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    1) 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 efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    2) 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

    Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.
    Figure Legend Snippet: Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Techniques Used: Activity Assay, Cell Culture, Derivative Assay, Incubation, Southern Blot, Plasmid Preparation, Marker

    Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.
    Figure Legend Snippet: Titration analysis of T5 Exo and PSD. (A) Two micrograms of genomic DNA samples from HBV-free HepG2 hNTCP cells was incubated with T5 Exo or PSD in time-dependent (1, 2, 4, and 16 h) and dose-dependent (1 and 5 U) manners. After digestion, products were visualized on an agarose gel, and the expression level of the human β-globin gene was measured as a representative readout to show digestion degree of genomic DNA. (B) Two micrograms of pSHH2.1 plasmid was incubated with T5 Exo or PSD similarly, and the remaining plasmid in products was determined by pp466-541 or directly visualized on an agarose gel.

    Techniques Used: Titration, Incubation, Agarose Gel Electrophoresis, Expressing, Plasmid Preparation

    T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.
    Figure Legend Snippet: Detection of cccDNA and validation of Myrcludex B in 96-well plate format. (A) HepG2 hNTCP cells seeded in a 96-well plate were infected at an mge/cell of 1,000. Myrcludex B was coadministered, tenofovir was added postinoculation, and IFN-α-2a was applied during and after infection. (B) Cells were treated with each antiviral at eight doses (1:3.2 serial dilutions) in triplicates. (C) On day 7 p.i., DNA samples were extracted together using a vacuum-based system. Crude DNA in 100 μl of elute was ethanol precipitated and resuspended with 10 μl of water. T5 Exo digestion was performed prior to cccDNA quantification by PCR. HBeAg levels during days 5 to 7 p.i. in the supernatant in all wells were measured.

    Techniques Used: Infection, Polymerase Chain Reaction

    cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.
    Figure Legend Snippet: cccDNA profiles in infections with increasing mge. (A) HepG2 hNTCP cells were infected with different amounts of virus inoculum (mge/cell of 30, 100, 300, 1,000, and 3,000) in parallel, and total DNA samples were prepared on day 10 p.i. Samples were hydrolyzed by T5 Exo (5 U, 60 min) at 37°C for 1 h, and cccDNA was determined using pp1040-1996. Total DNA copy numbers were also determined in undigested samples using pp466-541. (B) Within the same infections, secreted HBsAg values from day 7 to 10 p.i. were detected. (C) On day 10 p.i., intracellular HBcAg expression levels (red) were visualized. As a control to verify NTCP-mediated entry of the virus, Myrcludex B (1 μM) was administered during the infection.

    Techniques Used: Infection, Expressing

    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

    3) 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

    Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.
    Figure Legend Snippet: Identification of exonucleases selectively digesting rcDNA. (A) Properties of exonucleases tested in this study. +, strong activity; -, no significant activity; +/-, reduced activity; ss, single stranded; ds, double stranded; endo, endonuclease activity; dNMP, deoxyribonucleoside monophosphate; oligos, oligonucleotides. (B) Copies (3 × 10 8 ) of cell culture-derived viral DNA containing rcDNA and dslDNA were incubated for 1 h at 37°C with PSD (5 U), BAL-31 (5 U), Exo I (5 U), Exo V (5 U), and T5 Exo (5 U). Mung bean nuclease (5 U), EcoRI (5 U), and DNase I (5 U) were included as controls. After heat inactivation, the products were subjected to Southern blotting. The plasmid pUCX3.2 served as a marker for indicating the expected sizes of rcDNA and cccDNA.

    Techniques Used: Activity Assay, Cell Culture, Derivative Assay, Incubation, Southern Blot, Plasmid Preparation, Marker

    4) 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

    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 efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.
    Figure Legend Snippet: T5 Exo efficiently removes rcDNA and genomic DNA from DNA preparation. (A) Copies (3 × 10 8 ) of virion DNA from purified HBV virions were incubated with PSD (5 U), T5 Exo (5 U), EcoRI (5 U), or DNase I (5 U) at 37°C for 1 h and further subjected to Southern blotting. pUCX3.2 plasmid (3.2 kb) was loaded as well to indicate the positions of rcDNA and cccDNA. (B) (Top) Two micrograms of purified 3.2-kb linear HBV monomer released from the pSHH2.1 plasmid by EcoRI digestion was incubated with indicated units of T5 Exo or PSD at 37°C for 1 h. (Middle) A mixture of 3.2-kb open circular DNA (2 μg) that was artificially nicked by Nb.BtsI endonuclease and 3.2-kb supercoiled pUCX3.2 plasmid (2 μg) was subjected to T5 Exo or PSD digestion at 37°C for 1 h. (Bottom) Two micrograms of genomic DNA from uninfected HepG2 hNTCP cells was similarly treated with T5 Exo or PSD. All digestion products are shown on agarose gels, and for relative quantification, band density of untreated samples is set as 100%. (C) Copies (10 8 ) of virion DNA or pUCX3.2 plasmid were digested with T5 Exo (5 U) or PSD (5 U) in the absence (0 μg) or presence (2 μg) of genomic DNA (as shown above; 1% agarose gel) at 37°C for 1 h, and the products were loaded for Southern blotting (bottom). (D) Virion DNA (rcV) or pSHH2.1 plasmid was incubated with T5 Exo (5 U) or PSD (10 U) at 37°C for 1 h, and products were further analyzed by pp466-541 (left) or pp1040-1996 (right), respectively. ns, no significance. (E) Total DNA samples from HBV-infected HepG2 hNTCP cells (days 1, 2, 3, 6, and 9 p.i. and day 0 without inocula) were incubated with T5 Exo (5 U) or PSD (10 U) as described above, and cccDNA (left) and total DNA (right) copies were quantified by respective primers.

    Techniques Used: Purification, Incubation, Southern Blot, Plasmid Preparation, Agarose Gel Electrophoresis, Infection

    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

    Related Articles

    Stable Transfection:

    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
    Article Snippet: HepG2, HepaRG, Hepa1-6, Hepa56D, and HeLa cells stably expressing hNTCP were cultivated as described previously ( , ). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Cytometry:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: Paragraph title: Flow cytometry host cell reactivation assay ... The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004).

    Blocking Assay:

    Article Title: Lagging strand replication shapes the mutational landscape of the genome
    Article Snippet: Terminal transferase (NEB) was then used to block any free 3′ ends with ddATP for 2 h at 37°C, with 20 U of TdT per μg of DNA. .. After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per μg of library) or Nb.BtsI (NEB; 10 U per μg) for 2 h at 37°C.

    Incubation:

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
    Article Snippet: Sequential removal of PCR primer ends with on-bead BspQI digestion Post hybridization, the 3′-end PCR primer containing BtsI recognition site is digested with 50 U Nb.BtsI at 37°C for 2 h (total reaction volume 100 µl; 1x NEB, 1x BSA) followed by capture of cDNA on streptavidin coated magnetic beads. .. Once washed the beads are suspended in 100 µl binding solution (2 M NaCl, 1 mM EDTA, 10 mM Tris-HCl pH 7.5) to which is added 100 µl of the Nb.BtsI digestion reaction and incubated at room temperature for 15 min with gentle shaking.

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004). .. After phenol chloroform extraction the DNA was incubated with 5 U of ExoIII (NEB M0206S) per µg of DNA at 37 °C for 1 h in NEB buffer 1 (NEB B7001) to digest the nicked strand.

    Article Title: Novel Method For Quantifying Radiation-Induced Single-Strand-Break Yields In Plasmid DNA Highlights Tenfold Discrepancy
    Article Snippet: .. For Nb.BtsI digestion, DNA was incubated (37°C for 18 hrs) in 1X NEBuffer 4 (New England Biolabs, Inc.); for Nt. ..

    Article Title: Novel Method For Quantifying Radiation-Induced Single-Strand-Break Yields In Plasmid DNA Highlights Tenfold Discrepancy
    Article Snippet: Nicking endonucleases Nb.BsrDI, Nb.BtsI, Nt.BstNBI and Nt.AlwI (New England Biolabs, Beverly, MA) were used to create 3 HT-pUC19 (20 μg) plasmid DNA molecules with known number of SSBs (2, 3, 4 and 10, respectively). .. For Nb.BtsI digestion, DNA was incubated (37°C for 18 hrs) in 1X NEBuffer 4 (New England Biolabs, Inc.); for Nt.

    Expressing:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: Flow cytometry host cell reactivation assay Flow cytometry host cell reactivation assay (FM-HCR) assay was performed using plasmids for expression of the fluorescent proteins EGFP and mPlum subcloned into the pmaxCloning. .. The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004).

    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
    Article Snippet: HepG2, HepaRG, Hepa1-6, Hepa56D, and HeLa cells stably expressing hNTCP were cultivated as described previously ( , ). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Modification:

    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
    Article Snippet: HepG2hNTCP , Hepa1-6hNTCP , Hepa56DhNTCP , and HeLahNTCP cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM nonessential amino acids. .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Hybridization:

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
    Article Snippet: .. Sequential removal of PCR primer ends with on-bead BspQI digestion Post hybridization, the 3′-end PCR primer containing BtsI recognition site is digested with 50 U Nb.BtsI at 37°C for 2 h (total reaction volume 100 µl; 1x NEB, 1x BSA) followed by capture of cDNA on streptavidin coated magnetic beads. .. 50 µl of beads (250 pmol binding capacity) is used to immobilize 5′-biotinylated cDNA.

    Flow Cytometry:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: Paragraph title: Flow cytometry host cell reactivation assay ... The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004).

    Southern Blot:

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Paragraph title: Two-dimensional agarose gel electrophoresis and Southern blotting ... The supercoiled DNA ladder was nicked using Nb.BtsI (New England BioLabs) to generate the relaxed circular DNA standard.

    Ligation:

    Article Title: Lagging strand replication shapes the mutational landscape of the genome
    Article Snippet: The trP1 adapter (see below) was attached using NEBNext Quick Ligation with 120 pmol of adapter per μg of DNA for 14-18 h at 16°C. .. After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per μg of library) or Nb.BtsI (NEB; 10 U per μg) for 2 h at 37°C.

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer. .. The products were then loaded on a 1% agarose gel and visualized with GelRed.

    Cell Culture:

    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
    Article Snippet: HepG2hNTCP , Hepa1-6hNTCP , Hepa56DhNTCP , and HeLahNTCP cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 2 mM l -glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 1 mM nonessential amino acids. .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Generated:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: .. The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004). .. After phenol chloroform extraction the DNA was incubated with 5 U of ExoIII (NEB M0206S) per µg of DNA at 37 °C for 1 h in NEB buffer 1 (NEB B7001) to digest the nicked strand.

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: Substrates for mismatch repair in human nuclear extracts were generated as described previously ( ). .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    other:

    Article Title: Topo IV is the topoisomerase that knots and unknots sister duplexes during DNA replication
    Article Snippet: DNA treatments To induce single-stranded breaks, DNA was digested with Nb.BsmI, Nb.BtsI, Nt.BbvCI (New England Biolabs) or Nt.Bpu10I (Fermentas) for 30 min at 37°C.

    Host-Cell Reactivation:

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: Paragraph title: Flow cytometry host cell reactivation assay ... The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004).

    Sequencing:

    Article Title: A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine
    Article Snippet: The plasmid was nicked with Nb.Bts I (New England Biolabs) to generate a single-strand break in the transcribed strand. .. Twenty micrograms of the ssDNA were combined with 9 μl of a 10 mM oligonucleotide sequence containing a thymine dimer localized at bases 614 and 615 of the plasmid [5΄ TCAGGGCGGAXGGTTGC 3΄, where X denotes a thymine dimer] (Trilink Biotech) and 1× Pfu Polymerase Buffer (Thermo Scientific).

    Article Title: Lagging strand replication shapes the mutational landscape of the genome
    Article Snippet: Paragraph title: EmRiboSeq library preparation and sequencing ... After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per μg of library) or Nb.BtsI (NEB; 10 U per μg) for 2 h at 37°C.

    Binding Assay:

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
    Article Snippet: Sequential removal of PCR primer ends with on-bead BspQI digestion Post hybridization, the 3′-end PCR primer containing BtsI recognition site is digested with 50 U Nb.BtsI at 37°C for 2 h (total reaction volume 100 µl; 1x NEB, 1x BSA) followed by capture of cDNA on streptavidin coated magnetic beads. .. 50 µl of beads (250 pmol binding capacity) is used to immobilize 5′-biotinylated cDNA.

    Magnetic Beads:

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
    Article Snippet: .. Sequential removal of PCR primer ends with on-bead BspQI digestion Post hybridization, the 3′-end PCR primer containing BtsI recognition site is digested with 50 U Nb.BtsI at 37°C for 2 h (total reaction volume 100 µl; 1x NEB, 1x BSA) followed by capture of cDNA on streptavidin coated magnetic beads. .. 50 µl of beads (250 pmol binding capacity) is used to immobilize 5′-biotinylated cDNA.

    Isolation:

    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
    Article Snippet: PHH were isolated from liver specimens obtained after partial hepatectomy and following written informed consent of the patients (approved by the ethics commission of Hannover Medical School/Ethik-Kommission der MHH, no. 252-2008). .. T5 Exo (M0363), BAL-31 nuclease (M0213), exonuclease I (M0293), exonuclease III (M0206), exonuclease V (RecBCD, M0345), mung bean nuclease (M0250), EcoRI (R0101), and Nb.BtsI (R0707) were purchased from New England Biolabs.

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer. .. The products were then loaded on a 1% agarose gel and visualized with GelRed.

    Purification:

    Article Title: A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine
    Article Snippet: The plasmid was nicked with Nb.Bts I (New England Biolabs) to generate a single-strand break in the transcribed strand. .. The nicked strand was then digested with ExoIII (New England Biolabs), and the remaining single-stranded circular DNA (ssDNA) was purified using a 1% agarose gel.

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli
    Article Snippet: Proteins, chemicals, and reagents E. coli LacI and mutants were purified by the method of Chen and Matthews (E. coli strains containing the plasmid overexpressing LacI and mutants was kindly provided by K. S. Matthews at Rice University). .. Restriction enzymes Nt.BbvCI, Nb.BbvCI, Nb.BtsI, T4 DNA ligase, and E. coli DNA gyrase were purchased from New England Biolabs (Beverly, MA, USA).

    Article Title: Lagging strand replication shapes the mutational landscape of the genome
    Article Snippet: .. After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per μg of library) or Nb.BtsI (NEB; 10 U per μg) for 2 h at 37°C. .. RNase H2 purification and reaction conditions were as previously described .

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: The nicked substrate was purified from 1% agarose gel run in TAE buffer supplemented with 0.5 μg/ml ethidium bromide (EtBr), using the Promega Wizard SV Gel & PCR Cleanup Kit (Promega, Madison, USA). .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    Polymerase Chain Reaction:

    Article Title: Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries
    Article Snippet: .. Sequential removal of PCR primer ends with on-bead BspQI digestion Post hybridization, the 3′-end PCR primer containing BtsI recognition site is digested with 50 U Nb.BtsI at 37°C for 2 h (total reaction volume 100 µl; 1x NEB, 1x BSA) followed by capture of cDNA on streptavidin coated magnetic beads. .. 50 µl of beads (250 pmol binding capacity) is used to immobilize 5′-biotinylated cDNA.

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: The nicked substrate was purified from 1% agarose gel run in TAE buffer supplemented with 0.5 μg/ml ethidium bromide (EtBr), using the Promega Wizard SV Gel & PCR Cleanup Kit (Promega, Madison, USA). .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    Article Title: Comparison of DNA decatenation by Escherichia coli topoisomerase IV and topoisomerase III: implications for non-equilibrium topology simplification
    Article Snippet: .. The 4-kb multi-nicked DNA molecules were prepared by PCR of pFC94 ( ) from position 5422–9520 with a biotin-labelled forward primer and digoxigenin-labelled reverse primer (Eurofins MWG Operon) and were nicked with four nicking enzymes: Nb.BsmI, Nt.AIwI, Nb.BtsI and Nt.BtsNBI (New England Biolabs), generating 14 nicks located over the middle third of the DNA. ..

    Construct:

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: Briefly, the heteroduplexes containing a G/T mismatch within an AclI restriction site in the 46-bp polylinker of a pGEM13Zf(+) derivative were constructed by primer extension, using the mismatch-containing oligonucleotide (G/T: 5′-AGA CGT CTG TC G ACG TTG GGA AGC TTG AG-3′) as primer (the mispaired residue is highlighted in bold) and the single-stranded phagemid DNA carrying one Nt.BstNBI nicking site 363 bp 5′ from the mismatch, one Nb.BtsI nicking site 184 bp 3′ from the mismatch or both as template. .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    Plasmid Preparation:

    Article Title: A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine
    Article Snippet: .. The plasmid was nicked with Nb.Bts I (New England Biolabs) to generate a single-strand break in the transcribed strand. .. The nicked strand was then digested with ExoIII (New England Biolabs), and the remaining single-stranded circular DNA (ssDNA) was purified using a 1% agarose gel.

    Article Title: DNA supercoiling, a critical signal regulating the basal expression of the lac operon in Escherichia coli
    Article Snippet: Proteins, chemicals, and reagents E. coli LacI and mutants were purified by the method of Chen and Matthews (E. coli strains containing the plasmid overexpressing LacI and mutants was kindly provided by K. S. Matthews at Rice University). .. Restriction enzymes Nt.BbvCI, Nb.BbvCI, Nb.BtsI, T4 DNA ligase, and E. coli DNA gyrase were purchased from New England Biolabs (Beverly, MA, USA).

    Article Title: Alkyladenine DNA glycosylase associates with transcription elongation to coordinate DNA repair with gene expression
    Article Snippet: .. The repair mPlum plasmid (mPlum.Hx) was engineered by placing the AAG-specific base lesion hypoxanthine (Hx) lesion into the open reading frame of mPlum . mPlum.Hx Substrate Containing a Site-Specific Hypoxanthine (Hx) was generated as follows; single-stranded DNA was obtained by nicking the plasmid pmax:mPlum with Nb.BtsI (NEB R0707S) for 4 h at 37 °C in NEB Buffer 4 (NEB B7004). .. After phenol chloroform extraction the DNA was incubated with 5 U of ExoIII (NEB M0206S) per µg of DNA at 37 °C for 1 h in NEB buffer 1 (NEB B7001) to digest the nicked strand.

    Article Title: Novel Method For Quantifying Radiation-Induced Single-Strand-Break Yields In Plasmid DNA Highlights Tenfold Discrepancy
    Article Snippet: In essence, 3 HT-pUC19 (20 μg) plasmid SC DNA samples were each digested with 100 units (10 units/μl) of one of the above mentioned nicking endonucleases in 100 μl volume under appropriate conditions. .. For Nb.BtsI digestion, DNA was incubated (37°C for 18 hrs) in 1X NEBuffer 4 (New England Biolabs, Inc.); for Nt.

    Article Title: Novel Method For Quantifying Radiation-Induced Single-Strand-Break Yields In Plasmid DNA Highlights Tenfold Discrepancy
    Article Snippet: .. Nicking endonucleases Nb.BsrDI, Nb.BtsI, Nt.BstNBI and Nt.AlwI (New England Biolabs, Beverly, MA) were used to create 3 HT-pUC19 (20 μg) plasmid DNA molecules with known number of SSBs (2, 3, 4 and 10, respectively). .. In essence, 3 HT-pUC19 (20 μg) plasmid SC DNA samples were each digested with 100 units (10 units/μl) of one of the above mentioned nicking endonucleases in 100 μl volume under appropriate conditions.

    Recombinant:

    Article Title: Lagging strand replication shapes the mutational landscape of the genome
    Article Snippet: .. After Ampure XP purification, beads were removed and DNA nicked using recombinant RNase H2 (10 pmol per μg of library) or Nb.BtsI (NEB; 10 U per μg) for 2 h at 37°C. .. RNase H2 purification and reaction conditions were as previously described .

    Agarose Gel Electrophoresis:

    Article Title: A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine
    Article Snippet: The plasmid was nicked with Nb.Bts I (New England Biolabs) to generate a single-strand break in the transcribed strand. .. The nicked strand was then digested with ExoIII (New England Biolabs), and the remaining single-stranded circular DNA (ssDNA) was purified using a 1% agarose gel.

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: The nicked substrate was purified from 1% agarose gel run in TAE buffer supplemented with 0.5 μg/ml ethidium bromide (EtBr), using the Promega Wizard SV Gel & PCR Cleanup Kit (Promega, Madison, USA). .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    Article Title: Programmed genome rearrangements in Oxytricha produce transcriptionally active extrachromosomal circular DNA
    Article Snippet: Paragraph title: Two-dimensional agarose gel electrophoresis and Southern blotting ... The supercoiled DNA ladder was nicked using Nb.BtsI (New England BioLabs) to generate the relaxed circular DNA standard.

    CTG Assay:

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair
    Article Snippet: Briefly, the heteroduplexes containing a G/T mismatch within an AclI restriction site in the 46-bp polylinker of a pGEM13Zf(+) derivative were constructed by primer extension, using the mismatch-containing oligonucleotide (G/T: 5′-AGA CGT CTG TC G ACG TTG GGA AGC TTG AG-3′) as primer (the mispaired residue is highlighted in bold) and the single-stranded phagemid DNA carrying one Nt.BstNBI nicking site 363 bp 5′ from the mismatch, one Nb.BtsI nicking site 184 bp 3′ from the mismatch or both as template. .. After primer extension, ligation and isolation of the desired supercoiled heteroduplex substrates on CsCl gradients, substrates were nicked with Nt.BstNBI and Nb.BtsI (New England Biolabs) as indicated by the manufacturer.

    Gel Extraction:

    Article Title: A novel role for transcription-coupled nucleotide excision repair for the in vivo repair of 3,N4-ethenocytosine
    Article Snippet: The plasmid was nicked with Nb.Bts I (New England Biolabs) to generate a single-strand break in the transcribed strand. .. The ssDNA was extracted from the agarose gel using a Gel Extraction Kit (Qiagen).

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    New England Biolabs mung bean endonuclease
    Mung Bean Endonuclease, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 90/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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