 4-kb EcoRV fragment, and 1.3-kb HindIII fragment. Bars at left indicate positions of λ HindIII DNA length standards in kilobases.
    Figure Legend Snippet: Identification and Mapping of the AVR-Pita Telomere by Using Virulent Mutants. Genomic DNAs were digested with EcoRI (A) or NcoI or SacI (B) , electrophoresed on 0.7% agarose gels, blotted to a Hybond-N membrane, and hybridized with the 32 P-labeled telomere repeat oligonucleotide 5′-(AACCCT) 4 -3′. (A) Lanes 1 and 2 contain the DNAs of 6043 (virulent on Yashiro-mochi) and 4224-7-8 ( AVR-Pita ). DNAs loaded in the remaining lanes are from three pairs of avirulent strains and virulent mutants derived from them: 4360-17-1/CP917 (lanes 3 and 4); 4375-R-6/CP983 (lanes 5 and 6); and 4375-R-26/CP984 (lanes 7 and 8). The arrow marks the band corresponding to Tel 5. Bars at left indicate positions of λ HindIII DNA fragments used as length standards; from the top, they are 23.1, 9.4, 6.6, 4.4, 2.3, and 2.0 kb. (B) Distal restriction fragments of the AVR-Pita telomere were identified by restriction digestion and hybridization of genomic DNAs from two avirulent strain/mutant pairs (4375-R-26/CP984 in lanes 1 and 2, and 4360-17-1/CP917 in lanes 3 and 4). No changes were observed in telomere fragments > 4.4 kb, so these fragments are not shown. The telomeric 2.0-kb NcoI fragment and the telomeric 0.8-kb SacI fragment (arrows) were altered in each of the two independent mutants. Not shown are data identifying the distal 9-kb SalI fragment, 6.9-kb BamHI fragment, 6.5-kb BglII fragment, 4-kb EcoRV fragment, and 1.3-kb HindIII fragment. Bars at left indicate positions of λ HindIII DNA length standards in kilobases.

    Techniques Used: Labeling, Derivative Assay, Hybridization, Mutagenesis

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Identification of a novel proliferation-inducing determinant using lentiviral expression cloning
    Article Snippet: .. All PCR reactions were conducted using DyNAzyme.EXT polymerase (Finnzymes, Oulu, Finland). pND-A2 and pND-A8 represent nested deletions of pPCR102-2 which were performed by restricting pPCR102-2 with KpnI and BamHI followed by Exonuclease III (NEB, Beverly, MA) mediated digestion at 37°C for 1, 2 and 3 min, respectively. ..

    Concentration Assay:

    Article Title: High-Discrimination Factor Nanosensor Based on Tetrahedral DNA Nanostructures and Gold Nanoparticles for Detection of MiRNA-21 in Live Cells
    Article Snippet: .. For both human DNase I (Thermo Scientific) and exonuclease III (New England Biolabs) digestion, a common concentration of 3 U/mL was used to digest 2 nM Au-TDNNs samples in PBS. .. FAM fluorescence signals were monitored and recorded over a period of 3 h at 30 min intervals and at 37 °C.

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    New England Biolabs exonuclease iii exoiii
    Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease <t>III</t> footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of <t>ExoIII</t> activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.
    Exonuclease Iii Exoiii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exo iii
    Square wave voltammograms obtained at the gold electrode immobilized with the DNA strands. ( A ) In the absence of ER, (a) before, and (b) after the DNA strands are digested by <t>Exo</t> <t>III;</t> ( B ) In the presence of 100 nM ER, (a) before, and (b) after the DNA strands are digested by Exo III. Curves c–e are for the control experiments by using 500 nM bovine serum albumin (BSA), thrombin and α-fetoprotein (AFP) instead of 100 nM ER. Buffer: 10 mM phosphate-buffered saline (PBS) buffer (PH 7.4).
    Exo Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exo iii buffer
    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with <t>Exo</t> I and Exo <t>III</t> (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.
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    Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Journal: Nucleic Acids Research

    Article Title: Attenuation of DNA charge transport by compaction into a nucleosome core particle

    doi: 10.1093/nar/gkl030

    Figure Lengend Snippet: Structural characterization of AQ-157TG rNCPs. ( A ) Exonuclease III footprinting of AQ-157TG rNCPs (lane 1) and free AQ-157TG (lane 2). The restriction of ExoIII activity to the ∼10 bp proximal to AQ in the AQ-157TG rNCPs is evident. ( B ) Autoradiogram of hydroxyl radical footprinting on AQ-157TG rNCPs (lanes 1 and 2) and free AQ-157TG (lane 3). ( C ) Partial scan of the footprint in B of both free AQ-157TG (bottom) and AQ-157TG rNCPs (top). The 10 bp periodic cutting in the rNCPs is apparent.

    Article Snippet: T4 Polynucleotide Kinase (PNK), T4 DNA Ligase (T4 Lig) and Exonuclease III (ExoIII) were purchased from New England Biolabs.

    Techniques: Footprinting, Activity Assay

    Square wave voltammograms obtained at the gold electrode immobilized with the DNA strands. ( A ) In the absence of ER, (a) before, and (b) after the DNA strands are digested by Exo III; ( B ) In the presence of 100 nM ER, (a) before, and (b) after the DNA strands are digested by Exo III. Curves c–e are for the control experiments by using 500 nM bovine serum albumin (BSA), thrombin and α-fetoprotein (AFP) instead of 100 nM ER. Buffer: 10 mM phosphate-buffered saline (PBS) buffer (PH 7.4).

    Journal: International Journal of Molecular Sciences

    Article Title: An Exonuclease III Protection-Based Electrochemical Method for Estrogen Receptor Assay

    doi: 10.3390/ijms140510298

    Figure Lengend Snippet: Square wave voltammograms obtained at the gold electrode immobilized with the DNA strands. ( A ) In the absence of ER, (a) before, and (b) after the DNA strands are digested by Exo III; ( B ) In the presence of 100 nM ER, (a) before, and (b) after the DNA strands are digested by Exo III. Curves c–e are for the control experiments by using 500 nM bovine serum albumin (BSA), thrombin and α-fetoprotein (AFP) instead of 100 nM ER. Buffer: 10 mM phosphate-buffered saline (PBS) buffer (PH 7.4).

    Article Snippet: Exo III and NEB buffer I was obtained from New England Biolabs (Ipswich, WA, USA).

    Techniques:

    Exo III-treated minicircular DNA revealed after PFGE and 2D-gel electrophoresis. Exo III-treated whole-DNA extract from H. triquetra was subjected to PFGE ( A ) and 2D-gel ( B ), and detected with 1.1-kb psbA NCR fragment [and psbA gene fragment in (B) only]. (A) The 6–8 kb minicircular DNA bands (APBs) were removed in the Exo III lane, while the putative 3-kb psbA minicircle band was diminished in intensity. (B) Hybridization signals of larger than 2 kb were removed after Exo III treatment as revealed in both 2D-gels detected by psbA NCR and gene fragments. Extra spots indicated by the arrows were observed in these two images when comparing with the controls. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Journal: Nucleic Acids Research

    Article Title: The replication of plastid minicircles involves rolling circle intermediates

    doi: 10.1093/nar/gkp063

    Figure Lengend Snippet: Exo III-treated minicircular DNA revealed after PFGE and 2D-gel electrophoresis. Exo III-treated whole-DNA extract from H. triquetra was subjected to PFGE ( A ) and 2D-gel ( B ), and detected with 1.1-kb psbA NCR fragment [and psbA gene fragment in (B) only]. (A) The 6–8 kb minicircular DNA bands (APBs) were removed in the Exo III lane, while the putative 3-kb psbA minicircle band was diminished in intensity. (B) Hybridization signals of larger than 2 kb were removed after Exo III treatment as revealed in both 2D-gels detected by psbA NCR and gene fragments. Extra spots indicated by the arrows were observed in these two images when comparing with the controls. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Article Snippet: The effect of Exo III to the treated DNA was tested by PFGE and Southern blot hybridization as described.

    Techniques: Two-Dimensional Gel Electrophoresis, Electrophoresis, Hybridization

    Effects of DNA ligase/Klenow fragment on minicircular DNA. Total DNA from H. triquetra was treated with T4 DNA ligase, as well as Klenow fragment followed with T4 DNA ligase, prior to Exo III digestion. Treated DNAs were resolved by PFGE and detected the minicircular DNA signals by Southern Blot. The two upper arrows indicated APBs in the untreated control lane. The two lower arrows indicated the putative monomer of psbA minicircle signals of ∼2–3 kb. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Journal: Nucleic Acids Research

    Article Title: The replication of plastid minicircles involves rolling circle intermediates

    doi: 10.1093/nar/gkp063

    Figure Lengend Snippet: Effects of DNA ligase/Klenow fragment on minicircular DNA. Total DNA from H. triquetra was treated with T4 DNA ligase, as well as Klenow fragment followed with T4 DNA ligase, prior to Exo III digestion. Treated DNAs were resolved by PFGE and detected the minicircular DNA signals by Southern Blot. The two upper arrows indicated APBs in the untreated control lane. The two lower arrows indicated the putative monomer of psbA minicircle signals of ∼2–3 kb. DNA markers were linear DNAs from a commercial source (Invitrogen Corporation).

    Article Snippet: The effect of Exo III to the treated DNA was tested by PFGE and Southern blot hybridization as described.

    Techniques: Southern Blot

    Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Journal: Frontiers in Microbiology

    Article Title: Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    doi: 10.3389/fmicb.2015.01385

    Figure Lengend Snippet: Schematic of RCA of circularized padlock probes . (A) S. brasiliensis (Sbra-RCA) padlock probe design. (B) CAL is amplified by PCR with primers CAL -Fw and CAL -Rv; PCR products are submitted to ligation. Circularization of padlock probes occurs only if both probe arms hybridize correctly to the target sequence. (C) Upon specific hybridization, the phosphorylated 5′ end and the free hydroxyl at the 3′ end of the probe are joined by Pfu DNA ligase. After ligation, non-circularized probes and single-stranded primers are removed with Exo I and Exo III (optional step). (D) Circularized padlock probes serve as DNA template and signal amplification occurs via RCA, using Bst DNA polymerase and the primers RCA1 and RCA2. DNA synthesis occurs continuously for 1 h under isothermal amplification (65°C). DNA products are detected by gel electrophoresis or directly with SYBR Green I.

    Article Snippet: The exonucleolysis mix consisted of 0.5 μL Exo I (New England BioLabs, Ipswich, MA, USA), 1 μL 10 × Exo I Buffer (New England BioLabs), 0.2 μL Exo III (New England BioLabs), 1 μL 10 × Exo III Buffer (New England BioLabs), and 7.3 μL ultra-pure water.

    Techniques: Amplification, Polymerase Chain Reaction, Ligation, Sequencing, Hybridization, DNA Synthesis, Nucleic Acid Electrophoresis, SYBR Green Assay