exonuclease i  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    Name:
    Exonuclease I E coli
    Description:
    Exonuclease I E coli 15 000 units
    Catalog Number:
    m0293l
    Price:
    281
    Size:
    15 000 units
    Category:
    Exonucleases
    Buy from Supplier


    Structured Review

    New England Biolabs exonuclease i
    Exonuclease I E coli
    Exonuclease I E coli 15 000 units
    https://www.bioz.com/result/exonuclease i/product/New England Biolabs
    Average 99 stars, based on 1903 article reviews
    Price from $9.99 to $1999.99
    exonuclease i - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood"

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr424

    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.
    Figure Legend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Techniques Used: Activity Assay, Hybridization, Generated

    2) Product Images from "Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA"

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0016925

    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    Figure Legend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Techniques Used: Polyacrylamide Gel Electrophoresis

    3) Product Images from "Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals"

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    Journal: International Journal of Molecular Sciences

    doi: 10.3390/ijms17091457

    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.
    Figure Legend Snippet: Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Techniques Used: Hybridization, Nucleic Acid Electrophoresis, Electrophoresis, Blocking Assay, Incubation, Labeling

    Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .
    Figure Legend Snippet: Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Techniques Used: Northern Blot, Hybridization

    4) Product Images from "Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies"

    Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies

    Journal: The Journal of Molecular Diagnostics : JMD

    doi: 10.1016/j.jmoldx.2019.01.008

    Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).
    Figure Legend Snippet: Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).

    Techniques Used: Next-Generation Sequencing, Concentration Assay, Sequencing, Polymerase Chain Reaction, Incubation, Amplification, Purification, Selection

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw274

    DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 3′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) DNA substrates bearing different types of 3′ ends and labeled by 32 P at the third nucleotide from the 3′ end were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with four sets of data. ( C ) Assay for detecting biotin at the 3′ end of ss-DNA. The 32 P-labeled 3′ ddC or biotin DNA with short 3′ ss-overhangs was pre-incubated with buffer or avidin and then treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel. ( D ) Avidin was not removed from the 3′ end of resection intermediates. 3′ avidin DNA was incubated in extracts for the indicated times, isolated, supplemented with buffer or avidin, and treated with E. coli ExoI. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.
    Figure Legend Snippet: DNA with 5′ damaged nucleotides or bulky adducts is channeled to resection. ( A ) 32 P -labeled DNA substrates bearing different types of 5′ ends were incubated with Xenopus egg extracts for the indicated times. The products were analyzed on a 1% TAE-agarose gel and detected by exposing the dried gel to X-ray film. Avidin is bound to DNA ends via biotin. ( B ) Plot of the percentages of substrates converted into supercoiled monomer products at 180′. The averages and standard deviations were calculated with five sets of data. ( C ) Resection of 5′ avidin DNA proceeds in the 5′→3′ direction. 5′ avidin DNA was incubated with extracts for 30 min and re-isolated. They were incubated with buffer or avidin and then treated with E. coli ExoI or RecJ. The products were analyzed on a 1% TAE-agarose gel.

    Techniques Used: Labeling, Incubation, Agarose Gel Electrophoresis, Avidin-Biotin Assay, Isolation

    6) 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

    7) Product Images from "Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq"

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    Journal: Nature protocols

    doi: 10.1038/s41596-018-0055-0

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).
    Figure Legend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Techniques Used:

    8) Product Images from "Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair"

    Article Title: Dual daughter strand incision is processive and increases the efficiency of DNA mismatch repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw411

    Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.
    Figure Legend Snippet: Acceleration of daughter strand unwinding and degradation by GATC sites flanking the mismatch. ( A ) Agarose gel analysis of nicking and unwinding of 0.5 nM circular DNA containing a single G/T mismatch at different positions and one or two GATC sites by 10 nM MutS, 10 nM MutL, 5 nM MutH, 5 nM UvrD, 200 nM Ssb and 0.1 units of ExoI. Early time points (2 and 4 min) showed the conversion of the closed circular DNA (lower band) to open-circular DNA (upper band) due to nicking by MutH. Later time points showed unwinding of nicked daughter strand by UvrD and degradation by ExoI starting from the 3′ end as indicated in the schematic drawings above the gel panels. ( B ) Quantification of the fraction of unnicked and nicked DNA for GT#1, GT#1b, GT#2 and GT#2b (mean ± SD, n = 3) with fit according to the unwinding model. Kinetic parameters obtained from the fit are tabulated in Supplementary Table S4. ( C ) Unwinding and excision of GT#1b pre-nicked with MutH alone (left panel), with MutH and Cas9 at site CrB such that nicks were on the same side of the mismatch (middle panel), and with MutH and Cas9 at site CrA such that the nicks flank the mismatch (right panel). ( D ) Quantification of unwinding (mean ± SD, n = 3) and fit with a function describing a single exponential increase. Kinetic parameters obtained from the fits are tabulated in Supplementary Table S4.

    Techniques Used: Agarose Gel Electrophoresis

    9) Product Images from "Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast"

    Article Title: Pooled clone collections by multiplexed CRISPR-Cas12a-assisted gene tagging in yeast

    Journal: bioRxiv

    doi: 10.1101/476804

    Next-generation sequencing (NGS) library preparation with unique molecular identifiers (UMIs) for molecule counting. ( a ) Quantitative NGS of molecular recombineering intermediates used for CASTLING. The PCR-amplified oligonucleotide pools (left side) or the undigested (concatemeric) SICs yielded by rolling circle amplification (RCA, right side) are quantitated using a random hexameric oligonucleotide sequence (N 6 ) as UMI. This N 6 UMI is incorporated in two rounds of primer annealing and elongation (1 st PCR). Unincorporated primers are then destroyed by Exonuclease I treatment, followed by heat inactivation of Exonuclease I. In a final PCR (30 cycles), technical sequences required for Illumina NGS such as P5 and P7 along with barcodes to distinguish different samples are attached. ( b ) For quantitative Anchor-Seq of the yeast libraries, adapters with random octameric nucleotides (N 8 ) are ligated to the fragmented and end-repaired genomic DNA. Next, the fragments which contain SIC-derived sequences (the figure shows the junction of the ORF and the tag) are selectively amplified in a PCR with a SIC-specific and a reverse primer. During the initial cycle, the SIC-specific primer generates the binding site of the reverse primer by replication of an asymmetric sequence. This allows for these genomic fragments to be exponentially amplified. An elongation-inhibiting group (Spacer C3, SpC3) on the complementary strand of the ligated adapters prohibits non-specific amplification of fragments without the SIC-specific sequence of interest.
    Figure Legend Snippet: Next-generation sequencing (NGS) library preparation with unique molecular identifiers (UMIs) for molecule counting. ( a ) Quantitative NGS of molecular recombineering intermediates used for CASTLING. The PCR-amplified oligonucleotide pools (left side) or the undigested (concatemeric) SICs yielded by rolling circle amplification (RCA, right side) are quantitated using a random hexameric oligonucleotide sequence (N 6 ) as UMI. This N 6 UMI is incorporated in two rounds of primer annealing and elongation (1 st PCR). Unincorporated primers are then destroyed by Exonuclease I treatment, followed by heat inactivation of Exonuclease I. In a final PCR (30 cycles), technical sequences required for Illumina NGS such as P5 and P7 along with barcodes to distinguish different samples are attached. ( b ) For quantitative Anchor-Seq of the yeast libraries, adapters with random octameric nucleotides (N 8 ) are ligated to the fragmented and end-repaired genomic DNA. Next, the fragments which contain SIC-derived sequences (the figure shows the junction of the ORF and the tag) are selectively amplified in a PCR with a SIC-specific and a reverse primer. During the initial cycle, the SIC-specific primer generates the binding site of the reverse primer by replication of an asymmetric sequence. This allows for these genomic fragments to be exponentially amplified. An elongation-inhibiting group (Spacer C3, SpC3) on the complementary strand of the ligated adapters prohibits non-specific amplification of fragments without the SIC-specific sequence of interest.

    Techniques Used: Next-Generation Sequencing, Polymerase Chain Reaction, Amplification, Sequencing, Derivative Assay, Binding Assay

    10) Product Images from "Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System"

    Article Title: Towards the Construction of Expressed Proteomes Using a Leishmania tarentolae Based Cell-Free Expression System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0014388

    Use of Species-Independent Translational Sequence (SITS) in Overlap Extension (OE) PCR-mediated assembly of templates for in vitro translation. (A) Schematic representation of SITS structure. (B) Scheme of purification-free OE-PCR for synthesis of DNA templates for cell-free translation. PCR amplification of individual fragments with partially overlapping sequences. 5′ NTR – 5′ not transcribed regions, 3′ UTR – 3′ untranslated region. (C) Removal of residual primers from PCR reaction by exonuclease I treatment. (D) Fragments are fused and amplified by Overlap Extension PCR in the presence of the flanking primers.
    Figure Legend Snippet: Use of Species-Independent Translational Sequence (SITS) in Overlap Extension (OE) PCR-mediated assembly of templates for in vitro translation. (A) Schematic representation of SITS structure. (B) Scheme of purification-free OE-PCR for synthesis of DNA templates for cell-free translation. PCR amplification of individual fragments with partially overlapping sequences. 5′ NTR – 5′ not transcribed regions, 3′ UTR – 3′ untranslated region. (C) Removal of residual primers from PCR reaction by exonuclease I treatment. (D) Fragments are fused and amplified by Overlap Extension PCR in the presence of the flanking primers.

    Techniques Used: Sequencing, Overlap Extension Polymerase Chain Reaction, In Vitro, Purification, Polymerase Chain Reaction, Amplification

    11) 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

    12) 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

    13) Product Images from "A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding"

    Article Title: A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq687

    Experimental setting for genome-wide analysis of DNA-crosslinking probability mediated by TMP PB. In a typical experiment, exponentially growing yeast cells were split in three identical fractions. In the first fraction, cells were incubated with TMP and irradiated to produce a limited amount of intracellular DNA crosslinks (one crosslink per 10 kb). Cellular DNA was extracted and fragmented (2-kb average length). In the second fraction, cellular DNA was extracted, fragmented (2-kb average length) and then incubated with TMP and irradiated to produce one crosslink per 10 kb. Genomic DNA fragments from the above in vivo and in vitro experiments were thermally denatured to convert un-crosslinked segments into ssDNA. Exonuclease I degraded then ssDNA chains from their 3′-end, such that only crosslinked duplexes (dsDNA) and DNA chains with a blocked 3′-end remained. Exonuclease λ digested then ssDNA and dsDNA and from their 5′-ends. As a result, un-crosslinked fragments were degraded and crosslinked fragments were converted into a pair of ssDNA chains bridged by a TMP linkage. Random priming along these chains produced radiolabeled sequences that were hybridized on DNA arrays . The third fraction of the yeast culture was used to conduct a genome-wide analysis of ongoing transcription (GRO). In vivo radiolabeled RNA was purified and hybridized on arrays of the same lot used to analyze the TMP-mediated DNA crosslinks.
    Figure Legend Snippet: Experimental setting for genome-wide analysis of DNA-crosslinking probability mediated by TMP PB. In a typical experiment, exponentially growing yeast cells were split in three identical fractions. In the first fraction, cells were incubated with TMP and irradiated to produce a limited amount of intracellular DNA crosslinks (one crosslink per 10 kb). Cellular DNA was extracted and fragmented (2-kb average length). In the second fraction, cellular DNA was extracted, fragmented (2-kb average length) and then incubated with TMP and irradiated to produce one crosslink per 10 kb. Genomic DNA fragments from the above in vivo and in vitro experiments were thermally denatured to convert un-crosslinked segments into ssDNA. Exonuclease I degraded then ssDNA chains from their 3′-end, such that only crosslinked duplexes (dsDNA) and DNA chains with a blocked 3′-end remained. Exonuclease λ digested then ssDNA and dsDNA and from their 5′-ends. As a result, un-crosslinked fragments were degraded and crosslinked fragments were converted into a pair of ssDNA chains bridged by a TMP linkage. Random priming along these chains produced radiolabeled sequences that were hybridized on DNA arrays . The third fraction of the yeast culture was used to conduct a genome-wide analysis of ongoing transcription (GRO). In vivo radiolabeled RNA was purified and hybridized on arrays of the same lot used to analyze the TMP-mediated DNA crosslinks.

    Techniques Used: Genome Wide, Incubation, Irradiation, In Vivo, In Vitro, Produced, Purification

    14) Product Images from "AID–RNA polymerase II transcription-dependent deamination of IgV DNA"

    Article Title: AID–RNA polymerase II transcription-dependent deamination of IgV DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz821

    Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.
    Figure Legend Snippet: Experimental protocol used to analyze AID-catalyzed dC deamination on IGHV3-23*01 during transcription by human Pol II. ( A ) Pol II ± DSIF elongation complexes were assembled on a DNA–RNA ‘scaffolded bubble’ substrate and preincubated with AID. Transcription was initiated by the addition rNTP substrates, and the elongation reaction was performed at 30°C (Methods). Following transcription, Exonuclease I (Exo I) was added to digest ssDNA. TS and NTS DNAs were separately barcoded and subjected next-generation sequencing analysis using Maximum Depth Sequencing (MDS) ( 39 ) to assess AID-mediated dC deamination. ( B ) Transcription in the presence of AID and DSIF was visualized as 32 P-labeled RNA primer elongation bands that extend for the full length of the IgV DNA (198 nt). A strong transcription pause region is located ∼11 nt downstream of the scaffold bubble, and is followed by six C residues on the TS, in which deaminations are observed to occur at as many as three contiguous C sites – see also Supplemental Figure S9. ( C ) Distribution of Pol II extended transcripts. Percentage (mean ± standard deviation) of scaffold bubble proximal transcripts (1–12 nt from the end of the scaffold bubble to a run of six consecutive Cs) and full-length transcript (198 nt) were quantified by GE Healthcare ImageQuant software. A sketch depicting the transcribed IgV substrate and the scaffold bubble containing a 20 nt RNA primer strand is shown at the top.

    Techniques Used: Next-Generation Sequencing, Sequencing, Labeling, Standard Deviation, Software

    15) Product Images from "RPA accumulation during class switch recombination represents 5′-3′ DNA end resection during the S-G2/M phase of the cell cycle"

    Article Title: RPA accumulation during class switch recombination represents 5′-3′ DNA end resection during the S-G2/M phase of the cell cycle

    Journal: Cell reports

    doi: 10.1016/j.celrep.2012.12.006

    RPA interacts with resected ssDNA downstream of AID or I- Ppo I endonuclease (A) RPA association with + and − DNA strands at the Grap gene locus from Igκ AID-53BP1 −/− activated B cells. RPKM values for the specified window (chr11:61,399,129-61,572,079) are provided in parenthesis. (B) RPA bound to Igh locus. As in (A), ChIP-Seq signals were resolved into upper and lower strands. Technical replicates were treated with E. coli ExoI or RecJ exonucleases nuclease prior to deep-sequencing library preparation. (C) PolII binding to + and − strands at the Grap locus. (D) Technical IP replicates from panels A and C samples incubated in the presence of ExoI. (E) Composite diagram showing RPA profiles at 19 I- Ppo I mouse genomic sites in 53BP1 −/− MEFs transduced with retroviruses expressing the I- Ppo I homing endonuclease.
    Figure Legend Snippet: RPA interacts with resected ssDNA downstream of AID or I- Ppo I endonuclease (A) RPA association with + and − DNA strands at the Grap gene locus from Igκ AID-53BP1 −/− activated B cells. RPKM values for the specified window (chr11:61,399,129-61,572,079) are provided in parenthesis. (B) RPA bound to Igh locus. As in (A), ChIP-Seq signals were resolved into upper and lower strands. Technical replicates were treated with E. coli ExoI or RecJ exonucleases nuclease prior to deep-sequencing library preparation. (C) PolII binding to + and − strands at the Grap locus. (D) Technical IP replicates from panels A and C samples incubated in the presence of ExoI. (E) Composite diagram showing RPA profiles at 19 I- Ppo I mouse genomic sites in 53BP1 −/− MEFs transduced with retroviruses expressing the I- Ppo I homing endonuclease.

    Techniques Used: Recombinase Polymerase Amplification, Chromatin Immunoprecipitation, Sequencing, Binding Assay, Incubation, Transduction, Expressing

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

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

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008459

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

    Techniques Used: Southern Blot, Marker, Countercurrent Chromatography

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

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

    17) Product Images from "Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions"

    Article Title: Reovirus-mediated induction of ADAR1 (p150) minimally alters RNA editing patterns in discrete brain regions

    Journal: Molecular and cellular neurosciences

    doi: 10.1016/j.mcn.2014.06.001

    Deep sequencing strategy for multiplex quantification of editing profiles ) were used for PCR amplification (5 cycles) before digestion of the remaining single-stranded primers using Exonuclease I. A second round of amplification (25 cycles) was performed with universal primers in which the oligonucleotide contained sequences matching the T3 promoter, one of 24 unique 6-nt barcode sequences (yellow) for sample identification, as well as an adapter sequence (Adapter A; green) or sequences matching the T7 and an adapter sequence (Adapter B; purple) for high-throughput single-end sequencing on the Illumina platform.
    Figure Legend Snippet: Deep sequencing strategy for multiplex quantification of editing profiles ) were used for PCR amplification (5 cycles) before digestion of the remaining single-stranded primers using Exonuclease I. A second round of amplification (25 cycles) was performed with universal primers in which the oligonucleotide contained sequences matching the T3 promoter, one of 24 unique 6-nt barcode sequences (yellow) for sample identification, as well as an adapter sequence (Adapter A; green) or sequences matching the T7 and an adapter sequence (Adapter B; purple) for high-throughput single-end sequencing on the Illumina platform.

    Techniques Used: Sequencing, Multiplex Assay, Polymerase Chain Reaction, Amplification, High Throughput Screening Assay

    18) Product Images from "Methods and Compositions for Amplification and Detection of microRNAs (miRNAs) and Noncoding RNAs (ncRNAs) Using the Signature Sequence Amplification Method (SSAM)"

    Article Title: Methods and Compositions for Amplification and Detection of microRNAs (miRNAs) and Noncoding RNAs (ncRNAs) Using the Signature Sequence Amplification Method (SSAM)

    Journal: Recent advances in DNA & gene sequences

    doi:

    Depiction of the clearance of free single stranded FON with enzymatic digestion by exonuclease I before the addition of SONs.
    Figure Legend Snippet: Depiction of the clearance of free single stranded FON with enzymatic digestion by exonuclease I before the addition of SONs.

    Techniques Used:

    19) Product Images from "Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys"

    Article Title: Murine and related chapparvoviruses are nephro-tropic and produce novel accessory proteins in infected kidneys

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1008262

    Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.
    Figure Legend Snippet: Map of the MKPV genome. (A-B) Maps of the MKPV/MuCPV strains from Centenary Institute (CI, accession MH670587), Memorial Sloan Kettering Cancer Center (MSKCC, accession MH670588) and New York City basements (wild-NY, MF175078). “Bowties” indicate terminal repeats (TR). (A) Single nucleotide variations (SNV) between the CI, MSKCC and wild-NY accessions. Vertical lines—differences between accessions. Half height vertical lines—polymorphisms within an accession. ▼; 2 bp insertion in the CI strain. ▲; 1 bp insertion in a CI sub-strain. Dashed lines—missing extremities in MSKCC and wild-NY accessions, which consist of the exterior inverted repeats in the full-length CI sequence. (B-C) Alternative splicing allows production of the polypeptides p10, p15, NS1, NS2, NP and VP. Black, brown or blue shading indicate the relative reading frames of ORFs. p15, p10 and NP could theoretically be produced from multiple transcripts. Orange or red indicate peptides present in LC-MS/MS datasets PXD014938 (this paper) or PXD010540 [ 9 ], respectively. Exon or intron sequences flanking splice sites are shown in black or red text, respectively. (C) Quantitation of spliced MKPV reads in RNAseq data pooled from two MKPV-infected kidneys. Columns indicate splice site usage (left y-axis); heights of arcs (right y-axis) indicate the abundance of specific splice combinations. See S4 Table for more information. (D-E) Detection of spliced transcripts by RT-dependent PCR, using primers mapped in A-B. Input templates were MKPV-infected (D) kidney DNA or (E) DNAse/ExoI-treated kidney RNA, converted (+RT) or mock-converted (-RT) to cDNA. RT-PCR products corresponding to transcripts 1 to 4 are indicated by white numbers. (F) Mapping of transcription start and stop sites by RACE. See S1 Fig for RACE details. Major 5’ and 3’ RACE products, indicated by black arrows and corresponding to transcripts 2 to 4 or polyadenylation signals A and B, were gel-purified and Sanger sequenced. Other RACE products mentioned in the text are indicated by white arrows.

    Techniques Used: Sequencing, Produced, Liquid Chromatography with Mass Spectroscopy, Quantitation Assay, Infection, Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Purification

    20) 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

    21) Product Images from "Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing"

    Article Title: Human PSF concentrates DNA and stimulates duplex capture in DMC1-mediated homologous pairing

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr1229

    PSF stimulates dsDNA capture by the DMC1–ssDNA complex. ( A ) Schematic representation of the dsDNA capturing assay. Asterisks indicate the 32 P-labeled ends of the dsDNA. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), and 32 P-labeled dsDNA 49-mer (1.5 µM) in the presence of the 83-mer poly dT ssDNA conjugated to the beads. The dsDNA captured by the DMC1–ssDNA complex was detected. Lane 1 indicates a negative control reaction without the proteins. Lanes 2–4 represent the experiments with DMC1 alone, PSF alone, and both DMC1 and PSF, respectively. Portions including 80% of the dsDNA recovered in the ssDNA bound fraction were analyzed by native PAGE (upper panel), and 10% portions of the dsDNA samples remaining in the ssDNA unbound fraction were analyzed by native PAGE (lower panel). ( C ) The ssDNA protection assay with DMC1. The DMC1–ssDNA complexes were treated with exonuclease I in the presence or absence of PSF. The resulting ssDNAs were analyzed by native PAGE. Lane 1 indicates a negative control experiment without proteins and exonuclease I. Lanes 3, 6, 9 and 12 indicate experiments with 0.3 µM PSF, and lanes 4, 7, 10 and 13 indicate experiments with 0.6 µM PSF. Lanes 2, 5, 8 and 11 indicate experiments without PSF. The DMC1 concentrations were 0 µM (lanes 2–4), 0.5 µM (lanes 5–7), 1 µM (lanes 8–10) and 2 µM (lanes 11–13). ( D ) The ssDNA protection assay with RAD51. The reaction was conducted by the same method as in panel C, except RAD51 was used instead of DMC1. ( E ) Schematic representation of the synaptic complex formation assay. ( F ) The synaptic complex formation reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), in the presence of the 70-mer ssDNA and superhelical dsDNA. Lane 1 indicates a negative control experiment without proteins and PstI restriction enzyme. Lanes 2 and 6 represent the experiments without proteins. Lanes 2–5 and 6–9 indicate experiments with the homologous ssDNA and heterologous ssDNA, respectively. Lanes 3 and 7 indicate the experiments with DMC1 alone. Lanes 4 and 8 indicate the experiments with PSF alone. Lanes 5 and 9 indicate the experiments with both DMC1 and PSF. NC and SC represent nicked circular and superhelical dsDNA molecules. ( G ) Graphic representation of the synaptic complex formation assay shown in panel F. The band intensities of the protected DNAs were quantitated, and the average values of three independent experiments are shown with the SD values.
    Figure Legend Snippet: PSF stimulates dsDNA capture by the DMC1–ssDNA complex. ( A ) Schematic representation of the dsDNA capturing assay. Asterisks indicate the 32 P-labeled ends of the dsDNA. ( B ) The reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), and 32 P-labeled dsDNA 49-mer (1.5 µM) in the presence of the 83-mer poly dT ssDNA conjugated to the beads. The dsDNA captured by the DMC1–ssDNA complex was detected. Lane 1 indicates a negative control reaction without the proteins. Lanes 2–4 represent the experiments with DMC1 alone, PSF alone, and both DMC1 and PSF, respectively. Portions including 80% of the dsDNA recovered in the ssDNA bound fraction were analyzed by native PAGE (upper panel), and 10% portions of the dsDNA samples remaining in the ssDNA unbound fraction were analyzed by native PAGE (lower panel). ( C ) The ssDNA protection assay with DMC1. The DMC1–ssDNA complexes were treated with exonuclease I in the presence or absence of PSF. The resulting ssDNAs were analyzed by native PAGE. Lane 1 indicates a negative control experiment without proteins and exonuclease I. Lanes 3, 6, 9 and 12 indicate experiments with 0.3 µM PSF, and lanes 4, 7, 10 and 13 indicate experiments with 0.6 µM PSF. Lanes 2, 5, 8 and 11 indicate experiments without PSF. The DMC1 concentrations were 0 µM (lanes 2–4), 0.5 µM (lanes 5–7), 1 µM (lanes 8–10) and 2 µM (lanes 11–13). ( D ) The ssDNA protection assay with RAD51. The reaction was conducted by the same method as in panel C, except RAD51 was used instead of DMC1. ( E ) Schematic representation of the synaptic complex formation assay. ( F ) The synaptic complex formation reaction was conducted with DMC1 (4 µM) and/or PSF (0.6 µM), in the presence of the 70-mer ssDNA and superhelical dsDNA. Lane 1 indicates a negative control experiment without proteins and PstI restriction enzyme. Lanes 2 and 6 represent the experiments without proteins. Lanes 2–5 and 6–9 indicate experiments with the homologous ssDNA and heterologous ssDNA, respectively. Lanes 3 and 7 indicate the experiments with DMC1 alone. Lanes 4 and 8 indicate the experiments with PSF alone. Lanes 5 and 9 indicate the experiments with both DMC1 and PSF. NC and SC represent nicked circular and superhelical dsDNA molecules. ( G ) Graphic representation of the synaptic complex formation assay shown in panel F. The band intensities of the protected DNAs were quantitated, and the average values of three independent experiments are shown with the SD values.

    Techniques Used: Capturing Assay, Labeling, Negative Control, Clear Native PAGE, Tube Formation Assay

    22) Product Images from "MLGA--a rapid and cost-efficient assay for gene copy-number analysis"

    Article Title: MLGA--a rapid and cost-efficient assay for gene copy-number analysis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm651

    ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.
    Figure Legend Snippet: ( a ) Multiplex ligation-dependent genome amplification (MLGA), reaction scheme. (I) Genomic DNA is digested by restriction enzyme to generate targets with defined ends. (II) Each MLGA probe consists of two oligonucleotides, one selector oligo of 70–74 nt (green) and one general vector oligo of 34 nt (red). MLGA probe together with DNA-ligase forms circular DNA of target molecules after denaturation and hybridization. (III) To reduce background signal in the assay, undesirable, linear DNA is degraded by exonuclease I (Exo I). (IV) Multiplex PCR is facilitated by using universal primers that hybridize to a sequence in the vector. PCR products are analyzed using the Agilent Bioanalyzer 2100™ electrophoresis system. ( b ) Data from an MLGA set of 14 probes targeting loci on human chromosomes 13, 18, 21, X and Y. The upper graph shows the resulting elution diagrams from analyses of male and female DNA pools.

    Techniques Used: Multiplex Assay, Ligation, Amplification, Plasmid Preparation, Hybridization, Polymerase Chain Reaction, Sequencing, Electrophoresis

    23) Product Images from "Role of the RAD51–SWI5–SFR1 Ensemble in homologous recombination"

    Article Title: Role of the RAD51–SWI5–SFR1 Ensemble in homologous recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw375

    Functional characterization of SWI5–SFR1 dN202 complex. ( A ) To map the minimal complex unit of SWI5–SFR1, purified SWI5–SFR1 was incubated with proteinase K for the indicated times. The proteolytic products were resolved by 15% SDS-PAGE and stained by Coomassie Blue staining. The asterisk denotes the SFR1 dN202 . ( B ) Purified SWI5–SFR1 dN202 complex (3 μg) was subjected to 15% SDS-PAGE and Coomassie Blue staining. ( C ) SWI5–SFR1 dN202 was tested for RAD51 interaction by affinity pulldown as described in Figure 1D . ( D ) DNA strand exchange assay to monitor the stimulatory effect of SWI5–SFR1 by RAD51. ( I ) Schematic of the DNA strand exchange assay. The radiolabeled substrate and product are visualized and quantified by phosphorimaging analysis after PAGE. The asterisk denotes the 32 P label. ( II ) DNA strand exchange was conducted with the indicated amounts of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. ( E ) Exonuclease I protection assay to monitor the influence of SWI5–SFR1 on the stability of RAD51 filament. ( I ) Schematic of the exonuclease I protection assay. The RAD51 filament harboring 5′- 32 P-labeled DNA is challenged with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( II ) Treatment of the RAD51 presynaptic filament with exonuclease I in the presence of the indicated concentrations of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. (D-E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.
    Figure Legend Snippet: Functional characterization of SWI5–SFR1 dN202 complex. ( A ) To map the minimal complex unit of SWI5–SFR1, purified SWI5–SFR1 was incubated with proteinase K for the indicated times. The proteolytic products were resolved by 15% SDS-PAGE and stained by Coomassie Blue staining. The asterisk denotes the SFR1 dN202 . ( B ) Purified SWI5–SFR1 dN202 complex (3 μg) was subjected to 15% SDS-PAGE and Coomassie Blue staining. ( C ) SWI5–SFR1 dN202 was tested for RAD51 interaction by affinity pulldown as described in Figure 1D . ( D ) DNA strand exchange assay to monitor the stimulatory effect of SWI5–SFR1 by RAD51. ( I ) Schematic of the DNA strand exchange assay. The radiolabeled substrate and product are visualized and quantified by phosphorimaging analysis after PAGE. The asterisk denotes the 32 P label. ( II ) DNA strand exchange was conducted with the indicated amounts of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. ( E ) Exonuclease I protection assay to monitor the influence of SWI5–SFR1 on the stability of RAD51 filament. ( I ) Schematic of the exonuclease I protection assay. The RAD51 filament harboring 5′- 32 P-labeled DNA is challenged with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( II ) Treatment of the RAD51 presynaptic filament with exonuclease I in the presence of the indicated concentrations of SWI5–SFR1 or SWI5–SFR1 dN202 . The results were graphed. (D-E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

    Techniques Used: Functional Assay, Purification, Incubation, SDS Page, Staining, Polyacrylamide Gel Electrophoresis, Labeling, Standard Deviation

    SWI5 F83A/L85A-SFR1 is functionally impaired. ( A ) The effect of SWI5–SFR1 or SWI5 FL/AA –SFR1 on RAD51-mediated DNA strand exchange was examined. The results were graphed. ( B ) Exonuclease I protection assay was conducted with the indicated concentrations of SWI5–SFR1 and SWI5 FL/AA –SFR1. The results were graphed. ( C ) The average length of RAD51 filaments with negative staining was determined by electron microscopy (see Supplementary Figure S5). RAD51 was examined alone or with SWI5–SFR1 or SWI5 FL/AA –SFR1. The total 225 filaments were counted in each reaction. We note that the average length of the presynaptic filament in the presence of SWI5–SFR1 is much longer than expected. This could be due to the end-to-end association between two DNA molecules by RAD51 as described by Baumann et al. ( 35 ). ( D ) Thin-layer chromatography to monitor the hydrolysis of [γ- 32 P] ATP by RAD51 in the absence or presence of indicated concentrations of SWI5–SFR1 or SWI5 FL/AA –SFR1. The results were graphed. (A, B and D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.
    Figure Legend Snippet: SWI5 F83A/L85A-SFR1 is functionally impaired. ( A ) The effect of SWI5–SFR1 or SWI5 FL/AA –SFR1 on RAD51-mediated DNA strand exchange was examined. The results were graphed. ( B ) Exonuclease I protection assay was conducted with the indicated concentrations of SWI5–SFR1 and SWI5 FL/AA –SFR1. The results were graphed. ( C ) The average length of RAD51 filaments with negative staining was determined by electron microscopy (see Supplementary Figure S5). RAD51 was examined alone or with SWI5–SFR1 or SWI5 FL/AA –SFR1. The total 225 filaments were counted in each reaction. We note that the average length of the presynaptic filament in the presence of SWI5–SFR1 is much longer than expected. This could be due to the end-to-end association between two DNA molecules by RAD51 as described by Baumann et al. ( 35 ). ( D ) Thin-layer chromatography to monitor the hydrolysis of [γ- 32 P] ATP by RAD51 in the absence or presence of indicated concentrations of SWI5–SFR1 or SWI5 FL/AA –SFR1. The results were graphed. (A, B and D) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments.

    Techniques Used: Negative Staining, Electron Microscopy, Thin Layer Chromatography, Standard Deviation

    24) Product Images from "Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells"

    Article Title: Systematic analysis of human telomeric dysfunction using inducible telosome/shelterin CRISPR/Cas9 knockout cells

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.34

    Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P
    Figure Legend Snippet: Deletion of individual subunits impacts telomere length and overhang maintenance. The inducible KO cell lines were maintained in the presence (+) or absence (−) of doxycycline (Dox) and collected at different time points for the following assays. At least three experiments with independent doxycycline inductions were performed and the results were combined. ( a , b ) Genomic DNA was extracted from the cells for telomere restriction fragment (TRF) analysis using a 32 P-labeled telomere probe (TTAGGG) 3 . Telomere signals were quantified and processed using TeloRun, and average telomere length was calculated and graphed for each cell line in a . Representative gels of the TRF assay are shown in b . ( c ) The cells were collected 6 days after induction and immunostained using antibodies against each targeted protein and RPA1 along with a telomere PNA probe. 4,6-Diamidino-2-phenylindole (DAPI) was used to stain the nuclei. Three independent experiments were carried out with at least 100 cells examined for each experiment. Scale bars 10 μm. ( d ) The cells were harvested 6 days after induction for genomic DNA extraction. The DNA was then processed in the presence (+) or absence (−) of Exonuclease I (ExoI) for in-gel hybridization analysis of ss G overhangs. G overhangs were detected in the native gel using the 32 P-labeled (CCCTAA) 3 probe. Total telomeric DNA and Alu repeat signals were determined under denaturing conditions. ( e ) Overhang signals for each cell line from d were quantified and normalized against Alu repeat signals. Results from doxycycline-treated samples were compared with untreated samples and graphed as indicated. At least three independent experiments were performed for each cell line. Error bars indicate s.e. ( n =3). P- values were obtained using the Student’s t -test. ** P

    Techniques Used: Labeling, TRF Assay, Staining, DNA Extraction, Hybridization

    25) Product Images from "The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein"

    Article Title: The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00365-13

    Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane
    Figure Legend Snippet: Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane

    Techniques Used: Agarose Gel Electrophoresis

    26) 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

    27) Product Images from "A convenient system for highly specific and sensitive detection of miRNA expression"

    Article Title: A convenient system for highly specific and sensitive detection of miRNA expression

    Journal: RNA

    doi: 10.1261/rna.040220.113

    Schematic diagram of procedures. Extracted RNAs are hybridized in buffer with special hairpin DNA probes. Next, FD BamH I is added into the mixture to cut the probes. Following that, exonuclease I is pipetted into the mixture to digest the nonhybridized
    Figure Legend Snippet: Schematic diagram of procedures. Extracted RNAs are hybridized in buffer with special hairpin DNA probes. Next, FD BamH I is added into the mixture to cut the probes. Following that, exonuclease I is pipetted into the mixture to digest the nonhybridized

    Techniques Used:

    28) Product Images from "The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein"

    Article Title: The Mycoplasma gallisepticum Virulence Factor Lipoprotein MslA Is a Novel Polynucleotide Binding Protein

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00365-13

    Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane
    Figure Legend Snippet: Exonuclease I protection assay. Exonuclease I (0.04 U) was added 10 min after 20 pmol (dT) 30 and GST-MslA had been allowed to bind. Reaction products were analyzed by 2% agarose gel electrophoresis. The amount of GST-MslA in each reaction was 0 pmol (lane

    Techniques Used: Agarose Gel Electrophoresis

    29) Product Images from "A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples"

    Article Title: A novel use of random priming-based single-strand library preparation for whole genome sequencing of formalin-fixed paraffin-embedded tissue samples

    Journal: Nar Genomics and Bioinformatics

    doi: 10.1093/nargab/lqz017

    Work flow of the standard versus DDAT library preparation method. To generate WGS libraries from low-input, degraded DNA, the complete protocol starts with the addition of enzymes SMUG1 (single-strand-selective monofunctional uracil-DNA glycosylase) and Fpg (formamidopyrimidine [fapy]-DNA glycosylase) to the input DNA ( A and B ) that remove damaged bases such as deoxyuracil and 8-oxoguanine, caused by the FFPE treatment. A short denaturation step (B) is followed by the first strand synthesis; during this step, the genomic DNA, primers and Klenow fragment (3′ → 5′ exo-) are gradually heated from 4 to 37°C with a slow ramping speed of 4°C/min, which is an essential reaction condition (see ‘Discussion’ section), before incubation at 37°C for a further 1.5 h ( C ). The primers contain nine random nucleotides from the 3′-end, in addition to the standard Illumina adaptor sequence, and will anneal to complementary DNA sequences present in the DNA sample. After the first strand synthesis, any remaining primers or short ssDNA fragments are digested with exonuclease I and the dsDNA is purified with AMPure XP beads. Next, the dsDNA is denatured to carry out the second strand synthesis using a second adaptor primer also containing nine random nucleotides, with the same conditions as the first synthesis, followed by bead purification (C). Finally, 10 PCR cycles are carried out using standard Illumina p5 and p7 indexed primers ( D ). The library is purified and assessed using standard quality control methods.
    Figure Legend Snippet: Work flow of the standard versus DDAT library preparation method. To generate WGS libraries from low-input, degraded DNA, the complete protocol starts with the addition of enzymes SMUG1 (single-strand-selective monofunctional uracil-DNA glycosylase) and Fpg (formamidopyrimidine [fapy]-DNA glycosylase) to the input DNA ( A and B ) that remove damaged bases such as deoxyuracil and 8-oxoguanine, caused by the FFPE treatment. A short denaturation step (B) is followed by the first strand synthesis; during this step, the genomic DNA, primers and Klenow fragment (3′ → 5′ exo-) are gradually heated from 4 to 37°C with a slow ramping speed of 4°C/min, which is an essential reaction condition (see ‘Discussion’ section), before incubation at 37°C for a further 1.5 h ( C ). The primers contain nine random nucleotides from the 3′-end, in addition to the standard Illumina adaptor sequence, and will anneal to complementary DNA sequences present in the DNA sample. After the first strand synthesis, any remaining primers or short ssDNA fragments are digested with exonuclease I and the dsDNA is purified with AMPure XP beads. Next, the dsDNA is denatured to carry out the second strand synthesis using a second adaptor primer also containing nine random nucleotides, with the same conditions as the first synthesis, followed by bead purification (C). Finally, 10 PCR cycles are carried out using standard Illumina p5 and p7 indexed primers ( D ). The library is purified and assessed using standard quality control methods.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Incubation, Sequencing, Purification, Polymerase Chain Reaction

    30) Product Images from "Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice"

    Article Title: Fusion of the Cas9 endonuclease and the VirD2 relaxase facilitates homology-directed repair for precise genome engineering in rice

    Journal: Communications Biology

    doi: 10.1038/s42003-020-0768-9

    Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.
    Figure Legend Snippet: Chimeric Cas9-VirD2 fusions efficiently bind to repair template DNA and cleave the DNA target. a Expression and purification of Cas9-VirD2 and VirD2-Cas9 from BL21(DE3) cells. The HIS column-purified Cas9-VirD2 and VirD2-Cas9 fusion proteins were separated on SDS-PAGE. Both Cas9-VirD2 and VirD2-Cas9 with the exact size of 216 kDa were purified and 1 µg was loaded into the gel for separation. b Confirmation of the nicking and relaxase activity of the Cas9-VirD2 and VirD2-Cas9 fusions. The T-DNA vector with the RB and LB was incubated with increasing concentrations of Cas9-VirD2 and VirD2-Cas9. The complex was separated on a 1% TBE agarose gel. The red arrowhead indicates the conversion of the supercoiled plasmid to a completely relaxed gel-retarded DNA structure. c Confirmation of the covalent binding of Cas9-VirD2 and VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) RB sequence (T-RB-60b) and without RB (T-NRB-60b) were incubated with Cas9-VirD2 and VirD2-Cas9 in Mg 2+ buffer. After incubation, the sample mixture was boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB-60b) to Cas9-VirD2 and VirD2-Cas9. (d) Optimization of the covalent binding of VirD2-Cas9 fusions to the repair templates. ssDNA (60 nt) with RB sequence (T-RB) and without RB sequence (T-NRB) were incubated with VirD2-Cas9 in Mg 2+ buffer. After incubation for 5 min Exonuclease 1 was added to the sample mixture and incubated for another 25 min, the samples were boiled and separated on denaturing SDS-PAGE. The red arrowhead indicates binding of only the RB-containing repair templates (T-RB) to VirD2-Cas9. (e and f) Confirmation of the targeted endonuclease activity of Cas9-VirD2 and VirD2-Cas9. The purified Cas9-VirD2 or VirD2-Cas9 proteins and sgRNA with and without repair template were incubated with the target DNA. The reaction mixture was separated on a 2% agarose gel. Arrowheads indicate the proper digestion of the target by the Cas9-VirD2 and VirD2-Cas9-sgRNA complex in the presence and absence of the repair templates.

    Techniques Used: Expressing, Purification, SDS Page, Activity Assay, Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Binding Assay, Sequencing

    31) Product Images from "Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq"

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq

    Journal: Nature protocols

    doi: 10.1038/s41596-018-0055-0

    Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).
    Figure Legend Snippet: Detailed schematic overview of CIRCLE-seq method. Genomic DNA is randomly sheared to an average of ~300 bp, end-repaired, A-tailed, and ligated to uracil-containing stem-looped adapters. DNA molecules covalently closed with stem-looped adapters ligated to both ends are selected by treatment with a mixture of Lambda exonuclease I and E. coli ).

    Techniques Used:

    32) Product Images from "Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex"

    Article Title: Rad51 presynaptic filament stabilization function of the mouse Swi5-Sfr1 heterodimeric complex

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks305

    Functional significance of the RSfp motif in Sfr1. ( A ) Sfr1 (303 residues) harbors a RSfp motif in its N-terminus. This motif is deleted in the dN104Sfr1 mutant. ( B ) Purified Swi5–dN104Sfr1 complex (1.5 µg) was subjected to 13% SDS–PAGE and Coomassie Blue staining. ( C ) Purified Swi5–dN104Sfr1 mutant complex was tested for Rad51 interaction by affinity pulldown through the (His) 6 tag on dN104Sfr1. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. ( D ) DNA strand exchange was conducted with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. ( E ) The stability of the Rad51 presynaptic filament was tested by the exonuclease I assay with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, Swi5–Sfr1; S5-dN104S1, Swi5–dN104Sfr1.
    Figure Legend Snippet: Functional significance of the RSfp motif in Sfr1. ( A ) Sfr1 (303 residues) harbors a RSfp motif in its N-terminus. This motif is deleted in the dN104Sfr1 mutant. ( B ) Purified Swi5–dN104Sfr1 complex (1.5 µg) was subjected to 13% SDS–PAGE and Coomassie Blue staining. ( C ) Purified Swi5–dN104Sfr1 mutant complex was tested for Rad51 interaction by affinity pulldown through the (His) 6 tag on dN104Sfr1. The supernatant (S), wash (W) and SDS elute (E) from the pull-down reaction were analyzed by 13% SDS–PAGE with Coomassie Blue staining. ( D ) DNA strand exchange was conducted with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. ( E ) The stability of the Rad51 presynaptic filament was tested by the exonuclease I assay with the indicated concentration of Swi5–dN104Sfr1. The results were graphed. (D and E) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbols: S5S1, Swi5–Sfr1; S5-dN104S1, Swi5–dN104Sfr1.

    Techniques Used: Functional Assay, Mutagenesis, Purification, SDS Page, Staining, Concentration Assay, Standard Deviation

    Stabilization of the Rad51 presynaptic filament by Swi5–Sfr1. ( A ) Schematic of the exonuclease I protection assay for examining presynaptic filament stability. The Rad51 presynaptic filament harboring 5′- 32 P-labeled DNA is incubated with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( B ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5–Sfr1. The results were graphed. ( C ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5 (panel I), Sfr1 (panel II) or Swi5–Sfr1. The results were graphed. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.
    Figure Legend Snippet: Stabilization of the Rad51 presynaptic filament by Swi5–Sfr1. ( A ) Schematic of the exonuclease I protection assay for examining presynaptic filament stability. The Rad51 presynaptic filament harboring 5′- 32 P-labeled DNA is incubated with exonuclease I. The radiolabeled DNA and product are visualized and quantified by phosphorimaging analysis after PAGE. The 32 P label is denoted by the asterisk. ( B ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5–Sfr1. The results were graphed. ( C ) The Rad51 presynaptic filament was treated with exonuclease I in the absence or presence of the indicated concentration of Swi5 (panel I), Sfr1 (panel II) or Swi5–Sfr1. The results were graphed. (B and C) Error bars represent the standard deviation (±SD) calculated based on at least three independent experiments. Symbol: S5S1, Swi5–Sfr1.

    Techniques Used: Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Concentration Assay, Standard Deviation

    33) Product Images from "T-loops Refold after Telomerase Extension and Unfold Again at Late S/G2 for C-strand Fill-in"

    Article Title: T-loops Refold after Telomerase Extension and Unfold Again at Late S/G2 for C-strand Fill-in

    Journal: bioRxiv

    doi: 10.1101/869982

    Development of the T-loop Assay. A. Schematic of Exonuclease I action at telomeres. Exonuclease I digests ss DNA in the 3’→5’ direction. In linear telomeres (left image), Exo I removes the 3’ overhang. Digestion is inhibited in t-loops (right image) since the overhang is protected after insertion in the ds region of the telomere. B. Low temperature protects t-loops. HeLa cells were harvested under t-loop conditions (but without the EtBr used in Fig. 2E ), and Exo I digested at 4, 16, and 25°C Digestion at 4°C produced a small decrease in signal.. Maximal preservation would thus require the combined protective effects of both 4°C and EtBr. C. D. as in Exo I treatment of linear DNA shows a slightly decreased signal even at 4°C. 5ug DNA (HeLa cells, isolated with Quick Prep Buffer with ProK at 55°C) was digested with Exo I at 4, 16, and 25°C for 1 hr. A 50% decreased t-loop signal was seen at 4°C and becomes greater at increased temperaturesQuantitation of the experiment in B C. E. The 3’ end of t-loops is not protected above 5U Exo I. T-loops were digested with increasing amounts of Exo I at 4°C without EtBr. Although 80% of the signal was maintained after exposure to 5U, it was significantly reduced at higher concentrations. F. T-loop preparations using high salt and EtBr protect the ss signal. Triplicate samples of DNA harvested from H1299 and HeLa cells under t-loop (ProK at 4°C with high salt) or linear conditions (ProK at 55°C) were digested with 5U of Exo I per 5 ug DNA. We saw a consistent retention of signal in (+)Exo lanes in t-loop samples (ranging from 70-80%), and a consistent decrease in signal in linear telomeric samples (ranging from 30-40%) for both cell lines.
    Figure Legend Snippet: Development of the T-loop Assay. A. Schematic of Exonuclease I action at telomeres. Exonuclease I digests ss DNA in the 3’→5’ direction. In linear telomeres (left image), Exo I removes the 3’ overhang. Digestion is inhibited in t-loops (right image) since the overhang is protected after insertion in the ds region of the telomere. B. Low temperature protects t-loops. HeLa cells were harvested under t-loop conditions (but without the EtBr used in Fig. 2E ), and Exo I digested at 4, 16, and 25°C Digestion at 4°C produced a small decrease in signal.. Maximal preservation would thus require the combined protective effects of both 4°C and EtBr. C. D. as in Exo I treatment of linear DNA shows a slightly decreased signal even at 4°C. 5ug DNA (HeLa cells, isolated with Quick Prep Buffer with ProK at 55°C) was digested with Exo I at 4, 16, and 25°C for 1 hr. A 50% decreased t-loop signal was seen at 4°C and becomes greater at increased temperaturesQuantitation of the experiment in B C. E. The 3’ end of t-loops is not protected above 5U Exo I. T-loops were digested with increasing amounts of Exo I at 4°C without EtBr. Although 80% of the signal was maintained after exposure to 5U, it was significantly reduced at higher concentrations. F. T-loop preparations using high salt and EtBr protect the ss signal. Triplicate samples of DNA harvested from H1299 and HeLa cells under t-loop (ProK at 4°C with high salt) or linear conditions (ProK at 55°C) were digested with 5U of Exo I per 5 ug DNA. We saw a consistent retention of signal in (+)Exo lanes in t-loop samples (ranging from 70-80%), and a consistent decrease in signal in linear telomeric samples (ranging from 30-40%) for both cell lines.

    Techniques Used: Produced, Preserving, Isolation

    Cell cycle analysis of t-loops. A. FACS showing cells progressing throughout cell cycle. HeLa-hTERT cells, synchronized by double thymidine block, were released and followed at different time points. Aliquots of 0.5 million cells were analyzed by FACS to identify the specific phase of each cell cycle time point. B. T-loops are unfolded and refolded twice during S-phase. There was no decrease in the fraction of t-loops at any time of the cell cycle (other than at the S/G2 interface, when all of the t-loops unfold for fill-in of the extended overhangs). Each telomere replicates at a different specific time. T-loops must unfold for replication to reach the end. If telomeres remained unfolded, the exo I signal should decrease as S-phase progressed and more telomeres replicated. The maintenance of the signal suggests t-loops did not remain linear but were refolded after replication, and then unfolded again for end processing (C-strand fill-in). The denatured signal was compared to the signal from t-loop preparations to normalize for the amount of actual telomeric DNA that was loaded. One of two independent experiments is shown. C,E,E and F,G,H. FACS of HeLa-hTERT showing their cell cycle status after release for 4 or 8 hours, CsCl gradient separation of their replicated and unreplicated DNA, and sensitivity to exonuclease I. T:T represents unreplicated DNA (thymidine in both strands).The lack of sensitivity to exonuclease I (black arrows in E and H) indicates that t-loops that had replicated had been refolded and were thus resistant to digestion.
    Figure Legend Snippet: Cell cycle analysis of t-loops. A. FACS showing cells progressing throughout cell cycle. HeLa-hTERT cells, synchronized by double thymidine block, were released and followed at different time points. Aliquots of 0.5 million cells were analyzed by FACS to identify the specific phase of each cell cycle time point. B. T-loops are unfolded and refolded twice during S-phase. There was no decrease in the fraction of t-loops at any time of the cell cycle (other than at the S/G2 interface, when all of the t-loops unfold for fill-in of the extended overhangs). Each telomere replicates at a different specific time. T-loops must unfold for replication to reach the end. If telomeres remained unfolded, the exo I signal should decrease as S-phase progressed and more telomeres replicated. The maintenance of the signal suggests t-loops did not remain linear but were refolded after replication, and then unfolded again for end processing (C-strand fill-in). The denatured signal was compared to the signal from t-loop preparations to normalize for the amount of actual telomeric DNA that was loaded. One of two independent experiments is shown. C,E,E and F,G,H. FACS of HeLa-hTERT showing their cell cycle status after release for 4 or 8 hours, CsCl gradient separation of their replicated and unreplicated DNA, and sensitivity to exonuclease I. T:T represents unreplicated DNA (thymidine in both strands).The lack of sensitivity to exonuclease I (black arrows in E and H) indicates that t-loops that had replicated had been refolded and were thus resistant to digestion.

    Techniques Used: Cell Cycle Assay, FACS, Blocking Assay

    34) Product Images from "No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †"

    Article Title: No Overt Nucleosome Eviction at Deprotected Telomeres ▿No Overt Nucleosome Eviction at Deprotected Telomeres ▿ †

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.01764-07

    A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.
    Figure Legend Snippet: A novel assay for the nucleosomal organization near the telomere terminus. (A) Scheme of the last nucleosome assay. Nuclei are treated with MNase, and the isolated DNA fragments are incubated with a biotinylated oligonucleotide representing the C-rich telomeric DNA strand, biotin-(CCCTAA) 6 . Magnetic streptavidin beads are used to pull down DNA fragments containing a telomeric (TTAGGG) n 3′ overhang. The supernatant contains fragments of bulk nucleosomes, while the pulldown contains nucleosomal fragments ending at the telomere terminus. Fragments are fractionated on a 1% agarose gel and subjected to Southern blot hybridization with a 32 P-(CCCTAA) 4 probe. (B) Scheme for the construction of a model telomeric fragment to test the last nucleosome assay. A BglII/KpnI fragment excised from pSXneo.25(T 2 AG 3 ) (dsTel) was annealed and ligated to a single-stranded telomeric oligonucleotide (ssTel) containing the complementary BglII recognition sequence at its 5′ end. CIP, calf intestinal phosphatase. (C) Southern blot detection of the telomeric signal ligation products of dsTel and ssTel (lane 1), dsTel only (lane 2), or ssTel only (lane 3). (D) Southern blot detection of the telomeric signal after annealing the DNA products from lanes 1 and 2 shown in panel C with biotin-(CCCTAA) 6 and separating the supernatant (sup) and pulldown (ppt) following incubation with streptavidin beads. Two percent of the supernatant was loaded next to 50% of the total pulldown. Sizes in base pairs are marked to the left of panels C and D. (E) Detection of the telomeric signal associated with the last nucleosomes in wild-type MEFs. The last nucleosome assay was performed as described above, and the supernatant (2% of total fraction) containing the bulk nucleosomes was loaded in the first eight lanes, while nucleosomal fragments containing the telomeric overhang (50% of total pulldown) were loaded in the last eight lanes. Roman numerals represent oligonucleosomes formed by partial digestion. MNase concentrations in U/ml are shown. (F) Detection of the telomeric signal pulled down by the last nucleosome assay following ExoI treatment of DNA from MNase-digested nuclei. DNA fragments isolated after MNase digestion were mixed and treated with ExoI (300 U in 300 μl), removing the 3′ overhang. ExoI-treated and untreated samples were then subjected to the last nucleosome assay. Two percent of the supernatant (bulk nucleosomes) was loaded next to 50% of the total pulldown (last nucleosomes). Percentage of total TTAGGG signal pulled down was calculated with the formula (2 × pulldown signal)/[(2 × pulldown signal) + (50 × supernatant signal)]. +, present; −, absent.

    Techniques Used: Isolation, Incubation, Agarose Gel Electrophoresis, Southern Blot, Hybridization, Sequencing, Ligation

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
    Article Snippet: .. After first-stage PCR, 1 μL of exonuclease I (20 U/μL; New England BioLabs) was added to the reactions and incubated at 37°C for 30 minutes. .. After the completion of the first-stage PCR or exonuclease I treatment, PCR products were purified with 1× volume of SPRI beads and eluted in 20 μL of 10 mmol/L Tris-HCl (pH 8.0).

    Staining:

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA
    Article Snippet: .. In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done. ..

    Incubation:

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood
    Article Snippet: .. After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added. ..

    Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies
    Article Snippet: .. After first-stage PCR, 1 μL of exonuclease I (20 U/μL; New England BioLabs) was added to the reactions and incubated at 37°C for 30 minutes. .. After the completion of the first-stage PCR or exonuclease I treatment, PCR products were purified with 1× volume of SPRI beads and eluted in 20 μL of 10 mmol/L Tris-HCl (pH 8.0).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Defining CRISPR-Cas9 genome-wide nuclease activities with CIRCLE-seq
    Article Snippet: .. M0293L) Lambda Exonuclease (New England BioLabs, cat.no. .. M0262L) Plasmid-Safe ATP-dependent DNase (Epicentre, cat.no.

    Avidin-Biotin Assay:

    Article Title: The structure of ends determines the pathway choice and Mre11 nuclease dependency of DNA double-strand break repair
    Article Snippet: .. To detect the presence of 3′ biotin on 3′ ss-overhangs or resection intermediates, the DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with Escherichia coli ExoI (NEB, MA) at 22ºC for 60 min. To analyze the intermediates of the 5′ biotin-avidin DNA, DNA was treated with E. coli ExoI (0.2 u/μl, NEB, MA) or RecJ (0.3 u/μl; NEB, MA) at 22°C for 60 min. To detect the presence of 5′ biotin, DNA was pre-incubated with ELB buffer or avidin on ice for 5 min, and then treated with T7 Exo (0.6 unit/μl; NEB, MA) at 22°C for 60 min. .. Reactions were analyzed by 1% TAE-agarose gel electrophoresis and the gels were first stained with SYBR Gold (Invitrogen, CA, USA) and then dried for exposure to film.

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs exonuclease i
    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following <t>Exonuclease</t> I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.
    Exonuclease I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 561 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/exonuclease i/product/New England Biolabs
    Average 99 stars, based on 561 article reviews
    Price from $9.99 to $1999.99
    exonuclease i - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    Image Search Results


    Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Journal: PLoS ONE

    Article Title: Circular Single-Stranded Synthetic DNA Delivery Vectors for MicroRNA

    doi: 10.1371/journal.pone.0016925

    Figure Lengend Snippet: Circularization of DNA templates (COLIGOs) for Rolling Circle Transcription. A . Synthetic 5′ phosphorylated linear DNA sequences were circularized using the thermostable TS2126 RNA ligase. B . Denaturing polyacrylamide gel electrophoresis (DPAGE) at four stages during miR-19am DNA circle synthesis. Lane 1, crude DNA IDT Ultramer synthesis of COLIGO 19am. Lane 2, after preparative DPAGE. Lane 3, crude circularization product. Lane 4, DNA circle template following Exonuclease I clean-up. Visualization using Stains-All. C . Verification of circular topology. Nicking of circular templates by S1 nuclease leads first to linear forms, which are then further digested to successively smaller linear forms.

    Article Snippet: In cases where the COLIGO was still contaminated by > 5% of the linear oligonucleotide after elution (as determined by gel staining), an Exonuclease I (NEB) digest was done.

    Techniques: Polyacrylamide Gel Electrophoresis

    Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Schematic diagram of procedures of liquid hybridization and solid phase detection (LHSPD). ( 1 ) Liquid hybridization: The small RNA samples, hybridization buffer, and probe are mixed in a tube to make the probe hybridize with the specific RNA sequences and the non-hybridized sequences are digested by exonuclease I; ( 2 ) Gel electrophoresis: the products of the hybridization are separated by electrophoresis; ( 3 ) Transfer membrane; ( 4 ) UV crosslinking and membrane blocking; ( 5 ) Antibody incubation: alkaline phosphatase (AP)-anti-DIG antibody or AP-streptavidin or horseradish peroxidase (HRP)-streptavidin targeted the RNA-bound DIG-labeled probes or biotin-labeled probes respectively; ( 6 ) Hybridization signal detection: CDP-Star/luminol is used to detect the combination of antibody and target RNA.

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Hybridization, Nucleic Acid Electrophoresis, Electrophoresis, Blocking Assay, Incubation, Labeling

    Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Journal: International Journal of Molecular Sciences

    Article Title: Liquid Hybridization and Solid Phase Detection: A Highly Sensitive and Accurate Strategy for MicroRNA Detection in Plants and Animals

    doi: 10.3390/ijms17091457

    Figure Lengend Snippet: Specificity of LHSPD at different temperatures. ( A – D ) Hybridizations of 0.1 pmol ( DIG ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( E – H ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by LHSPD; ( I – L ) Hybridizations of 0.1 pmol ( Biotin ) -miD156rk with 1 pmol miD156s at different temperatures by traditional Northern hybridization. miD156 marked “+”, miD156 with one-base mismatch marked “−1”, miD156 with three-base mismatch marked “−3” and miD156 with five-base mismatch marked “−5”; ( M ) Hybridization performed in 0.25×, 0.5× and 1× Exonuclease I reaction buffer (New England Biolabs, Inc., Beijing, China) with 0.1 pmol ( Biotin ) -miD156rk ; ( N ) Hybridization performed in 0.25×, 0.5× and 1× PNE buffer with 0.1 pmol ( Biotin ) -miD156rk ; 20f represents 20 fmol of miD156 ; 10f represents 10 fmol of miD156 ; 5f represents 5 fmol of miD156 ; P represents control containing 1 pmol of ( Biotin ) -miD156rk .

    Article Snippet: After that, non-hybridized single-stranded DNA, including the probe, was digested with 1 U exonuclease I (New England BioLabs, Inc., M0293, Beijing, China) in the same tube according to the instruction protocol for 30 min at 37 °C.

    Techniques: Northern Blot, Hybridization

    Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Journal: Nucleic Acids Research

    Article Title: Homogeneous antibody-based proximity extension assays provide sensitive and specific detection of low-abundant proteins in human blood

    doi: 10.1093/nar/gkr424

    Figure Lengend Snippet: Exonuclease activity and hybridization length affects assay sensitivity. (A) VEGF assays were designed with different lengths of the hybridization site and compared with respect to sensitivity. A 9-nt hybridization site was found to give the best signal-to-noise levels and was selected for further studies. ( B ) Different DNA polymerases were tested with regards to their ability to generate good sensitivity in an IL-8-specific assay. T4 DNA polymerase I, DNA polymerase I and Klenow fragment exo + all possess a 3′→5′ exonuclease activity and performed well in the IL-8 detection. Klenow fragment exo − , on the other hand, generated a background signal that was almost at the level of the antigen-induced signal. When exogenous Exonuclease I was added to the reaction, the signal-to-noise level was restored.

    Article Snippet: After a 5-min incubation at 37°C, a 20 µl extension mix containing 66.8 mM Tris–HCl, 16.8 mM ammonium sulfate, 1 mM dithiothreitol, 33 mM magnesium chloride, 62.5 U/ml T4 DNA Polymerase [or 62.5 U/ml Klenow fragment exo(−), 125 U/ml Klenow fragment, 125 U/ml DNA Polymerase I (Fermentas), 250 U/ml Exonuclease I (New England Biolabs)] was added.

    Techniques: Activity Assay, Hybridization, Generated

    Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).

    Journal: The Journal of Molecular Diagnostics : JMD

    Article Title: Rational “Error Elimination” Approach to Evaluating Molecular Barcoded Next-Generation Sequencing Data Identifies Low-Frequency Mutations in Hematologic Malignancies

    doi: 10.1016/j.jmoldx.2019.01.008

    Figure Lengend Snippet: Identification of parameters crucial for improving the quality of molecular barcode–containing next-generation sequencing libraries. A: Exonuclease I treatment reduces the primer dimer concentration and improves the yield of sequencing libraries. First-stage PCR products were incubated with 1 μL of 10 mmol/L Tris-Cl (pH 8.0) or exonuclease I (20 U/μL) at 37°C for 30 minutes. B: Identification of an optimal number of second-stage PCR cycles for library preparation. The first-stage PCR amplification was performed in TaqMan genotyping master mix. The products were then digested with exonuclease I. The second-stage PCR amplification with Ultra II Q5 mix was performed for 17, 20, 23, or 26 cycles. The second-stage PCR products were purified with solid-phase reversible immobilization beads and run on the Agilent 2100 DNA bioanalyzer. C: Size selection efficiently eliminated primer dimers. Genomic DNA mixes A (1% A375, 0.5% Raji, 0.1% NCI-1355, and 98.4% OCI-AML3 DNA; lanes 1, 2, 5, and 6, respectively) and B (1% NCI-1355, 0.5% Raji, 0.1% A375, and 98.4% OCI-AML3 DNA; lanes 3, 4, 7, and 8, respectively) were created and subjected to first-stage PCR amplification, exonuclease I treatment, and second-stage PCR amplification. The purified second-stage PCR products were used for double-size selection with 056×/0.85× volumes of solid-phase reversible immobilization beads, and the size-selected libraries were analyzed on the Agilent 2100 DNA bioanalyzer. Note that a 300- to 400-bp target-specific library is indicated by brackets . Green and purple bars indicate lower and upper markers, respectively. All samples were evaluated in duplicate ( B and C ) or in triplicate ( A ).

    Article Snippet: After first-stage PCR, 1 μL of exonuclease I (20 U/μL; New England BioLabs) was added to the reactions and incubated at 37°C for 30 minutes.

    Techniques: Next-Generation Sequencing, Concentration Assay, Sequencing, Polymerase Chain Reaction, Incubation, Amplification, Purification, Selection