exonuclease iii  (New England Biolabs)


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    Name:
    Exonuclease III
    Description:

    Catalog Number:
    M0206
    Price:
    252
    Category:
    DNA Modifying Enzymes
    Applications:
    DNA Manipulation
    Size:
    25000 units
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    Structured Review

    New England Biolabs exonuclease iii
    Exonuclease III

    https://www.bioz.com/result/exonuclease iii/product/New England Biolabs
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    exonuclease iii - by Bioz Stars, 2021-07
    97/100 stars

    Images

    1) Product Images from "DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION"

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    Journal: Virology

    doi: 10.1016/j.virol.2009.12.026

    Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.
    Figure Legend Snippet: Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.
    Figure Legend Snippet: Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.

    Techniques Used: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.
    Figure Legend Snippet: Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Techniques Used: Luciferase, Plasmid Preparation, Sequencing, Expressing, Transfection

    Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.
    Figure Legend Snippet: Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.

    Techniques Used: Activity Assay, Mutagenesis, Transfection, Expressing, Negative Control, Western Blot

    Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.
    Figure Legend Snippet: Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.

    Techniques Used: Luciferase, Plasmid Preparation, Binding Assay, Transfection, Expressing, Western Blot

    Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.
    Figure Legend Snippet: Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.

    Techniques Used: Mutagenesis, Staining, SDS Page, Purification, Fluorescence, Binding Assay, Expressing, Plasmid Preparation, Western Blot

    2) 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

    3) 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

    4) 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

    5) 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

    6) Product Images from "Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen"

    Article Title: Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen

    Journal: Protein Engineering, Design and Selection

    doi: 10.1093/protein/gzq057

    Asymmetric cell division and the role of SpoIIE. ( A ) In the pre-divisional cell (upper) and the mother cell (MC) following cell division (lower), SpoIIAA (AA) is phosphorylated and σ F is in a complex with SpoIIAB (AB 2 ). In the forespore (FS), σ F is free and AA and AB are in complex. SpoIIE (E) accumulates at the asymmetric septum, a double membrane structure. ( B ) The putative three domain structure of SpoIIE. The limits of the putative FtsZ-binding domain are uncertain.
    Figure Legend Snippet: Asymmetric cell division and the role of SpoIIE. ( A ) In the pre-divisional cell (upper) and the mother cell (MC) following cell division (lower), SpoIIAA (AA) is phosphorylated and σ F is in a complex with SpoIIAB (AB 2 ). In the forespore (FS), σ F is free and AA and AB are in complex. SpoIIE (E) accumulates at the asymmetric septum, a double membrane structure. ( B ) The putative three domain structure of SpoIIE. The limits of the putative FtsZ-binding domain are uncertain.

    Techniques Used: Binding Assay

    Assays of SpoIIE fragments. ( A ) SpoIIAA-phosphate dephosphorylation by the H1 and B1′ fragments monitored by native gel electrophoresis. Lane 1, SpoIIAA∼P (5 μg); lane 2, SpoIIAA (5 μg); lanes 3 and 4, SpoIIAA∼P (5 μg) incubated in the presence of the H1 fragment at 100:1 and 400:1 molar ratios, respectively; lanes 5 and 6, SpoIIAA∼P (5 μg) incubated in the presence of the B1′ fragment at 100:1 and 400:1 molar ratios, respectively. The conversion of SpoIIAA∼P to the lower mobility SpoIIAA species upon incubation with the SpoIIE fragments is evident. ( B ) Gel mobility shift assay of FtsZ binding by the H1 and B1′ fragments. Lane 1, FtsZ (7 μg); lane 2, FtsZ (7 μg) + 1 mM GTP; lane 3, SpoIIE H1 fragment (7 μg); lane 4, FtsZ (7 μg) + SpoIIE H1 (7 μg) + 1 mM GTP; lane 5, SpoIIE B1′ fragment (7 μg); lane 6, FtsZ (7 μg) + SpoIIE B1′ (7 μg) + 1 mM GTP; lane 7, FtsZ (7 μg) + 1mM GTP. There is no mobility shift evident from these gels other than the additional staining of material at the top of lane 4. ( C ) SEC-MALLS traces of the molecular mass and differential refractive index (dRI) versus time, of the eluate from a Superdex S200 column. The bold lines give molecular mass of the eluting species calculated from measurements of the refractive index and the multi-angle laser light scattering. Three traces for the (i) H1 (red), (ii) B1′ (green) and (iii) B2–B1 (blue) fragments are overlaid.
    Figure Legend Snippet: Assays of SpoIIE fragments. ( A ) SpoIIAA-phosphate dephosphorylation by the H1 and B1′ fragments monitored by native gel electrophoresis. Lane 1, SpoIIAA∼P (5 μg); lane 2, SpoIIAA (5 μg); lanes 3 and 4, SpoIIAA∼P (5 μg) incubated in the presence of the H1 fragment at 100:1 and 400:1 molar ratios, respectively; lanes 5 and 6, SpoIIAA∼P (5 μg) incubated in the presence of the B1′ fragment at 100:1 and 400:1 molar ratios, respectively. The conversion of SpoIIAA∼P to the lower mobility SpoIIAA species upon incubation with the SpoIIE fragments is evident. ( B ) Gel mobility shift assay of FtsZ binding by the H1 and B1′ fragments. Lane 1, FtsZ (7 μg); lane 2, FtsZ (7 μg) + 1 mM GTP; lane 3, SpoIIE H1 fragment (7 μg); lane 4, FtsZ (7 μg) + SpoIIE H1 (7 μg) + 1 mM GTP; lane 5, SpoIIE B1′ fragment (7 μg); lane 6, FtsZ (7 μg) + SpoIIE B1′ (7 μg) + 1 mM GTP; lane 7, FtsZ (7 μg) + 1mM GTP. There is no mobility shift evident from these gels other than the additional staining of material at the top of lane 4. ( C ) SEC-MALLS traces of the molecular mass and differential refractive index (dRI) versus time, of the eluate from a Superdex S200 column. The bold lines give molecular mass of the eluting species calculated from measurements of the refractive index and the multi-angle laser light scattering. Three traces for the (i) H1 (red), (ii) B1′ (green) and (iii) B2–B1 (blue) fragments are overlaid.

    Techniques Used: De-Phosphorylation Assay, Nucleic Acid Electrophoresis, Incubation, Mobility Shift, Binding Assay, Staining, Size-exclusion Chromatography

    7) 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

    8) Product Images from "Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China"

    Article Title: Sequence analysis of an Archaeal virus isolated from a hypersaline lake in Inner Mongolia, China

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-8-410

    Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleases. Lanes 1 and 4 , undigested controls; Lane 2, DNAse treated; Lane 3 RNase treated; Lane 5, exonuclease III treated. Panel b, 1% agarose 0.5× TBE pulse field gel; lanes 1 and 4 size markers (kbps), lanes 2 and 3 BJ1 virus genome. Panel c, Bam H1 enzyme digest of virus BJ1 genomic DNA, DNA size markers are shown on the left (kbps). The image has been overexposed to show the smaller bands.
    Figure Legend Snippet: Panel a. 0.8% TAE agarose gel showing virus BJ1 genome sensitivity to nucleases. Lanes 1 and 4 , undigested controls; Lane 2, DNAse treated; Lane 3 RNase treated; Lane 5, exonuclease III treated. Panel b, 1% agarose 0.5× TBE pulse field gel; lanes 1 and 4 size markers (kbps), lanes 2 and 3 BJ1 virus genome. Panel c, Bam H1 enzyme digest of virus BJ1 genomic DNA, DNA size markers are shown on the left (kbps). The image has been overexposed to show the smaller bands.

    Techniques Used: Agarose Gel Electrophoresis

    9) Product Images from "In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide"

    Article Title: In Vitro Infection with Hepatitis B Virus Using Differentiated Human Serum Culture of Huh7.5-NTCP Cells without Requiring Dimethyl Sulfoxide

    Journal: Viruses

    doi: 10.3390/v13010097

    Enhancement of HBV replication by human serum culture. Human serum culture increased HBV ( A ) pgRNA, ( B ) cccDNA, and ( C ) HBV surface antigen (HBsAg) levels from Huh7.5-NTCP cells. Huh7.5-NTCP cells were cultured in the media supplemented with FBS or HS and with or without the addition of DMSO during HBV infection. Samples were collected on day 14 ( A , B ) or day 7 ( C ) post-infection. Pregenomic RNA was measured using RT-qPCR from 10 ng of total RNA. Covalently closed circular DNA was quantified using q-PCR from 10 ng of gDNA. HBsAg was measured in a culture supernatant using enzyme-linked immunosorbent assay (ELISA). Average values with error bars (±SD) derived from three experiments are plotted. One-way analysis of variance (ANOVA) was used with the Bonferroni correction for multiple comparison test. * p
    Figure Legend Snippet: Enhancement of HBV replication by human serum culture. Human serum culture increased HBV ( A ) pgRNA, ( B ) cccDNA, and ( C ) HBV surface antigen (HBsAg) levels from Huh7.5-NTCP cells. Huh7.5-NTCP cells were cultured in the media supplemented with FBS or HS and with or without the addition of DMSO during HBV infection. Samples were collected on day 14 ( A , B ) or day 7 ( C ) post-infection. Pregenomic RNA was measured using RT-qPCR from 10 ng of total RNA. Covalently closed circular DNA was quantified using q-PCR from 10 ng of gDNA. HBsAg was measured in a culture supernatant using enzyme-linked immunosorbent assay (ELISA). Average values with error bars (±SD) derived from three experiments are plotted. One-way analysis of variance (ANOVA) was used with the Bonferroni correction for multiple comparison test. * p

    Techniques Used: Cell Culture, Infection, Quantitative RT-PCR, Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Derivative Assay

    10) 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

    11) Product Images from "An Integral Recognition and Signaling for Electrochemical Assay of Protein Kinase Activity and Inhibitor by Reduced Graphene Oxide-Polydopamine-Silver Nanoparticle-Ti4+ Nanocomposite"

    Article Title: An Integral Recognition and Signaling for Electrochemical Assay of Protein Kinase Activity and Inhibitor by Reduced Graphene Oxide-Polydopamine-Silver Nanoparticle-Ti4+ Nanocomposite

    Journal: Frontiers in Bioengineering and Biotechnology

    doi: 10.3389/fbioe.2020.603083

    (A) DPV responses obtained at different concentrations of PKA. The PKA concentrations from curve a to k were 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, and 50 U/mL, respectively. (B) Calibration curve of DPV response vs. PKA concentration. (C) The linear plot between DPV peak current and PKA concentration. (D) Electrochemical response comparison toward thrombin (1 μM), bovine serum albumin (BSA, 1 μM), Exo III (50 U/mL), hemoglobin (1 mg/mL) and PKA (10 U/mL), respectively.
    Figure Legend Snippet: (A) DPV responses obtained at different concentrations of PKA. The PKA concentrations from curve a to k were 0, 0.01, 0.05, 0.1, 0.2, 0.5, 1, 5, 10, 20, and 50 U/mL, respectively. (B) Calibration curve of DPV response vs. PKA concentration. (C) The linear plot between DPV peak current and PKA concentration. (D) Electrochemical response comparison toward thrombin (1 μM), bovine serum albumin (BSA, 1 μM), Exo III (50 U/mL), hemoglobin (1 mg/mL) and PKA (10 U/mL), respectively.

    Techniques Used: Concentration Assay

    Optimization of experimental conditions. (A) The immobilization concentration of kemptide. Various kemptide concentrations (10, 50, 100, 200, 300, and 500 μM) were applied. (B) ATP concentration optimization. A series of ATP concentrations including 10, 20, 40, 60, 80, 100, and 120 μM were used. (C) The optimization of PKA catalytic time. The employed PKA catalytic time was 0, 10, 30, 60, 90, 120, and 150 min, respectively. (D) The optimization of AgNO 3 concentration during the synthesis of rGO-PDA-AgNPs nanocomposite. Various AgNO 3 concentration (1, 5, 10, 20, and 50 mM) were studied. The error bars were based on at least three repetitive experiment results. The used PKA concentration was 10 U/mL.
    Figure Legend Snippet: Optimization of experimental conditions. (A) The immobilization concentration of kemptide. Various kemptide concentrations (10, 50, 100, 200, 300, and 500 μM) were applied. (B) ATP concentration optimization. A series of ATP concentrations including 10, 20, 40, 60, 80, 100, and 120 μM were used. (C) The optimization of PKA catalytic time. The employed PKA catalytic time was 0, 10, 30, 60, 90, 120, and 150 min, respectively. (D) The optimization of AgNO 3 concentration during the synthesis of rGO-PDA-AgNPs nanocomposite. Various AgNO 3 concentration (1, 5, 10, 20, and 50 mM) were studied. The error bars were based on at least three repetitive experiment results. The used PKA concentration was 10 U/mL.

    Techniques Used: Concentration Assay

    12) Product Images from "Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication"

    Article Title: Multiple roles of PP2A binding motif in hepatitis B virus core linker and PP2A in regulating core phosphorylation state and viral replication

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1009230

    Model of interactions between HBc linker and host protein phosphatase (kinase) in regulating multiple steps of viral replication. The HBc domains (NTD, linker, CTD) are shown in the middle as horizontal boxes, with the linker sequence highlighted. Also highlighted are the last residue of NTD (L140, part of the PP2A-B56 consensus binding motif) and the three CTD sites where phosphorylation state was monitored in this study. The linker sequence is proposed to regulate dynamic CTD phosphorylation and dephosphorylation to control different stages of viral replication, among other mechanisms. The red and green arrows on the top denote the recruitment of cellular phosphatase (PP) and kinase (KI), respectively, by the indicated linker/NTD sites to modulate the indicated CTD phosphorylation sites. Shown at the bottom is a simplified scheme of HBV replication cycle, with the different stages affected by the different CTD phosphorylation sites and PP2A/CDK2 highlighted. The stimulatory and inhibitory effects of certain linker mutations on CCC DNA formation during intracellular amplification and infection, respectively, are denoted by the green and red arrows. See text for details.
    Figure Legend Snippet: Model of interactions between HBc linker and host protein phosphatase (kinase) in regulating multiple steps of viral replication. The HBc domains (NTD, linker, CTD) are shown in the middle as horizontal boxes, with the linker sequence highlighted. Also highlighted are the last residue of NTD (L140, part of the PP2A-B56 consensus binding motif) and the three CTD sites where phosphorylation state was monitored in this study. The linker sequence is proposed to regulate dynamic CTD phosphorylation and dephosphorylation to control different stages of viral replication, among other mechanisms. The red and green arrows on the top denote the recruitment of cellular phosphatase (PP) and kinase (KI), respectively, by the indicated linker/NTD sites to modulate the indicated CTD phosphorylation sites. Shown at the bottom is a simplified scheme of HBV replication cycle, with the different stages affected by the different CTD phosphorylation sites and PP2A/CDK2 highlighted. The stimulatory and inhibitory effects of certain linker mutations on CCC DNA formation during intracellular amplification and infection, respectively, are denoted by the green and red arrows. See text for details.

    Techniques Used: Sequencing, Binding Assay, De-Phosphorylation Assay, Countercurrent Chromatography, Amplification, Infection

    Effect of linker mutations on infection. HBV inocula were prepared from HepG2 cells co-transfected with the HBc-expression construct plus the HBc-defective replicon as shown in Fig 6 , and used to infect HepG2-NTCP cells. PF DNA was extracted three days post-transfection and analyzed by Southern blot analysis, with (lane 1–6) or without (lane 7–12) pretreatment with exonuclease I and III (Exo I III). Quantitative results are shown in the graphs to the right. The small m. w. DNA smear present in certain linker mutants (lane 3–6) is indicated by the bracket. PF-RC, PF-RC DNA; CCC, CCC DNA; ND, not detected. **, P
    Figure Legend Snippet: Effect of linker mutations on infection. HBV inocula were prepared from HepG2 cells co-transfected with the HBc-expression construct plus the HBc-defective replicon as shown in Fig 6 , and used to infect HepG2-NTCP cells. PF DNA was extracted three days post-transfection and analyzed by Southern blot analysis, with (lane 1–6) or without (lane 7–12) pretreatment with exonuclease I and III (Exo I III). Quantitative results are shown in the graphs to the right. The small m. w. DNA smear present in certain linker mutants (lane 3–6) is indicated by the bracket. PF-RC, PF-RC DNA; CCC, CCC DNA; ND, not detected. **, P

    Techniques Used: Infection, Transfection, Expressing, Construct, Southern Blot, Countercurrent Chromatography

    Effect of PP2A inhibition on HBV infection. HepG2-NTCP cells were infected with HBV for one day. Following removal of the inoculum, the cells were mock treated or treated with the PP2A inhibitor Fostriecin (20 μM, 40 μM, 80 μM). HBV PF DNA was extracted three days post-infection and detection by Southern blot analysis. PF-RC, PF-RC DNA; CCC, CCC DNA. In the graph, PF-RC DNA and CCC DNA signals from Fostriecin-treated cells were compared to mock treatment, which was set to 1.00. *, P
    Figure Legend Snippet: Effect of PP2A inhibition on HBV infection. HepG2-NTCP cells were infected with HBV for one day. Following removal of the inoculum, the cells were mock treated or treated with the PP2A inhibitor Fostriecin (20 μM, 40 μM, 80 μM). HBV PF DNA was extracted three days post-infection and detection by Southern blot analysis. PF-RC, PF-RC DNA; CCC, CCC DNA. In the graph, PF-RC DNA and CCC DNA signals from Fostriecin-treated cells were compared to mock treatment, which was set to 1.00. *, P

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

    Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein. The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L - derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P
    Figure Legend Snippet: Analysis of CCC DNA from TT146/147AA in the presence and absence of the L protein. The full-length HBV replicon, with WT or TT146/147AA mutant HBc, or their L - derivative was transfected into HepG2 cells. Transfected cells were harvested seven days post-transfection. A. HBV NC-associated DNA (core DNA) was released by SDS-proteinase K digestion from cytoplasmic lysates and detected by Southern blot analysis. B. PF DNA was extracted by Hirt extraction and digested with Dpn I (lane 1–4) or Dpn I plus exonuclease I and III (lane 5–8). RC, RC DNA; SS, SS DNA; PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. Quantitative results from multiple experiments. Left, PF-RC DNA normalized to core RC DNA; right, CCC DNA normalized to core RC DNA. All normalized values from the WT were set to 1.0. **, P

    Techniques Used: Countercurrent Chromatography, Mutagenesis, Transfection, Southern Blot

    Effects of linker mutations on CCC DNA formation. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids ( A ), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA ( B ), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P
    Figure Legend Snippet: Effects of linker mutations on CCC DNA formation. HepG2 were co-transfected with indicated HBc expression constructs and the HBV genomic construct defective in HBc expression and HBV PF DNA was extracted from the transfected cells seven days after transfection. The extracted DNA was digested with Dpn I to degrade input plasmids ( A ), or Dpn I plus the exonuclease I and III to removal all DNA except closed circular DNA ( B ), before agarose gel electrophoresis and Southern blot analysis. Novel PF DNA smears detected from certain mutants are marked with the white asterisks to the left of the relevant lanes (S141D, S141R, L143A, TT146/147DD). PF-RC, PF-RC DNA; CCC, CCC DNA; cM, closed minus strand DNA. C. CCC DNA and PF-RC DNA signals of each mutant were compared with WT (top two panels). CCC DNA and PF-RC DNA are normalized to core RC DNA (middle two panels), and CCC DNA is normalized PF-RC DNA (bottom). All values from the WT were set to 1.0. *, P

    Techniques Used: Countercurrent Chromatography, Transfection, Expressing, Construct, Agarose Gel Electrophoresis, Southern Blot, Mutagenesis

    13) 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

    14) 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

    15) 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

    16) 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

    17) 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

    18) 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

    19) 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

    20) 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

    21) 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

    22) Product Images from "Flexible regulation of DNA displacement reaction through nucleic acid-recognition enzyme and its application in keypad lock system and biosensing"

    Article Title: Flexible regulation of DNA displacement reaction through nucleic acid-recognition enzyme and its application in keypad lock system and biosensing

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-10459-y

    General scheme of the Exo III-controlled, toehold-based DNA strand displacement strategies.
    Figure Legend Snippet: General scheme of the Exo III-controlled, toehold-based DNA strand displacement strategies.

    Techniques Used:

    ( A ) Time-dependent fluorescence spectra changes upon subjecting the Exo III activation module to only invading strand I 1 (a) and I 1 and variable concentrations of Exo III: (b) 0, (c) 1, (d) 2, (e) 3, (f) 5, (g) 10 units, and (h) only strand F 1 , [F 1 ] = [Q 1 ] = [I 1 ] = 50 nM. ( B ) Real-time fluorescence changes upon treatment of the Exo III inhibition module with I 2 and variable concentrations of Exo III: (a) 0, (b) 5, and (c) 10 units, [F 1 ] = [Q 2 ] = [I 2 ] = 50 nM.
    Figure Legend Snippet: ( A ) Time-dependent fluorescence spectra changes upon subjecting the Exo III activation module to only invading strand I 1 (a) and I 1 and variable concentrations of Exo III: (b) 0, (c) 1, (d) 2, (e) 3, (f) 5, (g) 10 units, and (h) only strand F 1 , [F 1 ] = [Q 1 ] = [I 1 ] = 50 nM. ( B ) Real-time fluorescence changes upon treatment of the Exo III inhibition module with I 2 and variable concentrations of Exo III: (a) 0, (b) 5, and (c) 10 units, [F 1 ] = [Q 2 ] = [I 2 ] = 50 nM.

    Techniques Used: Fluorescence, Activation Assay, Inhibition

    23) 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

    24) 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

    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 "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

    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 "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

    29) Product Images from "RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2"

    Article Title: RNA-DNA strand exchange by the Drosophila Polycomb complex PRC2

    Journal: Nature Communications

    doi: 10.1038/s41467-020-15609-x

    Pattern of RNA used by PRC2 on EcoRI-digested DNA templates. a – c Schematic of DNA and RNA templates for the vg ( a ), bxd ( b ) and dia ( c ) PREs. DNA template were linearized at one of the indicated sites. d, e . Schematic of RNA and DNA templates. RNA transcripts were produced from linear templates that end four bases before the EcoRI site so that the sequence indicated is not present in the RNAs. f , g Schematics of the four possible hybrids formed using the 2 RNAs and the sense ( f ) or anti-sense ( g ) DNA, indicating which two are observed. h – j show representative gels for each data set. Experiments were repeated three times, and all four sets for each PRE were always run in the same experiment. [PRC2] is 25–400 nM.
    Figure Legend Snippet: Pattern of RNA used by PRC2 on EcoRI-digested DNA templates. a – c Schematic of DNA and RNA templates for the vg ( a ), bxd ( b ) and dia ( c ) PREs. DNA template were linearized at one of the indicated sites. d, e . Schematic of RNA and DNA templates. RNA transcripts were produced from linear templates that end four bases before the EcoRI site so that the sequence indicated is not present in the RNAs. f , g Schematics of the four possible hybrids formed using the 2 RNAs and the sense ( f ) or anti-sense ( g ) DNA, indicating which two are observed. h – j show representative gels for each data set. Experiments were repeated three times, and all four sets for each PRE were always run in the same experiment. [PRC2] is 25–400 nM.

    Techniques Used: Produced, Sequencing

    Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.
    Figure Legend Snippet: Nuclease contamination cannot explain RNA–DNA hybrid formation by PRC2. a Scheme of experiment to test whether PRC2 generate a long ssDNA filament. Linear DNA was incubated with PRC2 or exonuclease III and purified. SSB protein was added to pre-treated DNA and samples were visualized by electron microscopy. b , c Representative EM pictures of DNA pre-treated with Exonuclease III ( b ) or with PRC2 ( c ). Arrows indicate DNA with ( b ) or without ( c ) SSB coating. d Scheme of experiment to test whether pre-incubation of DNA with PRC2 allows RNA–DNA hybrid formation in the absence of PRC2.is sufficient. e , f Representative gel ( e ) and quantification ( f ) of strand exchange reactions using DNA that was pre-treated with PRC2 (400 nM) as the substrate. n = 3. Graph shows the mean +/− S.E.M. Source data are provided in Source Data file.

    Techniques Used: Incubation, Purification, Electron Microscopy

    PRC2 has RNA–DNA strand exchange activity. a , b Nucleic acids used for RNA strand exchange. Plasmids with the vestigial ( vg ) PRE in either orientation are transcribed to produce sense ( a ) or anti-sense RNAs ( b ). Linearized plasmid is used as the DNA template. “Stop” indicates where the RNA ends relative to the DNA. c Strand exchange assay scheme. d – f Titration of PRC2 with either the sense ( d ) or anti-sense ( e ) vg PRE RNA, or a non-complementary RNA ( f ). g Quantification of three titrations; graphs show mean +/− S.D. of the the fraction of signal with 200 nM PRC2. h , i RNA strand invasion products are sensitive to RNaseH (lane 4) but resistant to RNaseA (lane 6). See also Supplementary Fig. 6 . Source data are provided in Source Data file.
    Figure Legend Snippet: PRC2 has RNA–DNA strand exchange activity. a , b Nucleic acids used for RNA strand exchange. Plasmids with the vestigial ( vg ) PRE in either orientation are transcribed to produce sense ( a ) or anti-sense RNAs ( b ). Linearized plasmid is used as the DNA template. “Stop” indicates where the RNA ends relative to the DNA. c Strand exchange assay scheme. d – f Titration of PRC2 with either the sense ( d ) or anti-sense ( e ) vg PRE RNA, or a non-complementary RNA ( f ). g Quantification of three titrations; graphs show mean +/− S.D. of the the fraction of signal with 200 nM PRC2. h , i RNA strand invasion products are sensitive to RNaseH (lane 4) but resistant to RNaseA (lane 6). See also Supplementary Fig. 6 . Source data are provided in Source Data file.

    Techniques Used: Activity Assay, Plasmid Preparation, Titration

    30) 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

    31) 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

    32) 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

    33) Product Images from "Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system"

    Article Title: Fidelity of prespacer capture and processing is governed by the PAM-mediated interactions of Cas1-2 adaptation complex in CRISPR-Cas type I-E system

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.RA119.009438

    Tailoring of Cas1-2 bound large DNA fragments by exonucleases generates integration-competent prespacers. A , denaturing PAGE depicting the nuclease treatment of Cas1-2 bound P63 DNA fragments. The presence (+) or absence (−) of each reaction component is indicated at the top of each lane . Positions corresponding to the substrate (P63) and T5 exo/ExoIII-digested DNA fragments (P63exo+) are indicated on the left , whereas oligonucleotide marker ( M ) positions are shown on the right. B , denaturing PAGE displaying the integration reactions employing various prespacers (P23[3′-5] ( lanes 1 and 2 and lanes 5 and 6 ), P63 ( lanes 3 and 7 ), and Cas1-2 protected DNA fragments (P63exo+) ( lanes 4 and 8 )) and CRISPR DNA substrates (CD-T* ( lanes 1–4 ) and CD-B* ( lanes 5–8 )) is shown. The presence (+) or absence (−) of each reaction component is indicated at the top of each lane . Positions corresponding to labeled DNA products that result from prespacer nucleophilic attack ( L and R ′) are displayed on the left . The DNA molecular weight marker ( M ) positions are shown on the right. C , schema illustrating the mechanism of Cas1-2–mediated protection of prespacer boundaries. Cas1-2 (in blue and brown ), T5 exonuclease ( magenta pie ), exonuclease III ( cyan pie ), and prespacer P63 ( red ladder ) are portrayed.
    Figure Legend Snippet: Tailoring of Cas1-2 bound large DNA fragments by exonucleases generates integration-competent prespacers. A , denaturing PAGE depicting the nuclease treatment of Cas1-2 bound P63 DNA fragments. The presence (+) or absence (−) of each reaction component is indicated at the top of each lane . Positions corresponding to the substrate (P63) and T5 exo/ExoIII-digested DNA fragments (P63exo+) are indicated on the left , whereas oligonucleotide marker ( M ) positions are shown on the right. B , denaturing PAGE displaying the integration reactions employing various prespacers (P23[3′-5] ( lanes 1 and 2 and lanes 5 and 6 ), P63 ( lanes 3 and 7 ), and Cas1-2 protected DNA fragments (P63exo+) ( lanes 4 and 8 )) and CRISPR DNA substrates (CD-T* ( lanes 1–4 ) and CD-B* ( lanes 5–8 )) is shown. The presence (+) or absence (−) of each reaction component is indicated at the top of each lane . Positions corresponding to labeled DNA products that result from prespacer nucleophilic attack ( L and R ′) are displayed on the left . The DNA molecular weight marker ( M ) positions are shown on the right. C , schema illustrating the mechanism of Cas1-2–mediated protection of prespacer boundaries. Cas1-2 (in blue and brown ), T5 exonuclease ( magenta pie ), exonuclease III ( cyan pie ), and prespacer P63 ( red ladder ) are portrayed.

    Techniques Used: Polyacrylamide Gel Electrophoresis, Marker, CRISPR, Labeling, Molecular Weight

    34) 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

    35) 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

    36) 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

    37) Product Images from "Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion"

    Article Title: Accelerating Post-SELEX Aptamer Engineering Using Exonuclease Digestion

    Journal: Journal of the American Chemical Society

    doi: 10.1021/jacs.0c09559

    Design and characterization of binding properties of OBAwt and six mutant derivatives. (A) Schematic of the exonuclease digestion assay based on Exo III and Exo I. (B) Secondary structure of ligand-bound aptamers, with mutated nucleotides relative to the OBAwt parent sequence highlighted in red. (C) Time-course plot of OBA3 digestion by Exo III and Exo I in the absence and presence of 25 μ M ochratoxin A (OTA). (D) The half-life ( t 1/2 ) ratio of the digestion reaction was used to determine the relative binding affinity of OBAwt and its six mutants to 100 μ M OTA or OTB. The red line indicates a t 1/2 ratio of 1, which means there was no inhibition of aptamer digestion.
    Figure Legend Snippet: Design and characterization of binding properties of OBAwt and six mutant derivatives. (A) Schematic of the exonuclease digestion assay based on Exo III and Exo I. (B) Secondary structure of ligand-bound aptamers, with mutated nucleotides relative to the OBAwt parent sequence highlighted in red. (C) Time-course plot of OBA3 digestion by Exo III and Exo I in the absence and presence of 25 μ M ochratoxin A (OTA). (D) The half-life ( t 1/2 ) ratio of the digestion reaction was used to determine the relative binding affinity of OBAwt and its six mutants to 100 μ M OTA or OTB. The red line indicates a t 1/2 ratio of 1, which means there was no inhibition of aptamer digestion.

    Techniques Used: Binding Assay, Mutagenesis, Sequencing, Inhibition

    Design and characterization of a double-mutant aptamer. (A) Inversion of nucleotides G10 and T23 in construct A23T results in a double-mutant G10T-A23G. (B) Fluorescence time-course plot of G10T-A23G digestion by Exo III and Exo I in the absence and presence of 250 μ M ADE, AMP, ADP, and ATP. (C) The t 1/2 ratio (with log 2 scale) for ATPwt, A23T, and G10T-A23G in the presence vs the absence of 250 μ M of each ligand. The red line indicates a t 1/2 ratio of 1, reflecting no inhibition of aptamer digestion. Error bars represent the standard error of fitting.
    Figure Legend Snippet: Design and characterization of a double-mutant aptamer. (A) Inversion of nucleotides G10 and T23 in construct A23T results in a double-mutant G10T-A23G. (B) Fluorescence time-course plot of G10T-A23G digestion by Exo III and Exo I in the absence and presence of 250 μ M ADE, AMP, ADP, and ATP. (C) The t 1/2 ratio (with log 2 scale) for ATPwt, A23T, and G10T-A23G in the presence vs the absence of 250 μ M of each ligand. The red line indicates a t 1/2 ratio of 1, reflecting no inhibition of aptamer digestion. Error bars represent the standard error of fitting.

    Techniques Used: Mutagenesis, Construct, Fluorescence, Inhibition

    Exonuclease-based fluorescence profiling of ATPwt binding to various targets. (A) Time-course plot of ATPwt digestion by Exo III and Exo I in the absence and presence of various ribonucleotides at a concentration of 250 μ M. (B) The t 1/2 ratio was used to determine the relative binding affinity to each ligand at 100 and 250 μ M. The red line indicates a t 1/2 ratio of 1, which reflects no inhibition of aptamer digestion.
    Figure Legend Snippet: Exonuclease-based fluorescence profiling of ATPwt binding to various targets. (A) Time-course plot of ATPwt digestion by Exo III and Exo I in the absence and presence of various ribonucleotides at a concentration of 250 μ M. (B) The t 1/2 ratio was used to determine the relative binding affinity to each ligand at 100 and 250 μ M. The red line indicates a t 1/2 ratio of 1, which reflects no inhibition of aptamer digestion.

    Techniques Used: Fluorescence, Binding Assay, Concentration Assay, Inhibition

    Exonuclease-based fluorescence profiling of thrombin-binding aptamers. Time-course plot of digestion of 500 nM (A) Tasset, (B) Bock, and (C) Bock-hang by Exo III and Exo I in the absence (black) and presence of 500 nM human α -thrombin (red) or human factor X (brown). (D) The half-life ( t 1/2 ) ratio of the digestion reaction was used to determine relative aptamer binding affinity to α -thrombin and factor X. The y -axis is log 2 -scaled. The red line indicates a t 1/2 ratio of 1, which means there was no inhibition of aptamer digestion.
    Figure Legend Snippet: Exonuclease-based fluorescence profiling of thrombin-binding aptamers. Time-course plot of digestion of 500 nM (A) Tasset, (B) Bock, and (C) Bock-hang by Exo III and Exo I in the absence (black) and presence of 500 nM human α -thrombin (red) or human factor X (brown). (D) The half-life ( t 1/2 ) ratio of the digestion reaction was used to determine relative aptamer binding affinity to α -thrombin and factor X. The y -axis is log 2 -scaled. The red line indicates a t 1/2 ratio of 1, which means there was no inhibition of aptamer digestion.

    Techniques Used: Fluorescence, Binding Assay, Inhibition

    38) 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

    Related Articles

    Incubation:

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION
    Article Snippet: To measure the amount of replicated pFLORI31 or pFLORI40, 25 μl of total genomic DNA was digested with 10 units of DpnI (New England Biolabs) for 16 hrs in a final volume of 30 μl. .. The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes. .. The primers used to amplify an 80 bp-portion of the firefly luciferase gene present on the pFLORI40 (SV40 ori), pFLORI31 (HPV31 ori) or pCI-Fluc (No ori) plasmids as well as the probe used to detect the amplicon were synthesized according to unpublished sequences validated by Dr Iain Morgan (personal communication).

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq
    Article Snippet: Fragmented DNA was phosphorylated at 37 °C for 30 min in a reaction consisting of 22 μl DNA, 0.5 μl T4 PNK (T4 Polynucleotide Kinase, NEB, M0201S), 2.5 μl T4 DNA Ligase Buffer with 10 mM dATP, then purified with Oligo Clean & Concentrator Kit (Zymo, D4060). .. Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min. .. Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Hybridization:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Therefore, treated DNA was then tested on the sensitivity to Exo III. .. Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III. .. Presence of minicircle DNA was revealed by Southern blotting by the psbA NCR probe.

    Transfection:

    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
    Article Snippet: The insoluble materials containing viral replicative intermediates and most of the cellular DNA were removed by centrifugation, and the resultant supernatant containing viral cccDNA was extracted with phenol. .. To remove the transfected linear viral DNA, the nucleic acid obtained was treated with Exonuclease III (New England Biolabs) at 37°C for 1 h, followed by treatment with mung bean nuclease (New England Biolabs) at 37°C for 30 min. ..

    other:

    Article Title: The replication of plastid minicircles involves rolling circle intermediates
    Article Snippet: Exo III catalyzes a stepwise removal of mononucleotides from the 3′-hydroxyl termini of linear duplex DNA.

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    New England Biolabs exonuclease iii
    Validation of the HPV31 <t>DNA</t> replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using <t>three</t> different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.
    Exonuclease Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs exonuclease iii e coli
    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq <t>(three</t> experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.
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    Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the HPV31 DNA replication assay using E2 mutants (A) DNA replication activities of the indicated E2 mutants were tested using three different amounts of expression vector (1, 2.5 and 5 ng). Cells transfected without E2 expression vector (No E2) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 5 ng of E2 wild-type. (B) Anti-Flag Western blots showing the expression of E2 mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the HPV31 DNA replication assay using E1 mutants (A) DNA replication activities of the indicated E1 mutants were tested using three different amounts of expression vector (2.5, 5 and 10 ng). Cells transfected without E1 expression vector (No E1) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 10 ng of the E1 wild-type. (B) Anti-Flag Western blots showing the expression of E1 mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Expressing, Plasmid Preparation, Transfection, Negative Control, Activity Assay, Western Blot, Mutagenesis

    Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Principle of the luciferase SV40 DNA replication assay (A) Schematic representation of the three plasmids used in the assay. The name of each plasmid is written on the left. The location of the SV40 origin of replication is represented by a black box with the position of the core (grey) and 21 bp-repeat regions (black) enlarged above. The nucleotide (nt) sequence boundaries of the origin are indicated. The locations of the CMV promoter and intron are indicated by dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of LT are indicated by white boxes. Amino acid boundaries of each protein are indicated below each box. (B) Schematic representation of the assay. A plasmid expressing SV40 LT (pLT) is co-transfected in cells along with a second plasmid containing the SV40 origin of replication (pFLORI40) and a firefly luciferase reporter gene. A third plasmid expressing Renilla luciferase (pRL) is also transfected as an internal control to normalize for variations in transfection efficiency. Viral DNA replication is measured using a dual-luciferase assay, at different times post-transfection.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Luciferase, Plasmid Preparation, Sequencing, Expressing, Transfection

    Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Validation of the SV40 DNA replication assay using T antigen mutants (A) Replication activity of mutant T antigens in C33A cells. Each mutant was tested in three different amounts of pLT (2.5, 6.25 and 12.5 ng). Cells transfected without LT expression vectors (No LT) was used as a negative control. Replication activity is reported as a percentage (%) of the Fluc/Rluc ratio obtained with 12.5 ng of the LT wild-type, which was set at 100. (B) Western blot showing the expression of the different T antigen mutant proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Activity Assay, Mutagenesis, Transfection, Expressing, Negative Control, Western Blot

    Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Principle of the luciferase-based HPV31 DNA replication assay (A) Schematic representation of the three HPV plasmids used in the luciferase assay. The name of each plasmid is written on the left. The location of the CMV promoter and 3F epitope are indicated as dark and light grey boxes, respectively. The coding regions of firefly and Renilla luciferase as well as those of codon-optimized (co) E1 and E2 are indicated by white boxes. Amino acid boundaries are indicated below each box. The location (black box) and nucleotide boundaries of the HPV31 origin of replication are indicated with the position of E1 (black) and E2 (grey) binding sites enlarged above. (B) DNA replication activities measured in C33A cells transfected with the indicated amount of E1 and E2 expression vectors together with 2.5 ng of pFLORI31 and 0.5 ng of pRL. DNA replication activities are expressed as Fluc/Rluc ratios and were determined at 24, 48, 72, 96 and 120 h post-transfection, as indicated. (C) Western blots showing the expression of E1 and E2 at different time points post-transfection. Tubulin was used as a loading control. Note that the time-dependent increase in tubulin levels reflects the fact that cells continued to proliferate over the course of this 5-day assay.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Luciferase, Plasmid Preparation, Binding Assay, Transfection, Expressing, Western Blot

    Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.

    Journal: Virology

    Article Title: DEVELOPMENT OF QUANTITATIVE AND HIGH-THROUGHPUT ASSAYS OF POLYOMAVIRUS AND PAPILLOMAVIRUS DNA REPLICATION

    doi: 10.1016/j.virol.2009.12.026

    Figure Lengend Snippet: Characterization of a dimerization-defective HPV31 E1 mutant (A) Coomassie-stained SDS-PAGE showing the purified wild-type and G230R E1 OBDs. 3 μg of each protein was loaded on the gel. (B-D) Fluorescence polarization DNA binding assays. Binding isotherms were performed with increasing concentrations of wild-type (filled squares) or G230R (open squares) OBD and 10 nM of fluorescent DNA probe either lacking (No E1BS probe (D)) or containing two E1 binding sites spaced by 3 bp (2 E1BS probe (B)) or 5 bp (2+2 E1BS probe (C)). Only a spacing of 3 bp allows dimerization of the OBD. Each binding isotherm was performed in triplicate. (E) DNA replication activities of wild-type and G230R E1 were tested using three different amounts of expression vector, as described in the text. (F) Anti-Flag Western blots showing the expression of E1 proteins. Tubulin was used as a loading control.

    Article Snippet: The digested DNA samples were then incubated for 30 minutes with 100 units of Exonuclease III (New England Biolabs) and the enzyme was heat inactivated at the end of the reaction by incubation at 70°C for 30 minutes.

    Techniques: Mutagenesis, Staining, SDS Page, Purification, Fluorescence, Binding Assay, Expressing, Plasmid Preparation, Western Blot

    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: Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III.

    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: Total H. triquetra DNA (which contained minicircle DNA as detected in the Southern hybridization) were sequentially treated with Klenow, T4 DNA ligase and Exo III.

    Techniques: Southern Blot

    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.

    Journal: Journal of Virology

    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

    doi:

    Figure Lengend 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.

    Article Snippet: To remove the transfected linear viral DNA, the nucleic acid obtained was treated with Exonuclease III (New England Biolabs) at 37°C for 1 h, followed by treatment with mung bean nuclease (New England Biolabs) at 37°C for 30 min.

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

    Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Journal: Nature Communications

    Article Title: Ultrasensitive and high-efficiency screen of de novo low-frequency mutations by o2n-seq

    doi: 10.1038/ncomms15335

    Figure Lengend Snippet: Performance of o2n-seq for detecting mutations with 1% and 0.1% allele frequency. ( a , b ) Sensitivity and FPR of mutation detection of o2n-seq (three experimental replicates, orange), Cir-seq (three experimental replicates, blue) and o2n-seq after filtering with frequency (o2n-seq-f, green) under different CSs criteria for the 1:100 mixture of E. coli (means±s.d.). Two-tailed Student's t -test was used for statistical analysis. ( c ) Mutation frequency distribution of FP and TP variants detected by o2n-seq under different CSs (1 × and 2 × ) for the 1:100 mixture of E. coli . 3 × -5 × CSs were showed in Supplementary Fig. 5 . ( d ) MAFs of TP mutations detected by o2n-seq for the 1:100 mixture of E. coli . The MAFs of three experimental replicates was plotted. The dashed horizontal line indicates the theoretical MAF (0.99%). ( e , f ) Sensitivity and FPR of mutation detection of o2n-seq by different CSs criteria (3 × −9 × ) under different total CSs coverage (5,000–25,000 × ) for the 1:1,000 mix of phix174 . The results of the other experimental replicate were shown in Supplementary Fig. 6 . Dash lines were used to display the overlapped results better.

    Article Snippet: Subsequently, 1 μl Exonuclease I (NEB, M0293S) and 1 μl Exonuclease III (NEB, M0206S) were added into the reaction and incubated at 37 °C for 1 h. The enzymes were inactivated at 80 °C for another 20 min.

    Techniques: Mutagenesis, Two Tailed Test