a3h catalyzed deaminations  (New England Biolabs)


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    Structured Review

    New England Biolabs a3h catalyzed deaminations
    β2-β2 strand amino acids mediate <t>A3H</t> dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    A3h Catalyzed Deaminations, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 88/100, based on 2201 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *"

    Article Title: Natural Polymorphisms and Oligomerization of Human APOBEC3H Contribute to Single-stranded DNA Scanning Ability *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M115.666065

    β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.
    Figure Legend Snippet: β2-β2 strand amino acids mediate A3H dimerization. Size exclusion chromatography profiles of A3H haplotype II ( Hap II ) and A3H haplotype V ( Hap V ) obtained from a 25-ml G200 Superdex Increase column ( A ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( B ). Analysis demonstrated that both A3H haplotype II and A3H haplotype V were able to form monomers ( M ), dimers ( D ), and tetramers ( T ) in solution. Both 150 and 300 μg ( inset graph ) of enzyme were resolved to investigate whether A3H tetramer formation was concentration-dependent. According to the calibration curve, the apparent molecular masses of peak fractions for A3H haplotype II were 24 kDa (monomer), 44 kDa (dimer), and 94 kDa (tetramer), and for A3H haplotype V, they were 23 kDa (monomer), 44 kDa (dimer), and 102 kDa (tetramer). C , sequence alignment of A3H haplotype II and A2 β2 strand amino acid sequences. Amino acids that were mutated are indicated in red . The size exclusion chromatography profiles of GST-A3H haplotype II ( GST-HapII ) and GST-A3H haplotype II R44A/Y46A ( GST-R44A/Y46A ) obtained from a 10-ml G200 Superdex column ( D ) were used to calculate the oligomerization states of the enzymes from a standard calibration curve ( E ). When 10 μg of enzyme was loaded onto the size exclusion column, GST-A3H haplotype II formed dimers in solution (apparent molecular mass of 77 kDa in peak fraction). This is in contrast to GST-A3H haplotype II R44A/Y46A, which formed monomers (apparent molecular mass of 37 kDa in peak fraction). F , the chromatograms from the 10-ml Sephadex 200 column ( D ) were constructed by analyzing the integrated gel band intensities of the protein in each fraction after resolution by SDS-PAGE. The gels show the peak fractions of GST-A3H haplotype II and GST-A3H haplotype II R44A/Y46A with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. G , the Sephadex 200 column (10-ml bed volume) demonstrates that GST is a dimer. The gel shows the peak fractions of GST with start and end volumes corresponding to the fractions that were resolved by SDS-PAGE. However, GST-tagged A3H does not dimerize through the GST tag based on data from GST-A3H haplotype II R44A/Y46A ( D ). AU , absorbance units.

    Techniques Used: Size-exclusion Chromatography, Concentration Assay, Sequencing, Construct, SDS Page

    2) Product Images from "Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid"

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    Journal: Nucleic Acids Research

    doi:

    N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.
    Figure Legend Snippet: N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Techniques Used: Activity Assay, High Performance Liquid Chromatography, Incubation, Labeling

    3) Product Images from "Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase"

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

    Journal: Oncotarget

    doi:

    Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.
    Figure Legend Snippet: Detection of true KRAS and EGFR mutations after UDG treatment The effect of UDG treatment on detection of various types of true mutations are examined using a set of FFPE DNA samples harbouring either KRAS or EGFR exon 19 deletions and exon 20 insertion mutations. All KRAS -mutant and EGFR -mutant samples are correctly identifiable by HRM or Sanger sequencing regardless of UDG treatment. The positions of KRAS mutations and representative nucleotides of EGFR mutations are indicated by a red asterisk. Panel A: Sequence traces of KRAS exon 2 before and after UDG treatment. Both TX23 and TX63 samples harbour KRAS c.35G > A mutations and HCT116 cell line DNA contains a KRAS c.38G > A mutation. Panel B: Sequence traces of EGFR exon 19 before and after UDG treatment. Both TX35 and H1650 harbour EGFR p.E746_A750del mutations and TX48 harbours a p.T751_I759delinsN mutation. Panel C: Sequence traces of EGFR exon 20 before and after UDG treatment. TX202, TX383 and TX440 samples harbour EGFR p.C775_R776insPA, p.H773_R776insYNPY, and p.D770_H773insGSVD, respectively. Panel D: Difference plots of low-level KRAS- mutant samples before (left) and after UDG treatment (right). KRAS mutations detected are c.35G > T (N1 9), c.35G > T (N1 46), c.35G > C (N1 53), c.34G > T (TX450). RPMI8226 cell line DNA contains a KRAS c.35G > C mutation.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Mutagenesis, Sequencing

    The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.
    Figure Legend Snippet: The effect of UDG treatment on sequence artefacts in AKT1 as assessed using LCN-HRM The frequency of sequence artefacts in the AKT1 sequence were assessed in three FFPE DNA samples (SCC7, SCC8, and SCC14) with and without UDG treatment using LCN-HRM. The melting profiles of 60 individual LCN-HRM products are presented in the negative first derivative plot. Positive LCN-HRM reactions are shown in red and wild-type reactions are shown in green. There is a marked reduction in the number of LCN-HRM positive reactions after UDG treatment in all three samples. In SCC7, a total of 34 reactions were positive without UDG treatment (Panel A), which is markedly reduced to 5 after UDG treatment (Panel B). In SCC8, 24 and 10 LCN-HRM reactions were positive without (Panel C) and with UDG treatment (Panel D), and 20 and 3 LCN-HRM positives are found without (Panel E) and with UDG treatment (Panel F) in SCC14.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded

    Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.
    Figure Legend Snippet: Sequence artefacts detected in FFPE DNA by Sanger sequencing Multiple non-reproducible sequence artefacts detected in the AKT1 sequence from FFPE DNA are shown. Panel A: Four sequence artefacts detected in the SCC8 sample without UDG treatment. Three of the sequence artefacts (c.81C > T, c.145G > A and c.153C > T) were found in the same amplicon from one replicate and the c.110G > A change was detected in the second replicate. Panel B: Four sequence artefacts detected in three FFPE DNA samples (SCC7, SCC11, and SCC14) after UDG treatment. c.122G > A and c.143G > A changes were detected in different replicates from the SCC7 sample. A c.125C > T (SCC11) and a c.175C > T (SCC14) change was found in a replicate of SCC11 and SCC14 respectively. All of the C:G > T:A changes that were found after UDG treatment were detected in the sequence context of CpG dinucleotides.

    Techniques Used: Sequencing, Formalin-fixed Paraffin-Embedded, Amplification

    UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.
    Figure Legend Snippet: UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.

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

    Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.
    Figure Legend Snippet: Uracil lesions in FFPE DNA leading to sequence artefacts and in vitro removal of uracil by uracil-DNA glycosylase Spontaneous cytosine deamination is a frequent DNA damage that takes place at a rate of 70 - 200 events per day in the human genome. In normal cells, the resulting uracil lesions are effectively removed by UDG. The resulting abasic sites are then repaired by the base excision DNA repair system. However, in biopsy specimen, if cytosine deamination occurs during sample collection, formalin fixation, and fixed tissue storage, the resulting uracil lesions cannot be repaired due to the absence of functional DNA repair proteins. When DNA is extracted from the tissue with uracil lesions and then used as template for PCR amplification, transitional C:G > T:A sequence artefacts are generated as uracil efficiently pairs with adenine. The generation of artefactual C:G > T:A transitions from the uracil lesions in FFPE DNA can be effectively eliminated by treating FFPE DNA with UDG in vitro prior to PCR amplification. Abasic sites generated by the removal of uracil bases may reduce the extension by DNA polymerase and strand breakage during the repetitive exposure to high temperature during PCR cycling. Thus, treatment of FFPE DNA with UDG prior to PCR amplification eliminates the generation of artefactual C:G > T:A transitions arising from uracil lesions.

    Techniques Used: Formalin-fixed Paraffin-Embedded, Sequencing, In Vitro, Functional Assay, Polymerase Chain Reaction, Amplification, Generated

    The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.
    Figure Legend Snippet: The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.

    Techniques Used: Formalin-fixed Paraffin-Embedded, HRM Assay, Polymerase Chain Reaction, Amplification, Concentration Assay

    4) Product Images from "Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase"

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase

    Journal: Oncotarget

    doi:

    UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.
    Figure Legend Snippet: UDG treatment reduces artefactual false positives by HRM Sequence artefacts arising from uracil lesions can cause false HRM positives by formation of heteroduplexes. Treatment of FFPE DNA prior to PCR amplification removes uracil lesions, resulting in markedly reducing false HRM positives. BRAF exon 15 and EGFR exon 19 HRM results of three representative samples are shown. Panel A: Normalised plot for BRAF exon 15 without UDG treatment. Panel B: Normalised plot for BRAF exon 15 with UDG treatment. Panel C: Normalised plot for EGFR exon 19 without UDG treatment. Panel D: Normalised plot for EGFR exon 19 with UDG treatment.

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

    The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.
    Figure Legend Snippet: The melting profiles of FFPE DNA before and after UDG treatment The melting profiles of the AKT1 HRM assay for three representative FFPE DNA samples (SCC8, SCC11, and SCC39) without (Panels A and B) and with UDG treatment using four different UDG concentrations (Panels C – F) are shown. The early melting profiles that are indicative of heteroduplex formation were seen in all three samples without UDG treatment. UDG treatment prior to PCR amplification resulted in a marked reduction of heteroduplex formation. Panel A: Normalised plot without UDG treatment. Panel B: First negative derivative plot without UDG treatment. Panels C – F: First negative derivative plots with a concentration of 0.1, 0.25, 0.5, and 1 UDG unit/reaction, respectively. The early melting region of the heteroduplexes is indicated with a blue arrow.

    Techniques Used: Formalin-fixed Paraffin-Embedded, HRM Assay, Polymerase Chain Reaction, Amplification, Concentration Assay

    5) Product Images from "Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9"

    Article Title: Efficient Genome Editing of a Facultative Thermophile Using Mesophilic spCas9

    Journal: ACS Synthetic Biology

    doi: 10.1021/acssynbio.6b00339

    Schematic overview of the basic pWUR_Cas9nt construct. (A) The non-codon-optimized cas9 sp gene was employed for the construction of the pWUR_Cas9nt vector, since S. pyogenes and B. smithii GC content and codon usage are highly similar. In the pNW33n-based basic construct, s pCas9 was placed under the control of P xynA . A Rho-independent terminator from B. subtilis ( 59 ) was introduced after the stop codon of the gene. The spCas9 module is followed by an sgRNA-expressing module that encompasses a spacer which does not target the genome of ET 138. The sgRNA module was placed under the transcriptional control of P pta from B. coagulans (without its RBS), which was followed by a second Rho-independent terminator from B. subtilis . 15 , 49 The spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into pNW33n through the BspHI and HindIII restriction sites. (B) To prevent double restriction sites and create a modular system, five silent point mutations (C192A, T387C, T1011A, C3126A, G354A) were introduced to the gene (depicted as *). The depicted restriction sites are unique in the construct and introduced to facilitate the exchange of genetic parts. The spacer was easily exchanged to targeting spacers via BsmBI restriction digestion or Gibson assembly. The basic construct did not contain any HR templates, but in cases where these were added, they were always inserted immediately upstream of the spCas9 module and downstream of the origin of replication. (C) Total RNA was isolated from ET 138 wild-type cells transformed with pWUR_Cas9nt or pNW33n and grown at 55, 45, and 37 °C. Six cDNA libraries were produced with rt-PCR and used as templates for PCR with cas9sp-specific primers that amplify a 255 bp region. The PCR results are depicted as follows: lane 1 corresponds to the marker (1kb+ DNA ladder, ThermoFisher), lanes 2–4 correspond to ET 138 wild-type cultures transformed with pWUR_Cas9nt and grown at 55, 42, or 37 °C, respectively, lanes 5–7 correspond to ET 138 wild-type cultures transformed with pNW33n and grown at 55, 42, or 37 °C, respectively, lanes 7, 8, 9, 11, 12 correspond to different negative controls, and lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.
    Figure Legend Snippet: Schematic overview of the basic pWUR_Cas9nt construct. (A) The non-codon-optimized cas9 sp gene was employed for the construction of the pWUR_Cas9nt vector, since S. pyogenes and B. smithii GC content and codon usage are highly similar. In the pNW33n-based basic construct, s pCas9 was placed under the control of P xynA . A Rho-independent terminator from B. subtilis ( 59 ) was introduced after the stop codon of the gene. The spCas9 module is followed by an sgRNA-expressing module that encompasses a spacer which does not target the genome of ET 138. The sgRNA module was placed under the transcriptional control of P pta from B. coagulans (without its RBS), which was followed by a second Rho-independent terminator from B. subtilis . 15 , 49 The spCas9 and sgRNA modules were synthesized as one fragment, which was subsequently cloned into pNW33n through the BspHI and HindIII restriction sites. (B) To prevent double restriction sites and create a modular system, five silent point mutations (C192A, T387C, T1011A, C3126A, G354A) were introduced to the gene (depicted as *). The depicted restriction sites are unique in the construct and introduced to facilitate the exchange of genetic parts. The spacer was easily exchanged to targeting spacers via BsmBI restriction digestion or Gibson assembly. The basic construct did not contain any HR templates, but in cases where these were added, they were always inserted immediately upstream of the spCas9 module and downstream of the origin of replication. (C) Total RNA was isolated from ET 138 wild-type cells transformed with pWUR_Cas9nt or pNW33n and grown at 55, 45, and 37 °C. Six cDNA libraries were produced with rt-PCR and used as templates for PCR with cas9sp-specific primers that amplify a 255 bp region. The PCR results are depicted as follows: lane 1 corresponds to the marker (1kb+ DNA ladder, ThermoFisher), lanes 2–4 correspond to ET 138 wild-type cultures transformed with pWUR_Cas9nt and grown at 55, 42, or 37 °C, respectively, lanes 5–7 correspond to ET 138 wild-type cultures transformed with pNW33n and grown at 55, 42, or 37 °C, respectively, lanes 7, 8, 9, 11, 12 correspond to different negative controls, and lane 10 corresponds to the positive control, for which pWUR_Cas9nt was used as the PCR template.

    Techniques Used: Construct, Plasmid Preparation, Expressing, Synthesized, Clone Assay, Isolation, Transformation Assay, Produced, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Marker, Positive Control

    6) Product Images from "Integrated digital error suppression for improved detection of circulating tumor DNA"

    Article Title: Integrated digital error suppression for improved detection of circulating tumor DNA

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3520

    Development of integrated digital error suppression (iDES) ( a ) Diagram depicting the use of CAPP-Seq barcode adapters to suppress errors. Here, CAPP-Seq adapters are ligated to a double-stranded (duplex) DNA molecule containing a real biological mutation in both strands as well as a non-replicated, asymmetric base change in only one strand ( top ). The combined application of insert and index barcodes allows for (i) error suppression and (ii) recovery of single stranded ( center ) and duplex ( bottom ) DNA molecules ( Supplementary Fig. 1a , Methods ). ( b ) Top : Heat map showing position-specific selector-wide error rates parceled into all possible base substitutions (rows) and organized by decreasing mean allele fractions (for each substitution type) across 12 cfDNA samples from healthy controls (columns; Supplementary Table 2 ). Background patterns are shown for different error suppression methods, including the combined application of barcoding and background polishing. Errors were defined as non-reference alleles excluding germline SNPs. Bottom : Selector-wide error metrics ( Methods ).
    Figure Legend Snippet: Development of integrated digital error suppression (iDES) ( a ) Diagram depicting the use of CAPP-Seq barcode adapters to suppress errors. Here, CAPP-Seq adapters are ligated to a double-stranded (duplex) DNA molecule containing a real biological mutation in both strands as well as a non-replicated, asymmetric base change in only one strand ( top ). The combined application of insert and index barcodes allows for (i) error suppression and (ii) recovery of single stranded ( center ) and duplex ( bottom ) DNA molecules ( Supplementary Fig. 1a , Methods ). ( b ) Top : Heat map showing position-specific selector-wide error rates parceled into all possible base substitutions (rows) and organized by decreasing mean allele fractions (for each substitution type) across 12 cfDNA samples from healthy controls (columns; Supplementary Table 2 ). Background patterns are shown for different error suppression methods, including the combined application of barcoding and background polishing. Errors were defined as non-reference alleles excluding germline SNPs. Bottom : Selector-wide error metrics ( Methods ).

    Techniques Used: Mutagenesis

    Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq was assessed using technical controls ( a – c ) and patients with NSCLC ( d – f ). ( a ) A DNA reference blend containing known alleles spanning a broad AF range was diluted to 5% in normal cfDNA and analyzed in replicate ( n =4) for both known variants ( n =29) and 279 negative control variants ( Supplementary Table 4 , Methods ). Left : Differential impact of barcoding, polishing, and iDES on genotyping results for a single representative replicate. Only variant calls with at least 2 supporting reads are shown. Asterisks highlight the complementary background profiles removed by barcoding and polishing. Note that all variant calls are ordered along the x -axis, first by validation status and then by AF. Identical calls are aligned vertically. Right : Performance metrics across all four replicates. Genotyping thresholds were determined as described in Methods . ( b ) AFs determined by iDES-enhanced CAPP-Seq in the 5% variant blend from panel a (observed) versus their concentrations determined by digital PCR (expected). Only variants in the reference blend with externally validated AFs targeted by our NSCLC selector are shown ( n =13; Supplementary Table 4 ). Data are expressed as means ± s.e.m ( n =4 replicates). ( c ) Heat map ( top ) and scatter plot ( bottom ) depicting candidate SNVs identified by noninvasive selector-wide genotyping of the 5% variant blend from panel a ( Supplementary Fig. 10 , Methods ). SNVs were tracked across three additional replicates and a ten-fold lower spike. Horizontal lines depict mean AFs. ( d – f ) Noninvasive tumor genotyping of NSCLC patients. ( d ) Bottom : The number of hotspot SNVs noninvasively detected in 24 pretreatment NSCLC cfDNA samples by four methods, including iDES (barcoding + polishing). All queried variants are listed in Supplementary Table 4 . Top: Positive predictive value (PPV) of each method (indicated below), based on the number of hotspot SNVs that were later confirmed in matching tumor biopsies. ( e ) The performance of iDES for noninvasive tumor genotyping of two plasma cohorts was assessed using observed allele fractions with a Receiver Operating Characteristic (ROC) plot. In the first cohort ( n =66 plasma samples from patients with matching tumor biopsies), hotspot variants from a predefined list of 292 variants were assessed ( Supplementary Table 4 ). Results are shown for the 46 plasma samples with at least one detectable mutation (‘All genes’, n =24 patients); specificity was assessed using variants that were detected but that could not be verified in the primary tumor. In the second cohort, EGFR hotspot variants were assessed in an extended cohort of 103 plasma samples from 41 EGFR-positive patients with NSCLC (‘ EGFR’ ). Specificity was assessed using 27 EGFR-wildtype subjects ( Methods ). The pie chart shows the distribution of detected EGFR variants. Only patients with genotyped tumors were analyzed. AUC, area under the curve. ( f ) Noninvasive genotyping of EGFR mutations in plasma samples from 37 patients with advanced NSCLC and with biopsy-confirmed EGFR mutations. Top: Performance of iDES-enhanced CAPP-Seq for the genotyping of actionable EGFR mutations ( n =36 patients; 1 of 37 patients did not have an actionable alteration). All performance metrics were assessed at the variant level. Bottom: Comparison of error-suppression methods for noninvasive tumor genotyping of the entire EGFR kinase domain in all patients with biopsy-confirmed EGFR SNVs ( n =29 of 37 patients). Performance metrics were assessed separately at the variant level and patient level (using 27 EGFR-wildtype subjects). Percentages indicate iDES performance only. Further details are provided in Methods . Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.
    Figure Legend Snippet: Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq was assessed using technical controls ( a – c ) and patients with NSCLC ( d – f ). ( a ) A DNA reference blend containing known alleles spanning a broad AF range was diluted to 5% in normal cfDNA and analyzed in replicate ( n =4) for both known variants ( n =29) and 279 negative control variants ( Supplementary Table 4 , Methods ). Left : Differential impact of barcoding, polishing, and iDES on genotyping results for a single representative replicate. Only variant calls with at least 2 supporting reads are shown. Asterisks highlight the complementary background profiles removed by barcoding and polishing. Note that all variant calls are ordered along the x -axis, first by validation status and then by AF. Identical calls are aligned vertically. Right : Performance metrics across all four replicates. Genotyping thresholds were determined as described in Methods . ( b ) AFs determined by iDES-enhanced CAPP-Seq in the 5% variant blend from panel a (observed) versus their concentrations determined by digital PCR (expected). Only variants in the reference blend with externally validated AFs targeted by our NSCLC selector are shown ( n =13; Supplementary Table 4 ). Data are expressed as means ± s.e.m ( n =4 replicates). ( c ) Heat map ( top ) and scatter plot ( bottom ) depicting candidate SNVs identified by noninvasive selector-wide genotyping of the 5% variant blend from panel a ( Supplementary Fig. 10 , Methods ). SNVs were tracked across three additional replicates and a ten-fold lower spike. Horizontal lines depict mean AFs. ( d – f ) Noninvasive tumor genotyping of NSCLC patients. ( d ) Bottom : The number of hotspot SNVs noninvasively detected in 24 pretreatment NSCLC cfDNA samples by four methods, including iDES (barcoding + polishing). All queried variants are listed in Supplementary Table 4 . Top: Positive predictive value (PPV) of each method (indicated below), based on the number of hotspot SNVs that were later confirmed in matching tumor biopsies. ( e ) The performance of iDES for noninvasive tumor genotyping of two plasma cohorts was assessed using observed allele fractions with a Receiver Operating Characteristic (ROC) plot. In the first cohort ( n =66 plasma samples from patients with matching tumor biopsies), hotspot variants from a predefined list of 292 variants were assessed ( Supplementary Table 4 ). Results are shown for the 46 plasma samples with at least one detectable mutation (‘All genes’, n =24 patients); specificity was assessed using variants that were detected but that could not be verified in the primary tumor. In the second cohort, EGFR hotspot variants were assessed in an extended cohort of 103 plasma samples from 41 EGFR-positive patients with NSCLC (‘ EGFR’ ). Specificity was assessed using 27 EGFR-wildtype subjects ( Methods ). The pie chart shows the distribution of detected EGFR variants. Only patients with genotyped tumors were analyzed. AUC, area under the curve. ( f ) Noninvasive genotyping of EGFR mutations in plasma samples from 37 patients with advanced NSCLC and with biopsy-confirmed EGFR mutations. Top: Performance of iDES-enhanced CAPP-Seq for the genotyping of actionable EGFR mutations ( n =36 patients; 1 of 37 patients did not have an actionable alteration). All performance metrics were assessed at the variant level. Bottom: Comparison of error-suppression methods for noninvasive tumor genotyping of the entire EGFR kinase domain in all patients with biopsy-confirmed EGFR SNVs ( n =29 of 37 patients). Performance metrics were assessed separately at the variant level and patient level (using 27 EGFR-wildtype subjects). Percentages indicate iDES performance only. Further details are provided in Methods . Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.

    Techniques Used: Negative Control, Variant Assay, Digital PCR, Mutagenesis

    7) Product Images from "The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat"

    Article Title: The aromatic amino acid hydroxylase genes AAH1 and AAH2 in Toxoplasma gondii contribute to transmission in the cat

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1006272

    Disruption of the AAH1 and AAH2 genes. (A) Schematic of the AAH2 knockout strategy in the wild-type ME49 Δhxg :: Luc strain (referred to as WT). A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH2 was co-transfected with the pΔaah2 :: HXG plasmid ( S2 Table ) and selected for with MPA +Xanthine to delete AAH2 to produce the clone Δaah2 :: HXG ( Δh2-HXG ). Subsequently, the HXG gene was replaced with either a clean fusion of the AAH2 5’ and 3’ UTRs ( pΔaah2 ) or an AAH2 cDNA construct ( pAAH2 ). ( S2 Table ) using 6-thioxanthine selection against the HXG locus to create the clean knockout clone Δaah2 ( Δh2 ) (upper panel) and the complement clone Δaah2 :: AAH2 ( Δh2-H2 ) (lower panel). Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions ( S3 Table ). (B) Schematic of the knockout strategy for AAH1 . A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH1 was co-transfected with the pΔaah1 :: DHFR-Ts repair construct ( S2 Table ) into WT or Δaah2 parasites to create the clones Δaah1 ( Δh1 ) and Δaah1Δaah2 ( Δh1Δh2 ). Transfectants were selected for via pyrimethamine resistance. Subsequently, using pΔuprt :: AAH1 :: HXG , a cDNA copy of AAH1 driven by its native 5’ and 3’ UTRs was complemented into the UPRT locus by means of the HXGPRT drug resistance marker selected for with MPA +Xanthine, negative selection against UPRT with FUDR, and a single-cutting CRISPR-Cas9 construct targeted to the UPRT gene ( S2 Table ), creating the complement clones Δaah1-AAH1 ( Δh1-H1 ) and Δaah1Δaah2-AAH1 ( Δh1Δh2-H1 ). Brown Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions ( S3 Table ). (C) PCR verification of successful ablation and complementation of knockouts. Expected product sizes: Tubulin (Tub): 0.378kb. AAH1 (H1): 0.745kb (Native), 0.278kb (cDNA). AAH2 (H2): 0.745kb (Native), 0.278kb (cDNA). (D) Growth assays of parasites seeded into 96-well plates and allowed to proliferate for 24 h, then quantified using a luciferase assay. The WT, Δh1 , Δh2, Δh1Δh2 , Δh1-H1 , Δh2-H2 , and Δh1Δh2-H1 parasites showed no significant difference in total growth (Kruskal-Wallis test, P = 0.0672, N = 3 per strain).
    Figure Legend Snippet: Disruption of the AAH1 and AAH2 genes. (A) Schematic of the AAH2 knockout strategy in the wild-type ME49 Δhxg :: Luc strain (referred to as WT). A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH2 was co-transfected with the pΔaah2 :: HXG plasmid ( S2 Table ) and selected for with MPA +Xanthine to delete AAH2 to produce the clone Δaah2 :: HXG ( Δh2-HXG ). Subsequently, the HXG gene was replaced with either a clean fusion of the AAH2 5’ and 3’ UTRs ( pΔaah2 ) or an AAH2 cDNA construct ( pAAH2 ). ( S2 Table ) using 6-thioxanthine selection against the HXG locus to create the clean knockout clone Δaah2 ( Δh2 ) (upper panel) and the complement clone Δaah2 :: AAH2 ( Δh2-H2 ) (lower panel). Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions ( S3 Table ). (B) Schematic of the knockout strategy for AAH1 . A CRISPR-Cas9 construct with guide RNAs targeted to the 5’ and 3’ UTRs of AAH1 was co-transfected with the pΔaah1 :: DHFR-Ts repair construct ( S2 Table ) into WT or Δaah2 parasites to create the clones Δaah1 ( Δh1 ) and Δaah1Δaah2 ( Δh1Δh2 ). Transfectants were selected for via pyrimethamine resistance. Subsequently, using pΔuprt :: AAH1 :: HXG , a cDNA copy of AAH1 driven by its native 5’ and 3’ UTRs was complemented into the UPRT locus by means of the HXGPRT drug resistance marker selected for with MPA +Xanthine, negative selection against UPRT with FUDR, and a single-cutting CRISPR-Cas9 construct targeted to the UPRT gene ( S2 Table ), creating the complement clones Δaah1-AAH1 ( Δh1-H1 ) and Δaah1Δaah2-AAH1 ( Δh1Δh2-H1 ). Brown Yellow Bars: CRISPR targeting sites. Black bars: PCR screening primer target regions ( S3 Table ). (C) PCR verification of successful ablation and complementation of knockouts. Expected product sizes: Tubulin (Tub): 0.378kb. AAH1 (H1): 0.745kb (Native), 0.278kb (cDNA). AAH2 (H2): 0.745kb (Native), 0.278kb (cDNA). (D) Growth assays of parasites seeded into 96-well plates and allowed to proliferate for 24 h, then quantified using a luciferase assay. The WT, Δh1 , Δh2, Δh1Δh2 , Δh1-H1 , Δh2-H2 , and Δh1Δh2-H1 parasites showed no significant difference in total growth (Kruskal-Wallis test, P = 0.0672, N = 3 per strain).

    Techniques Used: Knock-Out, CRISPR, Construct, Transfection, Plasmid Preparation, Selection, Polymerase Chain Reaction, Clone Assay, Marker, Luciferase

    8) Product Images from "Exploiting a Natural Auxotrophy for Genetic Selection"

    Article Title: Exploiting a Natural Auxotrophy for Genetic Selection

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.00762-12

    Shuttle plasmid pBR103. For the DNA sequence of pBR103, see Fig. S1 in the supplemental material. repA , replication region from pFNL10; ori , replication origin from p15A; bla , β-lactamase gene; lacZ , β-galactosidase gene.
    Figure Legend Snippet: Shuttle plasmid pBR103. For the DNA sequence of pBR103, see Fig. S1 in the supplemental material. repA , replication region from pFNL10; ori , replication origin from p15A; bla , β-lactamase gene; lacZ , β-galactosidase gene.

    Techniques Used: Plasmid Preparation, Sequencing

    9) Product Images from "Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells"

    Article Title: Vaccinia Virus Uracil DNA Glycosylase Has an Essential Role in DNA Synthesis That Is Independent of Its Glycosylase Activity: Catalytic Site Mutations Reduce Virulence but Not Virus Replication in Cultured Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.1.159-166.2003

    Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of
    Figure Legend Snippet: Fluorescence assay of uracil DNA glycosylase activity. RK13 cells were mock infected or infected with recombinant virus at 10 PFU/cell. After 8 h at 37°C, the cells were harvested and lysed. Fluorescence assays were performed using 5 μg of cell extract protein and 1 μg of supercoiled pUC-19 DNA containing (A and B) or lacking (C and D) uracil residues. At the indicated times, aliquots of the reaction mixture were removed into pH 12 buffer, and fluorescence was measured before and after the mixtures were boiled. The percent fluorescence represents the amount of supercoiled pUC-19 remaining. The presence of nicks in the uracil containing pUC 19, prepared from E. coli CJ236 ( dut ung ) cells, accounted for the values of

    Techniques Used: Fluorescence, Activity Assay, Infection, Recombinant

    10) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    11) Product Images from "Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa"

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8557

    Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.
    Figure Legend Snippet: Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.

    Techniques Used: Amplification, Molecular Weight, Marker, Negative Control

    12) Product Images from "Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa"

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8557

    Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.
    Figure Legend Snippet: Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.

    Techniques Used: Amplification, Molecular Weight, Marker, Negative Control

    13) Product Images from "APOBEC3A is a prominent cytidine deaminase in breast cancer"

    Article Title: APOBEC3A is a prominent cytidine deaminase in breast cancer

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.1008545

    APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.
    Figure Legend Snippet: APOBEC3A is the predominant cytidine deaminase in BRCA cell lines lacking APOBEC3B. (A) The mutation profile of AU565 and SKBR3 BRCA cell lines. (B) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression in AU565 (black) and SKBR3 (grey). Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean (SEM) of these measurements. n.d. indicates “not detected.” Similar results were obtained using TBP instead of HPRT1 as the internal reference gene ( S2 Table ). (C) Schematic of in vitro cytidine deaminase assay. (D) AU565, AU565 cells containing a CRISPR-Cas9 mediated disruption of APOBEC3A (-/-), and (E) SKBR3 BRCA cell lines either un-transduced or expressing scramble control, A3A shRNA, or A3B shRNA were tested for cytidine deaminase activity on a hairpin or linear substrate containing a YTCA APOBEC target motif. Each cell line was additionally transduced to express a vector control or uracil glycosylase inhibitor (UGI) as indicated. 40 μg of total protein was incubated with 0.25 μM of hairpin substrate for 24 hrs at 37°C, prior to heating the samples at 95°C for 10 min and separating substrate from cleavage product on a denaturing polyacrylamide gel. Knockdown specificity was confirmed by qRT-PCR and equal protein amount in each reaction was confirmed via α-GAPDH western.

    Techniques Used: Mutagenesis, Expressing, In Vitro, CRISPR, shRNA, Activity Assay, Plasmid Preparation, Incubation, Quantitative RT-PCR, Western Blot

    Correlation of A3A expression with APOBEC-induced mutations in multiple cancer types. RSEM normalized RNA-seq values for A3A (blue) and A3B (red) were obtained for 410 bladder (BLCA), 197 cervical (CESC), and 549 head and neck (HNSC) cancers assessed by the Cancer Genome Atlas. Expression of each APOBEC was normalized to HPRT1 and compared to the minimum estimate of APOBEC-induced mutations in the corresponding samples. A3A expression strongly correlates with mutagenesis in each tumor type by Pearson correlation analysis even when only evaluating APOBEC mutated tumors.
    Figure Legend Snippet: Correlation of A3A expression with APOBEC-induced mutations in multiple cancer types. RSEM normalized RNA-seq values for A3A (blue) and A3B (red) were obtained for 410 bladder (BLCA), 197 cervical (CESC), and 549 head and neck (HNSC) cancers assessed by the Cancer Genome Atlas. Expression of each APOBEC was normalized to HPRT1 and compared to the minimum estimate of APOBEC-induced mutations in the corresponding samples. A3A expression strongly correlates with mutagenesis in each tumor type by Pearson correlation analysis even when only evaluating APOBEC mutated tumors.

    Techniques Used: Expressing, RNA Sequencing Assay, Mutagenesis

    APOBEC3A is the predominant cytidine deaminase in BT474 cells expressing APOBEC3B. (A) The mutation profile of BT474 cells. (B) The number of APOBEC-induced (black bars) and non-APOBEC-induced (grey bars) mutations in BT474 cells. (C) mRNA expression levels of individual APOBEC3 family members relative to HPRT1 expression in BT474 as measured by qRT-PCR. Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean of these measurements. n.d. indicates “not detected.” (D) In vitro cytidine deaminase assay (conducted similarly to Fig 1D and 1E ) of whole-cell extracts generated BT474 cells or BT474 cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. Deaminase reactions were supplemented with either 2 units UGI (NEB #M0281S) or an equal volume of 50% glycerol. Specificity of each shRNA was confirmed by qRT-PCR, and equal protein amounts used in deaminase assays were verified by α-GAPDH western.
    Figure Legend Snippet: APOBEC3A is the predominant cytidine deaminase in BT474 cells expressing APOBEC3B. (A) The mutation profile of BT474 cells. (B) The number of APOBEC-induced (black bars) and non-APOBEC-induced (grey bars) mutations in BT474 cells. (C) mRNA expression levels of individual APOBEC3 family members relative to HPRT1 expression in BT474 as measured by qRT-PCR. Bars indicate the mean values of 3 replicate measurements. Error bars indicate the standard error of the mean of these measurements. n.d. indicates “not detected.” (D) In vitro cytidine deaminase assay (conducted similarly to Fig 1D and 1E ) of whole-cell extracts generated BT474 cells or BT474 cells transduced with lentiviral vectors to express scramble control, A3A-targeting, or A3B targeting shRNAs. Deaminase reactions were supplemented with either 2 units UGI (NEB #M0281S) or an equal volume of 50% glycerol. Specificity of each shRNA was confirmed by qRT-PCR, and equal protein amounts used in deaminase assays were verified by α-GAPDH western.

    Techniques Used: Expressing, Mutagenesis, Quantitative RT-PCR, In Vitro, Generated, Transduction, shRNA, Western Blot

    APOBEC3A expression correlates with the abundance of APOBEC-induced mutations in primary BRCA tumors, despite the similarity between APOBEC3A and APOBEC3B transcripts. (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and
    Figure Legend Snippet: APOBEC3A expression correlates with the abundance of APOBEC-induced mutations in primary BRCA tumors, despite the similarity between APOBEC3A and APOBEC3B transcripts. (A) The pairwise alignment of the A3A (blue) and A3B (red) transcripts shows a central region of high sequence identity with two unique regions in each transcript. Green, yellow, and red indicate 100%, ≥30%, and

    Techniques Used: Expressing, Sequencing

    APOBEC3A mRNA transcript levels correlate with the extent of APOBEC-induced mutations in BRCA cell lines. The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in S2 Table . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP ( S2 Table ). Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).
    Figure Legend Snippet: APOBEC3A mRNA transcript levels correlate with the extent of APOBEC-induced mutations in BRCA cell lines. The average mutation profiles of (A) 14 non-APOBEC-mutated and (B) 14 APOBEC-mutated BRCA cell lines. Specific cell lines in each category are defined in S2 Table . (C) mRNA expression level of individual APOBEC3 family members relative to HPRT1 expression was measured by qRT-PCR in non-APOBEC-mutated (N) and APOBEC-mutated (M) BRCA cell lines. Similar results were obtained comparing APOBEC expression to TBP ( S2 Table ). Each circle represents the mean of 3 replicate measurements for an individual cell line. Horizontal bars indicate the median expression for each APOBEC3 family member among the non-APOBEC-mutated or APOBEC-mutated cell lines. Data points corresponding to cell lines without detectable expression of individual APOBECs are not shown on the graph but are included in the calculation of the median. Statistical significance for differences in the expression of a given APOBEC family member between non-APOBEC-mutated and APOBEC-mutated lines was assessed by Mann-Whitney Summed Rank test. ** indicates p = 0.0067. Correlations between A3A expression (blue dots) or A3B expression (red dots) measured by qRT-PCR and the minimum estimate of APOBEC-induced mutations for each of the 28 BRCA cell lines were determined by a Pearson correlation test using mutation lists obtained from (D) the Cancer Cell Line Encyclopedia and (E) the Catalogue of Somatic Mutations in Cancer (COSMIC).

    Techniques Used: Mutagenesis, Expressing, Quantitative RT-PCR, MANN-WHITNEY

    14) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.
    Figure Legend Snippet: A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Techniques Used: Activity Assay, Transfection, Derivative Assay, Labeling, Countercurrent Chromatography, Recombinant, Incubation, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

    15) Product Images from "T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity"

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.0030135

    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.
    Figure Legend Snippet: A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Techniques Used: Activity Assay, Transfection, Derivative Assay, Labeling, Countercurrent Chromatography, Recombinant, Incubation, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

    16) Product Images from "The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient"

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkm1053

    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.
    Figure Legend Snippet: L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Techniques Used: Purification, Incubation, Activity Assay, Primer Extension Assay

    17) Product Images from "Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries"

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-54044-x

    ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.
    Figure Legend Snippet: ( A ) Sequence design for the investigation of uracil and abasic site cleavage on microarrays. Each sequence consists of a dT 15 -linker, then a 30mer with either no dUs (control) or an increasing number of dU-incorporations (from 1 to 9) replacing dTs in the following sequence: 5′-TTA CCA TAG AAT CAT GTG CCA TAC ATC ATC-3′. At the 5′-end, a control 25mer is synthesized (QC25), serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). The cleavage process was monitored by recording the hybridization-based fluorescence intensity before and after the UDG-mediated cleavage of uracil nucleotides. ( B ) Small excerpt (ca. 7% of the total synthesis area) of fluorescence scans before and after enzyme exposure. The scans show the fluorescence intensity, resulting from hybridization to a labelled, complementary oligonucleotide. The microarrays were scanned at 5 µm resolution. ( C ) Decrease in fluorescence intensity for the UDG-mediated uracil excision (thus generating abasic sites) as a function of the number of dU nucleotide incorporations per DNA substrate. The actual cleavage efficiencies correlate with the loss of fluorescence intensity resulting from DNA substrate cleavage. The array was incubated for one hour with UDG and the generated abasic sites were subsequently cleaved under alkaline conditions. The decrease in fluorescence intensity was recorded and normalized to the control strand (U0). The normalized intensities, indicated in arbitrary units, were plotted over the number of dUs per DNA substrate. Error bars are SD.

    Techniques Used: Sequencing, Synthesized, Hybridization, Fluorescence, Incubation, Generated

    Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.
    Figure Legend Snippet: Schematic illustration of the sequence design for the investigation of E. coli UDG sequence dependences on single- ( A ) and double-stranded DNA substrates. ( B ) In order to investigate the UDG sequence dependence, a single dU is incorporated into a DNA strand and enclosed by 3 permuted bases on each side. A The design for the study of UDG sequence dependence on single-stranded DNA substrates consists of a 15mer dT-linker, a single dU enclosed by 3 permuted bases on each side, followed by a 5′ 25mer sequence (QC25) serving as target for the hybridization to its 3′-Cy3-labelled complementary oligonucleotide (QC25c). B For the study of UDG sequence dependence on double-stranded DNA substrates, the sequences were designed to form a hairpin loop. The resulting strands consisted of a 15mer dT-linker, a 11-nt stem, equivalent to the single-stranded design, containing the variable region flanked by dG·dC base pairs, a 4-nt loop followed by the complementary 11nt strand. At the 5′ end, a hybridizable 25mer target sequence (QC25) was synthesized.

    Techniques Used: Sequencing, Hybridization, Synthesized

    (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.
    Figure Legend Snippet: (Left) Schematic illustration of the UDG-mediated generation of single nucleotide gaps on nucleic acid strands. In a first step, UDG catalyzes the excision of uracil, leading to the formation of an abasic site. This AP-site can then either be cleaved by the lyase activity of specific endonucleases, or chemically. The USER enzyme, a mixture of UDG and Endonuclease VIII, combines AP-site formation and cleavage in a single solution. (Right) Molecular structures indicating the generation of a single nucleotide gap/strand cleavage via a β- and a subsequent δ-elimination reaction. First, UDG hydrolyzes the glycosidic bond from the uracil-containing DNA strand. The ribose at the apyrimidinic site lacks a glycosidic bond and is therefore highly unstable and converts rapidly into its reactive open-chain aldehyde, its hemiacetal or its hydrate form. The lyase activity of AP-endonucleases, or the exposure to either basic or acidic conditions, initiates a β-elimination reaction, resulting in the cleavage of the phosphodiester backbone 3′ to the AP-site and the formation of an α,β-unsaturated aldehyde. Subsequent δ-elimination induces DNA strand cleavage 5′ to the AP-site resulting in the generation of a single-nucleotide gap in dsDNA or strand cleavage in ssDNA.

    Techniques Used: Activity Assay

    Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.
    Figure Legend Snippet: Representative sequence motifs for the UDG-mediated uracil cleavage on double- ( A , B ) and single-stranded ( C , D ) DNA strands. Substrates were incubated with UDG for different time periods ranging from 5 seconds to 30 minutes (since the cleavage motifs showed little to no sequence dependence, only the 5s, 30s, 60s and 120s were determined for ssDNA). The sequence motifs were extracted from the 1% (41 of 4096 sequences) most cleaved ( A , C ) and least cleaved ( B , D ) sequences of the library.

    Techniques Used: Sequencing, Incubation

    18) Product Images from "Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair"

    Article Title: Identification of a conserved 5′-dRP lyase activity in bacterial DNA repair ligase D and its potential role in base excision repair

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw054

    Bsu LigD is endowed with an AP lyase activity. ( A ) Analysis of the capacity of BsuL igD to incise an internal natural abasic site. The [α 32 P]3′-labeled 2′-deoxyuridine-containing substrate was treated with 27 nM E. coli UDG (lane c ), leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either 5 nM h APE1 that cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or increasing concentrations of Bsu LigD (0, 29, 57 and 114 nM) for 1 h at 30°C, as described in Materials and Methods. After incubation samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) Analysis of the capacity of Bsu LigD to incise an internal tetrahydrofuran (H). The 3′ [α 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analogue tetrahydrofuran (H) was incubated in the presence of either h APE1, EndoIII or increasing concentrations of Bsu LigD as described above. Position corresponding to the products 16-mer 5′-dRP and 16-mer 5′-P is indicated. The figure is a composite image made from different parts of the same experiment.
    Figure Legend Snippet: Bsu LigD is endowed with an AP lyase activity. ( A ) Analysis of the capacity of BsuL igD to incise an internal natural abasic site. The [α 32 P]3′-labeled 2′-deoxyuridine-containing substrate was treated with 27 nM E. coli UDG (lane c ), leaving an intact AP site. The resulting AP-containing DNA was incubated in the presence of either 5 nM h APE1 that cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or increasing concentrations of Bsu LigD (0, 29, 57 and 114 nM) for 1 h at 30°C, as described in Materials and Methods. After incubation samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. ( B ) Analysis of the capacity of Bsu LigD to incise an internal tetrahydrofuran (H). The 3′ [α 32 P]3′-dAMP labeled oligonucleotide containing the lyase-resistant analogue tetrahydrofuran (H) was incubated in the presence of either h APE1, EndoIII or increasing concentrations of Bsu LigD as described above. Position corresponding to the products 16-mer 5′-dRP and 16-mer 5′-P is indicated. The figure is a composite image made from different parts of the same experiment.

    Techniques Used: Activity Assay, Labeling, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Left: Pae LigD is endowed with a 5′-dRP lyase activity. The assay was performed as indicated in Materials and Methods in the presence of either 3.5 nM of EndoIII or 60 nM of the indicated LigD in the absence (−) or presence (+) of 0.64 mM MnCl 2 . After incubation during 30 min at 30°C samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. Alk , alkaline hydrolysis of the 5′-dRP moiety. Right: the 5′-dRP lyase activity of Bsu LigD resides in the ligase domain. The assay was performed as in left panel in the presence of 216 nM LigDom. After incubation during 30 min at 30°C samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. The figure is a composite image made from different parts of the same experiment.
    Figure Legend Snippet: Left: Pae LigD is endowed with a 5′-dRP lyase activity. The assay was performed as indicated in Materials and Methods in the presence of either 3.5 nM of EndoIII or 60 nM of the indicated LigD in the absence (−) or presence (+) of 0.64 mM MnCl 2 . After incubation during 30 min at 30°C samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. Alk , alkaline hydrolysis of the 5′-dRP moiety. Right: the 5′-dRP lyase activity of Bsu LigD resides in the ligase domain. The assay was performed as in left panel in the presence of 216 nM LigDom. After incubation during 30 min at 30°C samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. The figure is a composite image made from different parts of the same experiment.

    Techniques Used: Activity Assay, Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    Bsu LigD performs non-metal-dependent release of the 5′-dRP moiety. Top: schematic representation of the substrates used in the assay and corresponding to a filled gap with a dangling 5′-dRP group in the downstream strand and either a 3′-OH (left) or dideoxy (right) terminus. Bottom: autodiagrams showing the release of the 5′-dRP group by Bsu LigD. Reactions were performed as described in Materials and Methods in the presence of either 3.5 nM EndoIII (lanes b and g ) or 57 nM Bsu LigD (lanes c, d, e, h and i ). After incubation during 30 min at 30°C, samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. Alk, alkaline hydrolysis of the 5′-dRP moiety. Lanes a and f , original substrate; lanes c and h , reactions performed in the absence of metal ions; lanes d and i , reactions performed in the presence of 0.64 mM MnCl 2 ; lane e , reaction carried out in the presence of 0.64 mM MnCl 2 and further incubation with alkali. Ctrl lane corresponds to a control of the initial DNA before starting the reaction. The figure is a composite image made from different parts of the same experiment.
    Figure Legend Snippet: Bsu LigD performs non-metal-dependent release of the 5′-dRP moiety. Top: schematic representation of the substrates used in the assay and corresponding to a filled gap with a dangling 5′-dRP group in the downstream strand and either a 3′-OH (left) or dideoxy (right) terminus. Bottom: autodiagrams showing the release of the 5′-dRP group by Bsu LigD. Reactions were performed as described in Materials and Methods in the presence of either 3.5 nM EndoIII (lanes b and g ) or 57 nM Bsu LigD (lanes c, d, e, h and i ). After incubation during 30 min at 30°C, samples were analyzed by 8 M urea-20% PAGE and autoradiography. Position of products is indicated. Alk, alkaline hydrolysis of the 5′-dRP moiety. Lanes a and f , original substrate; lanes c and h , reactions performed in the absence of metal ions; lanes d and i , reactions performed in the presence of 0.64 mM MnCl 2 ; lane e , reaction carried out in the presence of 0.64 mM MnCl 2 and further incubation with alkali. Ctrl lane corresponds to a control of the initial DNA before starting the reaction. The figure is a composite image made from different parts of the same experiment.

    Techniques Used: Incubation, Polyacrylamide Gel Electrophoresis, Autoradiography

    19) Product Images from "Phaeocystis globosa Virus DNA Polymerase X: a “Swiss Army knife”, Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair"

    Article Title: Phaeocystis globosa Virus DNA Polymerase X: a “Swiss Army knife”, Multifunctional DNA polymerase-lyase-ligase for Base Excision Repair

    Journal: Scientific Reports

    doi: 10.1038/s41598-017-07378-3

    AP-lyase activity of PgVPolX . The depicted [ 32 P]3′-labeled uracil-containing oligonucleotide (top) was treated with E. coli UDG, leaving an intact AP site. The resulting AP-containing DNA (1 nM) was incubated in the presence of either E. coli EndoIII that incises 3′ to the AP site, or the indicated PgV-PolX for 10 min at 30 °C, as described in Materials and Methods. Position of products is indicated.
    Figure Legend Snippet: AP-lyase activity of PgVPolX . The depicted [ 32 P]3′-labeled uracil-containing oligonucleotide (top) was treated with E. coli UDG, leaving an intact AP site. The resulting AP-containing DNA (1 nM) was incubated in the presence of either E. coli EndoIII that incises 3′ to the AP site, or the indicated PgV-PolX for 10 min at 30 °C, as described in Materials and Methods. Position of products is indicated.

    Techniques Used: Activity Assay, Labeling, Incubation

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

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

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkw1205

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

    Techniques Used: Fluorescence, Activity Assay

    21) Product Images from "Human Base Excision Repair Creates a Bias Toward -1 Frameshift Mutations *"

    Article Title: Human Base Excision Repair Creates a Bias Toward -1 Frameshift Mutations *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.118596

    Proposed single nucleotide deletion pathway catalyzed by BER enzymes. The pathway for AAG-initiated short-patch BER is shown on the left and the proposed pathway for single nucleotide deletion is shown on the right. X denotes a damaged nucleotide. Note that only the glycosylase and endonuclease reactions differ for the two pathways. After APE1 cleavage the 5′-dRP intermediate is chemically identical to the intermediate generated after single nucleotide incorporation in the short-patch BER pathway.
    Figure Legend Snippet: Proposed single nucleotide deletion pathway catalyzed by BER enzymes. The pathway for AAG-initiated short-patch BER is shown on the left and the proposed pathway for single nucleotide deletion is shown on the right. X denotes a damaged nucleotide. Note that only the glycosylase and endonuclease reactions differ for the two pathways. After APE1 cleavage the 5′-dRP intermediate is chemically identical to the intermediate generated after single nucleotide incorporation in the short-patch BER pathway.

    Techniques Used: Generated

    22) Product Images from "CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide"

    Article Title: CUX1 stimulates APE1 enzymatic activity and increases the resistance of glioblastoma cells to the mono-alkylating agent temozolomide

    Journal: Neuro-Oncology

    doi: 10.1093/neuonc/nox178

    CUT domains stimulate APE1 endonuclease activity in vitro. (A) Schematic of CUX1 recombinant proteins used in APE1 endonuclease assays. (B, C) APE1 endonuclease assays were carried out using purified APE1, in the presence of purified CUX1 recombinant proteins or controls (BSA or homeobox B3) at 50 nM or as indicated, and a radiolabeled probe containing an abasic site produced in 2 different ways: as a tetrahydrofuran (B) or through removal of a uracil residue by UDG (C). (D) Schematic of the APE1 assay that utilizes a molecular beacon probe containing a tetrahydrofuran site. The APE1 assay was carried out with purified APE1 in the presence of nothing, 50 nM BSA, or purified CUX1 recombinant proteins (C2C3HD, C1C2, and C3HD), as indicated.
    Figure Legend Snippet: CUT domains stimulate APE1 endonuclease activity in vitro. (A) Schematic of CUX1 recombinant proteins used in APE1 endonuclease assays. (B, C) APE1 endonuclease assays were carried out using purified APE1, in the presence of purified CUX1 recombinant proteins or controls (BSA or homeobox B3) at 50 nM or as indicated, and a radiolabeled probe containing an abasic site produced in 2 different ways: as a tetrahydrofuran (B) or through removal of a uracil residue by UDG (C). (D) Schematic of the APE1 assay that utilizes a molecular beacon probe containing a tetrahydrofuran site. The APE1 assay was carried out with purified APE1 in the presence of nothing, 50 nM BSA, or purified CUX1 recombinant proteins (C2C3HD, C1C2, and C3HD), as indicated.

    Techniques Used: Activity Assay, In Vitro, Recombinant, Purification, Produced

    23) Product Images from "The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *"

    Article Title: The Structural Location of DNA Lesions in Nucleosome Core Particles Determines Accessibility by Base Excision Repair Enzymes *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M112.441444

    Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI
    Figure Legend Snippet: Assessment of the removal of rotationally and translationally positioned uracils by UDG and APE1. A, NCPs containing a single uracil at different sites were incubated with UDG and APE1. Open symbols represent in uracils as follows: red square , NCP-UI

    Techniques Used: Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1
    Figure Legend Snippet: Polymerase β extension activity in NCPs near the dyad. A, representative gels for NCP-gO (+10) and NCP-gI (+4) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (+10) and NCP-gI (+4) were incubated with pol β and APE1

    Techniques Used: Activity Assay, Incubation

    Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated
    Figure Legend Snippet: Polymerase β extension activity in NCPs near DNA ends. A, representative gels for NCP-gO (−35) and NCP-gI (−49) pol β (100 n m ) extension in the absence of APE1. B, NCP-gO (−35) and NCP-gI (−49) were incubated

    Techniques Used: Activity Assay, Incubation

    UDG and APE1 Digestion
    Figure Legend Snippet: UDG and APE1 Digestion

    Techniques Used:

    24) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    25) Product Images from "Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair"

    Article Title: Incision of DNA-protein crosslinks by UvrABC nuclease suggests a potential repair pathway involving nucleotide excision repair

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.042700399

    Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna ) or Hae ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.
    Figure Legend Snippet: Preparation of site-specific DNA–protein crosslinks. ( A ) Sequence of the uracil-containing 60-mer oligodeoxynucleotide. ( B ) Urea-PAGE showing DNA substrate preparation. Lane 1, uracil-containing 60-mer; lane 2, uracil-containing 60-mer, digested with uracil DNA glycosylase and tested with T4-pdg (control of AP-site formation); reduced AP site-containing DNA before (lane 3) and after (lanes 4–6) purification; DPC-containing DNA before (lane 7) and after (lanes 8–10) purification. After purification, DNAs were subjected to the restriction endonuclease digestion with Sna ) or Hae ). ( C ) SDS/PAGE showing DPC-containing DNA substrates before (lane 1) and after (lane 2) Hae III digestion.

    Techniques Used: Sequencing, Polyacrylamide Gel Electrophoresis, Purification, SDS Page

    26) Product Images from "Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage"

    Article Title: Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn118

    True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.
    Figure Legend Snippet: True color fluorescence oligonucleotide assay. ( I ) Scheme for construction of dual-color fluorescently labeled oligonucleotides. The 51mer A strand contains a single uracil, whereas the opposing strand is synthesized from a central cassette (Bb, 21 bp) containing one of a number of lesion configurations, and two flanking sequences, Ba and Bc, each 15 bp. In the example shown, A contains one uracil residue, and is labeled at its 5′ end with 6-FAM; Ba is 3′ end-labeled with TAMRA, and the central Bb cassette contains one uracil residue. The components are annealed, ligated and treated with uracil DNA glycosylase to convert the uracil moieties to abasic sites. The action of Ape1 on the construct is then assessed. ( II ) True color denaturing gel (adjacent segments of the same gel, separated for clarity) with fluorescence of intact and Ape1-cleaved oligonucleotides. Constructs and pairs of gel lanes showing substrates (Lanes 1, 3 and 5) and products (Lanes 2, 4 and 6). Lanes 1 and 2: 51mer A1•B−5, where A1 is 5′-labeled with 6-FAM, and B-5 is 3″ TAMRA-labeled. Lane 1 intact substrate plus free, unligated TAMRA-labeled Ba); Lane 2, products of Ape1 cleavage of A1•B−5: 3′ end of B- TAMRA, 5′ end of A-FAM) plus unligated Ba. Lanes 3 and 4: A1•B−5 containing unlabelled A1 and dually labeled B-5 (3′ TAMRA and 5′ 6-FAM). Lane 3, intact substrate, a small quantity of the partial ligation product BaBb, plus unligated TAMRA-labeled Ba and 6-FAM-labeled Bc. Lane 4, Ape cleavage products: 3′ end of B, 5′ end of B plus Ba and Bc as in Lane 3. Lanes 5 and 6, Substrate and products as in Lanes 3 and 4, but Bc was 5′-labeled with JOE (6-carboxy-4′, 5′-dichloro-2′, 7′-dimethoxyfluorescein, light green) and 3′- labeled with TAMRA.

    Techniques Used: Fluorescence, Oligonucleotide Assay, Labeling, Synthesized, Construct, Ligation

    27) Product Images from "Characterization of interstrand DNA-DNA cross-links derived from abasic sites using bacteriophage ϕ29 DNA polymerase"

    Article Title: Characterization of interstrand DNA-DNA cross-links derived from abasic sites using bacteriophage ϕ29 DNA polymerase

    Journal: Biochemistry

    doi: 10.1021/acs.biochem.5b00482

    The dA-Ap (8) cross-link blocks primer extension by ϕ29 DNA polymerase The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is the 15 nt, 5′- 32 P-labeled primer, lane 2 is the 5′- 32 P-labeled full-length extension product, lanes 3–7 depict the results of primer extension reactions on substrates O – S , and lane 8 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA). The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.
    Figure Legend Snippet: The dA-Ap (8) cross-link blocks primer extension by ϕ29 DNA polymerase The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is the 15 nt, 5′- 32 P-labeled primer, lane 2 is the 5′- 32 P-labeled full-length extension product, lanes 3–7 depict the results of primer extension reactions on substrates O – S , and lane 8 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA). The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.

    Techniques Used: Labeling, Incubation

    Cross-links containing the abasic site in the template strand block primer extension by ϕ29 DNA polymerase extension, while an un-cross-linked abasic site in the template strand is a partial block The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA); lanes 2 and 3 are Maxam-Gilbert G- and A+G-reactions carried out on the 5′- 32 P-labeled full-length extension product; lane 4 is the 15 nt, 5′- 32 P-labeled primer; lane 5 is the 5′- 32 P-labeled full-length extension product; lane 6, primer extension on the single-strand substrate H containing dU in the template; lane 7, single-strand substrate I containing Ap in the template; lane 8, duplex substrate J containing dU in the template; lane 9, duplex substrate K containing an un-cross-linked Ap site in the template strand; lane 10, duplex substrate L containing reduced dG-Ap cross-link 5 with the Ap residue in the template strand; lane 11, duplex substrate X containing the dA-Ap cross-link in which the Ap residue is in the template strand.
    Figure Legend Snippet: Cross-links containing the abasic site in the template strand block primer extension by ϕ29 DNA polymerase extension, while an un-cross-linked abasic site in the template strand is a partial block The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA); lanes 2 and 3 are Maxam-Gilbert G- and A+G-reactions carried out on the 5′- 32 P-labeled full-length extension product; lane 4 is the 15 nt, 5′- 32 P-labeled primer; lane 5 is the 5′- 32 P-labeled full-length extension product; lane 6, primer extension on the single-strand substrate H containing dU in the template; lane 7, single-strand substrate I containing Ap in the template; lane 8, duplex substrate J containing dU in the template; lane 9, duplex substrate K containing an un-cross-linked Ap site in the template strand; lane 10, duplex substrate L containing reduced dG-Ap cross-link 5 with the Ap residue in the template strand; lane 11, duplex substrate X containing the dA-Ap cross-link in which the Ap residue is in the template strand.

    Techniques Used: Blocking Assay, Labeling, Incubation

    The effects of incubation time on the collection of products generated by ϕ29 DNA polymerase-mediated primer extension on substrates containing the dA-Ap cross-link 8 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 5–60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–18 depict primer extension reactions on duplexes O , Q , and S for the indicated times. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.
    Figure Legend Snippet: The effects of incubation time on the collection of products generated by ϕ29 DNA polymerase-mediated primer extension on substrates containing the dA-Ap cross-link 8 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 5–60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–18 depict primer extension reactions on duplexes O , Q , and S for the indicated times. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.

    Techniques Used: Incubation, Generated, Labeling

    The chemically-stable, reduced dG-Ap cross-link 5 blocks primer extension by ϕ29 DNA polymerase. The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 30 min at 24 °C. After reaction work-up, the primer extension products were analyzed by electrophoresis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–8 depict the results of primer extension on templates C – G while the primer extension products possess 3′-hydroxyl termini.
    Figure Legend Snippet: The chemically-stable, reduced dG-Ap cross-link 5 blocks primer extension by ϕ29 DNA polymerase. The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 30 min at 24 °C. After reaction work-up, the primer extension products were analyzed by electrophoresis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–8 depict the results of primer extension on templates C – G while the primer extension products possess 3′-hydroxyl termini.

    Techniques Used: Labeling, Incubation, Electrophoresis

    Higher dNTP concentrations favor extension of primers to the −1 position immediately preceding the reduced dG-Ap cross-link 5 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (0.01–1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 30 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–12 depict the results of primer extension on templates D–F in the presence of the indicated dNTP concentrations. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.
    Figure Legend Snippet: Higher dNTP concentrations favor extension of primers to the −1 position immediately preceding the reduced dG-Ap cross-link 5 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (0.01–1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 30 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–12 depict the results of primer extension on templates D–F in the presence of the indicated dNTP concentrations. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.

    Techniques Used: Labeling, Incubation

    The effects of incubation time on the collection of products generated by ϕ29 DNA polymerase-mediated primer extension on substrates containing the reduced dG-Ap cross-link 5 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 5–60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–18 depict primer extension reactions on templates C , E , and G for the indicated times. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.
    Figure Legend Snippet: The effects of incubation time on the collection of products generated by ϕ29 DNA polymerase-mediated primer extension on substrates containing the reduced dG-Ap cross-link 5 The 32 P-labeled primers were extended by incubation of the DNA substrates with ϕ29 DNA polymerase (10 units) and the four dNTPs (1 mM in each) in Tris-HCl (50 mM, pH 7.5), MgCl 2 (10 mM), (NH 4 ) 2 SO 4 (10 mM), DTT (4 mM), and bovine serum albumin (0.1 mg/mL) for 5–60 min at 24 °C. After reaction work-up, the primer extension products were subjected to electrophoretic analysis on a 20% denaturing polyacrylamide gel. Lane 1 is an iron-EDTA cleavage reaction on a synthetic standard of the full-length extension product (5′- 32 P-GAT CAC AGT GAG TAC AAT AGA ATA GAT GAA CTA AGA CAT ATA), lane 2 is the 15 nt, 5′- 32 P-labeled primer, lane 3 is the 5′- 32 P-labeled full-length extension product, and lanes 4–18 depict primer extension reactions on templates C , E , and G for the indicated times. The arrow corresponds to extension of the primer to the last base in the single-stranded region of the template.

    Techniques Used: Incubation, Generated, Labeling

    28) Product Images from "Cytidine deaminase efficiency of the lentiviral viral restriction factor APOBEC3C correlates with dimerization"

    Article Title: Cytidine deaminase efficiency of the lentiviral viral restriction factor APOBEC3C correlates with dimerization

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx066

    Analysis of A3C processivity on ssDNA oligonucleotides. Processivity of A3C was tested on ssDNA substrates that contain a fluorescein-labeled deoxythymidine (yellow star) between two 5΄TTC deamination motifs separated by different distances. (A and B) hA3C S188I is more processive than hA3C. ( A ) Deamination of a 60 nt ssDNA substrate with deamination targets spaced 5 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 42- and 23-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 5 nt labeled fragment. ( B ) Deamination of a 118 nt ssDNA substrate with deaminated cytosines spaced 63 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 63 nt labeled fragment. (C–F) cA3C and gA3C are more processive than hA3C. ( C ) Deamination of a 60 nt ssDNA substrate as for panel (A). ( D ) Deamination of a 118 nt ssDNA as for panel (B). ( E ) Deamination of a 69 nt ssDNA substrate with deamination targets spaced 14 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 51- and 32-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 14 nt labeled fragment. ( F ) Deamination of an 85 nt ssDNA substrate with deaminated cytosines spaced 30 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 67- and 48-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 30 nt labeled fragment. If no 5΄C and 3΄C band was detected, the processivity was denoted with N.D. (not detected). The measurements of enzyme processivity (processivity factor) and the S.D. are shown below the gels. All values are calculated from at least three independent experiments.
    Figure Legend Snippet: Analysis of A3C processivity on ssDNA oligonucleotides. Processivity of A3C was tested on ssDNA substrates that contain a fluorescein-labeled deoxythymidine (yellow star) between two 5΄TTC deamination motifs separated by different distances. (A and B) hA3C S188I is more processive than hA3C. ( A ) Deamination of a 60 nt ssDNA substrate with deamination targets spaced 5 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 42- and 23-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 5 nt labeled fragment. ( B ) Deamination of a 118 nt ssDNA substrate with deaminated cytosines spaced 63 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 100- and 81-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 63 nt labeled fragment. (C–F) cA3C and gA3C are more processive than hA3C. ( C ) Deamination of a 60 nt ssDNA substrate as for panel (A). ( D ) Deamination of a 118 nt ssDNA as for panel (B). ( E ) Deamination of a 69 nt ssDNA substrate with deamination targets spaced 14 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 51- and 32-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 14 nt labeled fragment. ( F ) Deamination of an 85 nt ssDNA substrate with deaminated cytosines spaced 30 nt apart. Single deaminations of the 5΄C and 3΄C are detected as the appearance of labeled 67- and 48-nt fragments, respectively; double deamination of both C residues on the same molecule results in a 30 nt labeled fragment. If no 5΄C and 3΄C band was detected, the processivity was denoted with N.D. (not detected). The measurements of enzyme processivity (processivity factor) and the S.D. are shown below the gels. All values are calculated from at least three independent experiments.

    Techniques Used: Labeling

    Dimerization influences processive ssDNA scanning. Processivity of A3C mutants was tested on ssDNA substrates and compared to the wild type enzyme. (A–C) Processivity factor values are shown for short-range movements based on deamination of a 60 nt ssDNA substrate with deamination targets spaced 5 nt apart and long-range movements based on deamination of a 118 nt substrate with deaminated cytosines spaced 63 nt apart for ( A ) hA3C, hA3C S188I and hA3C S188I/N115K, ( B ) cA3C, cA3C S188I and cA3C K115N and ( C ) gA3C, gA3C S188I and gA3C K115N. See Supplementary Figure S4 for a representative gel. ( D ) Intersegmental transfer ability of cA3C was determined by keeping an A3C/ssDNA ratio of 7:1 constant, but increasing the total reaction components. If the enzyme is able to undergo intersegmental transfer, the assay will result in an apparent decrease in the processivity factor with increasing concentrations of reaction components. The ssDNA substrate contained a fluorescein-labeled deoxythymidine (yellow star) between two deamination targets separated by 63 nt. The measurements of enzyme processivity (processivity factor) and the S.D. are shown below the gel. (E and F) Summary of intersegmental transfer assays shown in Supplementary Figure S5 . ( E ) The monomer/dimer forms of A3C (cA3C, gA3C, hA3C S188I) are better able to undergo intersegmental transfer than the ( F ) stable dimer forms of A3C (cA3C S188I, gA3C S188I, hA3C S188I/N115K). For comparison, the hatched line in (F) denotes the decrease in processivity observed for monomer/dimer forms of A3C in (E). All values are calculated from at least three independent experiments.
    Figure Legend Snippet: Dimerization influences processive ssDNA scanning. Processivity of A3C mutants was tested on ssDNA substrates and compared to the wild type enzyme. (A–C) Processivity factor values are shown for short-range movements based on deamination of a 60 nt ssDNA substrate with deamination targets spaced 5 nt apart and long-range movements based on deamination of a 118 nt substrate with deaminated cytosines spaced 63 nt apart for ( A ) hA3C, hA3C S188I and hA3C S188I/N115K, ( B ) cA3C, cA3C S188I and cA3C K115N and ( C ) gA3C, gA3C S188I and gA3C K115N. See Supplementary Figure S4 for a representative gel. ( D ) Intersegmental transfer ability of cA3C was determined by keeping an A3C/ssDNA ratio of 7:1 constant, but increasing the total reaction components. If the enzyme is able to undergo intersegmental transfer, the assay will result in an apparent decrease in the processivity factor with increasing concentrations of reaction components. The ssDNA substrate contained a fluorescein-labeled deoxythymidine (yellow star) between two deamination targets separated by 63 nt. The measurements of enzyme processivity (processivity factor) and the S.D. are shown below the gel. (E and F) Summary of intersegmental transfer assays shown in Supplementary Figure S5 . ( E ) The monomer/dimer forms of A3C (cA3C, gA3C, hA3C S188I) are better able to undergo intersegmental transfer than the ( F ) stable dimer forms of A3C (cA3C S188I, gA3C S188I, hA3C S188I/N115K). For comparison, the hatched line in (F) denotes the decrease in processivity observed for monomer/dimer forms of A3C in (E). All values are calculated from at least three independent experiments.

    Techniques Used: Labeling

    Monomeric A3C induces lower levels of mutagenesis than dimeric A3C. (A–G) An in vitro HIV replication assay was utilized to determine the enzymes abilities to catalyze deaminations during proviral DNA synthesis. (A–C) Spectra of mutations are plotted as the percentage of clones containing a G→A mutation at a particular location (nt) in the 368 nt prot-lacZ α construct for ( A ) hA3C, ( B ) cA3C or ( C ) gA3C. (D–F) Histograms illustrate the number of mutations that can be induced by ( D ) hA3C, ( E ) cA3C or ( F ) gA3C within individual clones. ( G ) Summarized G→A mutation frequency for A3C monomers (hA3C, cA3C K115N, gA3C K115N), monomers/dimers (hA3C S188I, cA3C, gA3C), and dimers (hA3C S188I/N115K, cA3C S188I, gA3C S188I). The graph denotes whether the A3C is from human (h), chimpanzee (c) or gorilla (g). Individual spectra and clonal mutation frequencies not included in Figure 5 are in Supplementary Figure S6 . ( H ) HIV Δvif infectivity was measured by β-galactosidase expression driven by the HIV-1 5΄LTR from HeLa CD4+ HIV-1 LTR-β-gal cells infected with HIV Δvif that was produced in the absence or presence of A3G or A3C orthologs. Relative decrease in virus infectivity is shown for A3G, A3C monomers (hA3C, cA3C K115N, gA3C K115N), A3C monomers/dimers (hA3C S188I, cA3C, gA3C) and A3C dimers (hA3C S188I/N115K, cA3C S188I, gA3C S188I). The graph denotes whether the A3C is from human (h), chimpanzee (c) or gorilla (g). Results normalized to the no A3 condition are shown with the Standard Deviation of the mean calculated from at least three independent experiments. Statistical significance of HIV Δvif restriction for each A3C ortholog was determined in comparison to the monomer form (hA3C, cA3C K115N or gA3C K115N). Designations for significant difference of values were *** P ≤ 0.001, ** P ≤ 0.01 or * P ≤ 0.05. ( I ) Immunoblotting for the HA tag was used to detect A3 enzymes expressed in cells and encapsidated into HIV Δvif virions. The cell lysate and virion loading controls were α-tubulin and p24, respectively. Quantification of the relative amount of A3 was normalized to hA3C.
    Figure Legend Snippet: Monomeric A3C induces lower levels of mutagenesis than dimeric A3C. (A–G) An in vitro HIV replication assay was utilized to determine the enzymes abilities to catalyze deaminations during proviral DNA synthesis. (A–C) Spectra of mutations are plotted as the percentage of clones containing a G→A mutation at a particular location (nt) in the 368 nt prot-lacZ α construct for ( A ) hA3C, ( B ) cA3C or ( C ) gA3C. (D–F) Histograms illustrate the number of mutations that can be induced by ( D ) hA3C, ( E ) cA3C or ( F ) gA3C within individual clones. ( G ) Summarized G→A mutation frequency for A3C monomers (hA3C, cA3C K115N, gA3C K115N), monomers/dimers (hA3C S188I, cA3C, gA3C), and dimers (hA3C S188I/N115K, cA3C S188I, gA3C S188I). The graph denotes whether the A3C is from human (h), chimpanzee (c) or gorilla (g). Individual spectra and clonal mutation frequencies not included in Figure 5 are in Supplementary Figure S6 . ( H ) HIV Δvif infectivity was measured by β-galactosidase expression driven by the HIV-1 5΄LTR from HeLa CD4+ HIV-1 LTR-β-gal cells infected with HIV Δvif that was produced in the absence or presence of A3G or A3C orthologs. Relative decrease in virus infectivity is shown for A3G, A3C monomers (hA3C, cA3C K115N, gA3C K115N), A3C monomers/dimers (hA3C S188I, cA3C, gA3C) and A3C dimers (hA3C S188I/N115K, cA3C S188I, gA3C S188I). The graph denotes whether the A3C is from human (h), chimpanzee (c) or gorilla (g). Results normalized to the no A3 condition are shown with the Standard Deviation of the mean calculated from at least three independent experiments. Statistical significance of HIV Δvif restriction for each A3C ortholog was determined in comparison to the monomer form (hA3C, cA3C K115N or gA3C K115N). Designations for significant difference of values were *** P ≤ 0.001, ** P ≤ 0.01 or * P ≤ 0.05. ( I ) Immunoblotting for the HA tag was used to detect A3 enzymes expressed in cells and encapsidated into HIV Δvif virions. The cell lysate and virion loading controls were α-tubulin and p24, respectively. Quantification of the relative amount of A3 was normalized to hA3C.

    Techniques Used: Mutagenesis, In Vitro, DNA Synthesis, Clone Assay, Construct, Infection, Expressing, Produced, Standard Deviation

    A3C dimerization is mediated through α-helix 6 or β-strand 4. (A–C) SEC profile for 10 μg of (A) cA3C, cA3C K115N and cA3C S188I; ( B ) gA3C, gA3C K115N and gA3C S188I and ( C ) hA3C, hA3C S188I, hA3C N115K and hA3C S188I/N115K from a 10 ml Superdex 200 column was used to calculate the oligomerization state of the enzyme from a standard calibration curve. An M denotes a monomer fraction and a D denotes a dimer fraction. ( A ) cA3C formed monomers and dimers (apparent molecular weights 19 kDa and 45 kDa, respectively), cA3C S188I formed a stable dimer (apparent molecular weight 45 kDa), and cA3C K115N formed monomers (apparent molecular weight 19 kDa). ( B ) The gA3C SEC profiles were similar to cA3C, except for gA3C K115N that was mainly monomers (apparent molecular weight 19 kDa), but also retained a small proportion of dimers (apparent molecular weight 45 kDa). ( C ) hA3C formed monomers in solution (apparent molecular weight 19 kDa), hA3C S188I and hA3C N115K were an equilibrium of monomers and dimers (apparent molecular weights 19 kDa and 45 kDa, respectively) and hA3C S188I/N115K was a stable dimer (apparent molecular weight 45 kDa). The chromatograms were constructed by analyzing the integrated gel-band intensities of each protein in each fraction after resolution by SDS-PAGE ( Supplementary Figure S3 ). ( D ) A3C enzymes were incubated in the absence or presence of 20 μM BS3 crosslinker and subsequently visualized with SDS-PAGE and immunoblotting. Monomeric A3C enzymes remained as monomers in the presence of crosslinker (cA3C K115N, hA3C). A3C enzymes that were able to form dimers according to SEC, were also present as monomers/dimers (cA3C, gA3C, gA3C K115N, hA3C S188I) or as dimers (cA3C S188I, gA3C S188I, hA3C S188I/N115K) in the presence of the crosslinker. Molecular weight standards are indicated. ( E ) Coimmunoprecipitation of A3C-V5 with A3C-HA. The A3C-HA and A3C-V5 were transfected in combination and the immunoprecipitation of cell lysates used either anti-HA antibody or Rabbit IgG (mock) and was immunoblotted with antibodies against α-tubulin, HA and V5. Cell lysates show the expression of α-tubulin, HA and V5. (F–H) The apparent K d of A3C enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy for ( E ) cA3C, cA3C S188I and cA3C K115N; ( F ) gA3C, gA3C S188I, and gA3C K115N and ( G ) hA3C, hA3C S188I, hA3C N115K and hA3C S188I/N115K. Apparent K d values are shown in the figure. Hill coefficients for cooperative binding curves are (E) cA3C, 1.6; cA3C S188I, 1.7; (F) gA3C, 1.8; gA3C S188I, 2.1; (G) hA3C S188I, 1.6; hA3C N115K, 1.5; hA3C S188I/N115K, 1.9. Error bars represent the S.D. from three independent experiments.
    Figure Legend Snippet: A3C dimerization is mediated through α-helix 6 or β-strand 4. (A–C) SEC profile for 10 μg of (A) cA3C, cA3C K115N and cA3C S188I; ( B ) gA3C, gA3C K115N and gA3C S188I and ( C ) hA3C, hA3C S188I, hA3C N115K and hA3C S188I/N115K from a 10 ml Superdex 200 column was used to calculate the oligomerization state of the enzyme from a standard calibration curve. An M denotes a monomer fraction and a D denotes a dimer fraction. ( A ) cA3C formed monomers and dimers (apparent molecular weights 19 kDa and 45 kDa, respectively), cA3C S188I formed a stable dimer (apparent molecular weight 45 kDa), and cA3C K115N formed monomers (apparent molecular weight 19 kDa). ( B ) The gA3C SEC profiles were similar to cA3C, except for gA3C K115N that was mainly monomers (apparent molecular weight 19 kDa), but also retained a small proportion of dimers (apparent molecular weight 45 kDa). ( C ) hA3C formed monomers in solution (apparent molecular weight 19 kDa), hA3C S188I and hA3C N115K were an equilibrium of monomers and dimers (apparent molecular weights 19 kDa and 45 kDa, respectively) and hA3C S188I/N115K was a stable dimer (apparent molecular weight 45 kDa). The chromatograms were constructed by analyzing the integrated gel-band intensities of each protein in each fraction after resolution by SDS-PAGE ( Supplementary Figure S3 ). ( D ) A3C enzymes were incubated in the absence or presence of 20 μM BS3 crosslinker and subsequently visualized with SDS-PAGE and immunoblotting. Monomeric A3C enzymes remained as monomers in the presence of crosslinker (cA3C K115N, hA3C). A3C enzymes that were able to form dimers according to SEC, were also present as monomers/dimers (cA3C, gA3C, gA3C K115N, hA3C S188I) or as dimers (cA3C S188I, gA3C S188I, hA3C S188I/N115K) in the presence of the crosslinker. Molecular weight standards are indicated. ( E ) Coimmunoprecipitation of A3C-V5 with A3C-HA. The A3C-HA and A3C-V5 were transfected in combination and the immunoprecipitation of cell lysates used either anti-HA antibody or Rabbit IgG (mock) and was immunoblotted with antibodies against α-tubulin, HA and V5. Cell lysates show the expression of α-tubulin, HA and V5. (F–H) The apparent K d of A3C enzymes from a 118 nt ssDNA was analyzed by steady-state rotational anisotropy for ( E ) cA3C, cA3C S188I and cA3C K115N; ( F ) gA3C, gA3C S188I, and gA3C K115N and ( G ) hA3C, hA3C S188I, hA3C N115K and hA3C S188I/N115K. Apparent K d values are shown in the figure. Hill coefficients for cooperative binding curves are (E) cA3C, 1.6; cA3C S188I, 1.7; (F) gA3C, 1.8; gA3C S188I, 2.1; (G) hA3C S188I, 1.6; hA3C N115K, 1.5; hA3C S188I/N115K, 1.9. Error bars represent the S.D. from three independent experiments.

    Techniques Used: Size-exclusion Chromatography, Molecular Weight, Construct, SDS Page, Incubation, Transfection, Immunoprecipitation, Expressing, Binding Assay

    Sequence alignment and structural analysis of A3C. ( A ) Sequence alignment of hA3C, cA3C and gA3C with amino acid differences shown in white. The sequence alignment was performed by a Clustal Omega multiple sequence alignment ( 75 ) and plotted using the program ESPript ( 76 ). ( B ) Surface representation of a hA3C dimer from the crystal structure (PDB: 3VOW). Amino acids unique to hA3C that are potentially involved in the dimer interface are shown in purple (α-helix 6, S188; β-strand 4, N115) and other amino acids unique to hA3C are shown in yellow (β-strand 3, K85; α-helix 3, D99).
    Figure Legend Snippet: Sequence alignment and structural analysis of A3C. ( A ) Sequence alignment of hA3C, cA3C and gA3C with amino acid differences shown in white. The sequence alignment was performed by a Clustal Omega multiple sequence alignment ( 75 ) and plotted using the program ESPript ( 76 ). ( B ) Surface representation of a hA3C dimer from the crystal structure (PDB: 3VOW). Amino acids unique to hA3C that are potentially involved in the dimer interface are shown in purple (α-helix 6, S188; β-strand 4, N115) and other amino acids unique to hA3C are shown in yellow (β-strand 3, K85; α-helix 3, D99).

    Techniques Used: Sequencing

    Models of A3C dimerization. ( A ) For hA3C, N115 (chain A) is 4.7 Å away from the backbone of R44 (chain B). ( B ) In contrast, a model of cA3C K115 (chain A) positions the backbone of R44 (chain B) only 3.0 Å away. This distance could indicate a new hydrogen bond with potential to stabilize the dimer between chains A and B. ( C ) In hA3C, S188, shown with van der Waal space filling dots, packs closely to F126 and N132, but does not clash with either. ( D ) In contrast, a model of hA3C shows how I188 would clash with F126 and N132 (arrows indicating overlap of van der Waal space filling dots). Conformational changes, potentially including a repositioning of the helix to enable formation of an A–B dimer, would be needed to accommodate this amino acid variant.
    Figure Legend Snippet: Models of A3C dimerization. ( A ) For hA3C, N115 (chain A) is 4.7 Å away from the backbone of R44 (chain B). ( B ) In contrast, a model of cA3C K115 (chain A) positions the backbone of R44 (chain B) only 3.0 Å away. This distance could indicate a new hydrogen bond with potential to stabilize the dimer between chains A and B. ( C ) In hA3C, S188, shown with van der Waal space filling dots, packs closely to F126 and N132, but does not clash with either. ( D ) In contrast, a model of hA3C shows how I188 would clash with F126 and N132 (arrows indicating overlap of van der Waal space filling dots). Conformational changes, potentially including a repositioning of the helix to enable formation of an A–B dimer, would be needed to accommodate this amino acid variant.

    Techniques Used: Variant Assay

    29) Product Images from "Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa"

    Article Title: Development of uracil-DNA-glycosylase-supplemented loop-mediated isothermal amplification coupled with nanogold probe (UDG-LAMP-AuNP) for specific detection of Pseudomonas aeruginosa

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2018.8557

    Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.
    Figure Legend Snippet: Optimization of dUTP to dTTP for loop-mediated isothermal amplification. The ratios of dUTP to dTTP were as follows: Lane 1, 100% dUTP; lane 2, 80% dUTP + 20% dTTP; lane 3, 60% dUTP + 40% dTTP; lane 4, 40% dUTP + 60% dTTP; lane 5, 20% dUTP + 80% dTTP; and lane 6, 100% dTTP. Lane M, molecular weight marker; lane N, negative control (no DNA template); dUTP, deoxyuridine triphosphate; dTTP, deoxythymidine triphosphate.

    Techniques Used: Amplification, Molecular Weight, Marker, Negative Control

    30) Product Images from "Protective efficacy and safety of liver stage attenuated malaria parasites"

    Article Title: Protective efficacy and safety of liver stage attenuated malaria parasites

    Journal: Scientific Reports

    doi: 10.1038/srep26824

    Generation and characterization of ANKA LISP2 (–) and N-LISP2 parasites. ( A ) LISP2 locus and differently modified gene loci as generated in previous studies (i, ii) and in the current study (iii, iv). The region encoding the N-terminus is colored in orange. Boxes not drawn to scale. ( B ) Schematic of the LISP2 replacement strategy using the 5′ and 3′UTRs of LISP2 to integrate a resistance cassette by double crossover into the genome of the P. bergh ei strain ANKA. Note that this resistance cassette (yellow) included a negative selection marker to enable recycling of the cassette. The locations of primers used for PCR in ( C ) are indicated. ( C ) Diagnostic PCR to investigate generation of LISP2 (–) parasites. Numbers below the gel indicate expected amplicon sizes. ( D ) Liver stages in HepG2 cells formed by ANKA WT and LISP2 (–) sporozoites 65 hours post infection labeled with antibodies against EXP-1 and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. ( E ) Schematic of the strategy used to integrate the N-terminus of LISP2 into the LISP2(–) parasite. The locations of primers used for PCR in ( F ) are indicated. The N-terminal fragment contained a start and stop codon. ( F ) Diagnostic PCR to investigate generation of N-LISP2 parasites. Numbers below the gel indicate expected amplicon sizes. KO: LISP2 (–) parasites, N-L: N-LISP2 parasites. ( G ) Meyer-Kaplan plot to indicate the number of blood stage positive mice after injection of sporozoites i.v. and by bite. 4 groups of 4 mice each were infected; of these 15 became blood stage patent.
    Figure Legend Snippet: Generation and characterization of ANKA LISP2 (–) and N-LISP2 parasites. ( A ) LISP2 locus and differently modified gene loci as generated in previous studies (i, ii) and in the current study (iii, iv). The region encoding the N-terminus is colored in orange. Boxes not drawn to scale. ( B ) Schematic of the LISP2 replacement strategy using the 5′ and 3′UTRs of LISP2 to integrate a resistance cassette by double crossover into the genome of the P. bergh ei strain ANKA. Note that this resistance cassette (yellow) included a negative selection marker to enable recycling of the cassette. The locations of primers used for PCR in ( C ) are indicated. ( C ) Diagnostic PCR to investigate generation of LISP2 (–) parasites. Numbers below the gel indicate expected amplicon sizes. ( D ) Liver stages in HepG2 cells formed by ANKA WT and LISP2 (–) sporozoites 65 hours post infection labeled with antibodies against EXP-1 and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. ( E ) Schematic of the strategy used to integrate the N-terminus of LISP2 into the LISP2(–) parasite. The locations of primers used for PCR in ( F ) are indicated. The N-terminal fragment contained a start and stop codon. ( F ) Diagnostic PCR to investigate generation of N-LISP2 parasites. Numbers below the gel indicate expected amplicon sizes. KO: LISP2 (–) parasites, N-L: N-LISP2 parasites. ( G ) Meyer-Kaplan plot to indicate the number of blood stage positive mice after injection of sporozoites i.v. and by bite. 4 groups of 4 mice each were infected; of these 15 became blood stage patent.

    Techniques Used: Modification, Generated, Selection, Marker, Polymerase Chain Reaction, Diagnostic Assay, Amplification, Infection, Labeling, Mouse Assay, Injection

    Characterization of a ANKA LISP2 (–) /uis3 (–) double knockout parasite line and comparison with single knockout lines. ( A ) Liver stages in HepG2 cells formed by WT and LISP2 (–) /uis3 (–) sporozoites 65 hours post infection as revealed by labelling with antibodies against EXP-1, and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. ( B ) Percentage of breakthrough infections (black) and infected mice per 1 million sporozoites injected (white) of the different parasite lines, see also Appendix Table SII. ( C ) Number of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoites. All data normalized to the mean of WT duplicates in each individual experiment. Raw data are shown in Supplementary Fig. S4 . ( D ) Sizes of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoites. ( E,F ) Relative liver load of two mice 40 and 56 hours post infection (hpi) of LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoite infected C57BL/6 mice. 18S rRNA abundance was normalized to the average value of 2 Pb ANKA infected mice for each time point. Note that at 56 hpi some WT parasites have already emerged from the liver, hence the lower relative levels.
    Figure Legend Snippet: Characterization of a ANKA LISP2 (–) /uis3 (–) double knockout parasite line and comparison with single knockout lines. ( A ) Liver stages in HepG2 cells formed by WT and LISP2 (–) /uis3 (–) sporozoites 65 hours post infection as revealed by labelling with antibodies against EXP-1, and MSP1; Hoechst reveals host and parasite DNA. Scale bar: 10 μm. ( B ) Percentage of breakthrough infections (black) and infected mice per 1 million sporozoites injected (white) of the different parasite lines, see also Appendix Table SII. ( C ) Number of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoites. All data normalized to the mean of WT duplicates in each individual experiment. Raw data are shown in Supplementary Fig. S4 . ( D ) Sizes of liver stages 24 and 48 hours post infection (hpi) of HepG2 cells with WT, LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoites. ( E,F ) Relative liver load of two mice 40 and 56 hours post infection (hpi) of LISP2 (–), uis3 (–) and LISP2 (–) /uis3 (–) sporozoite infected C57BL/6 mice. 18S rRNA abundance was normalized to the average value of 2 Pb ANKA infected mice for each time point. Note that at 56 hpi some WT parasites have already emerged from the liver, hence the lower relative levels.

    Techniques Used: Double Knockout, Knock-Out, Infection, Mouse Assay, Injection

    31) Product Images from "Integrated digital error suppression for improved detection of circulating tumor DNA"

    Article Title: Integrated digital error suppression for improved detection of circulating tumor DNA

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3520

    Development of integrated digital error suppression (iDES) ( a ) Diagram depicting the use of CAPP-Seq barcode adapters to suppress errors. Here, CAPP-Seq adapters are ligated to a double-stranded (duplex) DNA molecule containing a real biological mutation in both strands as well as a non-replicated, asymmetric base change in only one strand ( top ). The combined application of insert and index barcodes allows for (i) error suppression and (ii) recovery of single stranded ( center ) and duplex ( bottom ) DNA molecules ( Supplementary Fig. 1a , Methods ). ( b ) Top : Heat map showing position-specific selector-wide error rates parceled into all possible base substitutions (rows) and organized by decreasing mean allele fractions (for each substitution type) across 12 cfDNA samples from healthy controls (columns; Supplementary Table 2 ). Background patterns are shown for different error suppression methods, including the combined application of barcoding and background polishing. Errors were defined as non-reference alleles excluding germline SNPs. Bottom : Selector-wide error metrics ( Methods ).
    Figure Legend Snippet: Development of integrated digital error suppression (iDES) ( a ) Diagram depicting the use of CAPP-Seq barcode adapters to suppress errors. Here, CAPP-Seq adapters are ligated to a double-stranded (duplex) DNA molecule containing a real biological mutation in both strands as well as a non-replicated, asymmetric base change in only one strand ( top ). The combined application of insert and index barcodes allows for (i) error suppression and (ii) recovery of single stranded ( center ) and duplex ( bottom ) DNA molecules ( Supplementary Fig. 1a , Methods ). ( b ) Top : Heat map showing position-specific selector-wide error rates parceled into all possible base substitutions (rows) and organized by decreasing mean allele fractions (for each substitution type) across 12 cfDNA samples from healthy controls (columns; Supplementary Table 2 ). Background patterns are shown for different error suppression methods, including the combined application of barcoding and background polishing. Errors were defined as non-reference alleles excluding germline SNPs. Bottom : Selector-wide error metrics ( Methods ).

    Techniques Used: Mutagenesis

    Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq was assessed using technical controls ( a – c ) and patients with NSCLC ( d – f ). ( a ) A DNA reference blend containing known alleles spanning a broad AF range was diluted to 5% in normal cfDNA and analyzed in replicate ( n =4) for both known variants ( n =29) and 279 negative control variants ( Supplementary Table 4 , Methods ). Left : Differential impact of barcoding, polishing, and iDES on genotyping results for a single representative replicate. Only variant calls with at least 2 supporting reads are shown. Asterisks highlight the complementary background profiles removed by barcoding and polishing. Note that all variant calls are ordered along the x -axis, first by validation status and then by AF. Identical calls are aligned vertically. Right : Performance metrics across all four replicates. Genotyping thresholds were determined as described in Methods . ( b ) AFs determined by iDES-enhanced CAPP-Seq in the 5% variant blend from panel a (observed) versus their concentrations determined by digital PCR (expected). Only variants in the reference blend with externally validated AFs targeted by our NSCLC selector are shown ( n =13; Supplementary Table 4 ). Data are expressed as means ± s.e.m ( n =4 replicates). ( c ) Heat map ( top ) and scatter plot ( bottom ) depicting candidate SNVs identified by noninvasive selector-wide genotyping of the 5% variant blend from panel a ( Supplementary Fig. 10 , Methods ). SNVs were tracked across three additional replicates and a ten-fold lower spike. Horizontal lines depict mean AFs. ( d – f ) Noninvasive tumor genotyping of NSCLC patients. ( d ) Bottom : The number of hotspot SNVs noninvasively detected in 24 pretreatment NSCLC cfDNA samples by four methods, including iDES (barcoding + polishing). All queried variants are listed in Supplementary Table 4 . Top: Positive predictive value (PPV) of each method (indicated below), based on the number of hotspot SNVs that were later confirmed in matching tumor biopsies. ( e ) The performance of iDES for noninvasive tumor genotyping of two plasma cohorts was assessed using observed allele fractions with a Receiver Operating Characteristic (ROC) plot. In the first cohort ( n =66 plasma samples from patients with matching tumor biopsies), hotspot variants from a predefined list of 292 variants were assessed ( Supplementary Table 4 ). Results are shown for the 46 plasma samples with at least one detectable mutation (‘All genes’, n =24 patients); specificity was assessed using variants that were detected but that could not be verified in the primary tumor. In the second cohort, EGFR hotspot variants were assessed in an extended cohort of 103 plasma samples from 41 EGFR-positive patients with NSCLC (‘ EGFR’ ). Specificity was assessed using 27 EGFR-wildtype subjects ( Methods ). The pie chart shows the distribution of detected EGFR variants. Only patients with genotyped tumors were analyzed. AUC, area under the curve. ( f ) Noninvasive genotyping of EGFR mutations in plasma samples from 37 patients with advanced NSCLC and with biopsy-confirmed EGFR mutations. Top: Performance of iDES-enhanced CAPP-Seq for the genotyping of actionable EGFR mutations ( n =36 patients; 1 of 37 patients did not have an actionable alteration). All performance metrics were assessed at the variant level. Bottom: Comparison of error-suppression methods for noninvasive tumor genotyping of the entire EGFR kinase domain in all patients with biopsy-confirmed EGFR SNVs ( n =29 of 37 patients). Performance metrics were assessed separately at the variant level and patient level (using 27 EGFR-wildtype subjects). Percentages indicate iDES performance only. Further details are provided in Methods . Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.
    Figure Legend Snippet: Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq Noninvasive tumor genotyping with iDES-enhanced CAPP-Seq was assessed using technical controls ( a – c ) and patients with NSCLC ( d – f ). ( a ) A DNA reference blend containing known alleles spanning a broad AF range was diluted to 5% in normal cfDNA and analyzed in replicate ( n =4) for both known variants ( n =29) and 279 negative control variants ( Supplementary Table 4 , Methods ). Left : Differential impact of barcoding, polishing, and iDES on genotyping results for a single representative replicate. Only variant calls with at least 2 supporting reads are shown. Asterisks highlight the complementary background profiles removed by barcoding and polishing. Note that all variant calls are ordered along the x -axis, first by validation status and then by AF. Identical calls are aligned vertically. Right : Performance metrics across all four replicates. Genotyping thresholds were determined as described in Methods . ( b ) AFs determined by iDES-enhanced CAPP-Seq in the 5% variant blend from panel a (observed) versus their concentrations determined by digital PCR (expected). Only variants in the reference blend with externally validated AFs targeted by our NSCLC selector are shown ( n =13; Supplementary Table 4 ). Data are expressed as means ± s.e.m ( n =4 replicates). ( c ) Heat map ( top ) and scatter plot ( bottom ) depicting candidate SNVs identified by noninvasive selector-wide genotyping of the 5% variant blend from panel a ( Supplementary Fig. 10 , Methods ). SNVs were tracked across three additional replicates and a ten-fold lower spike. Horizontal lines depict mean AFs. ( d – f ) Noninvasive tumor genotyping of NSCLC patients. ( d ) Bottom : The number of hotspot SNVs noninvasively detected in 24 pretreatment NSCLC cfDNA samples by four methods, including iDES (barcoding + polishing). All queried variants are listed in Supplementary Table 4 . Top: Positive predictive value (PPV) of each method (indicated below), based on the number of hotspot SNVs that were later confirmed in matching tumor biopsies. ( e ) The performance of iDES for noninvasive tumor genotyping of two plasma cohorts was assessed using observed allele fractions with a Receiver Operating Characteristic (ROC) plot. In the first cohort ( n =66 plasma samples from patients with matching tumor biopsies), hotspot variants from a predefined list of 292 variants were assessed ( Supplementary Table 4 ). Results are shown for the 46 plasma samples with at least one detectable mutation (‘All genes’, n =24 patients); specificity was assessed using variants that were detected but that could not be verified in the primary tumor. In the second cohort, EGFR hotspot variants were assessed in an extended cohort of 103 plasma samples from 41 EGFR-positive patients with NSCLC (‘ EGFR’ ). Specificity was assessed using 27 EGFR-wildtype subjects ( Methods ). The pie chart shows the distribution of detected EGFR variants. Only patients with genotyped tumors were analyzed. AUC, area under the curve. ( f ) Noninvasive genotyping of EGFR mutations in plasma samples from 37 patients with advanced NSCLC and with biopsy-confirmed EGFR mutations. Top: Performance of iDES-enhanced CAPP-Seq for the genotyping of actionable EGFR mutations ( n =36 patients; 1 of 37 patients did not have an actionable alteration). All performance metrics were assessed at the variant level. Bottom: Comparison of error-suppression methods for noninvasive tumor genotyping of the entire EGFR kinase domain in all patients with biopsy-confirmed EGFR SNVs ( n =29 of 37 patients). Performance metrics were assessed separately at the variant level and patient level (using 27 EGFR-wildtype subjects). Percentages indicate iDES performance only. Further details are provided in Methods . Sn, sensitivity; Sp, specificity; PPV, positive predictive value; NPV, negative predictive value.

    Techniques Used: Negative Control, Variant Assay, Digital PCR, Mutagenesis

    32) Product Images from "H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast"

    Article Title: H2A histone-fold and DNA elements in nucleosome activate SWR1-mediated H2A.Z replacement in budding yeast

    Journal: eLife

    doi: 10.7554/eLife.06845

    Nucleosome structure showing critical H2A residues that effect SWR1 activity. ( A ) Left : The yeast nucleosome crystal structure 1ID3 in Protein Data Bank was modeled to show histones on one face of nucleosome. Histone H2A is yellow, H2B is black and H3, H4 are gray. The domains of H2A that affect SWR1 activity-M3A (cyan), M4 (magenta), and M5 (blue) are marked. Center and right : Buried residues of histone H2A are shown by removing other histones and rotating on X-axis by 45°. ( B ) The H2A surface residue G47 in 1ID3 is shown in magenta. Bottom left : Zoom-in view shows that G47 is at the bottom of a cleft. Bottom right : Replacing Glycine for Lysine in H2A.Z histone shows the long side-chain of Lysine filling the cleft. DOI: http://dx.doi.org/10.7554/eLife.06845.006
    Figure Legend Snippet: Nucleosome structure showing critical H2A residues that effect SWR1 activity. ( A ) Left : The yeast nucleosome crystal structure 1ID3 in Protein Data Bank was modeled to show histones on one face of nucleosome. Histone H2A is yellow, H2B is black and H3, H4 are gray. The domains of H2A that affect SWR1 activity-M3A (cyan), M4 (magenta), and M5 (blue) are marked. Center and right : Buried residues of histone H2A are shown by removing other histones and rotating on X-axis by 45°. ( B ) The H2A surface residue G47 in 1ID3 is shown in magenta. Bottom left : Zoom-in view shows that G47 is at the bottom of a cleft. Bottom right : Replacing Glycine for Lysine in H2A.Z histone shows the long side-chain of Lysine filling the cleft. DOI: http://dx.doi.org/10.7554/eLife.06845.006

    Techniques Used: Activity Assay

    Nucleosomal histone and DNA elements critical for SWR1 activity and model for SWR1-mediated H2A-H2B displacement. ( A ) Yeast nucleosome structure PDB 1ID3 was modeled to show one face of the nucleosome and the histone-fold elements that are critical for SWR1 activation. The SWR1 footprint is shown in blue. The gap-sensitive region, 17–22 nt from dyad, is shown in cyan. Residues of H2A that affect SWR1 activity are shown in magenta. ( B ) Nucleosome model showing histone-DNA and histone–histone interactions that hold H2A-H2B within the nucleosome. Also shown is the gap-sensitive region, where SWR1 interacts with nucleosome DNA leading to eviction of H2A/H2B and concomitant deposition of H2A.Z/H2B. DOI: http://dx.doi.org/10.7554/eLife.06845.011
    Figure Legend Snippet: Nucleosomal histone and DNA elements critical for SWR1 activity and model for SWR1-mediated H2A-H2B displacement. ( A ) Yeast nucleosome structure PDB 1ID3 was modeled to show one face of the nucleosome and the histone-fold elements that are critical for SWR1 activation. The SWR1 footprint is shown in blue. The gap-sensitive region, 17–22 nt from dyad, is shown in cyan. Residues of H2A that affect SWR1 activity are shown in magenta. ( B ) Nucleosome model showing histone-DNA and histone–histone interactions that hold H2A-H2B within the nucleosome. Also shown is the gap-sensitive region, where SWR1 interacts with nucleosome DNA leading to eviction of H2A/H2B and concomitant deposition of H2A.Z/H2B. DOI: http://dx.doi.org/10.7554/eLife.06845.011

    Techniques Used: Activity Assay, Activation Assay

    Position of SWR1 footprint on linker-distal face of nucleosome. The 601 DNA-containing nucleosome structure PDB 3MVD was modeled to highlight the position of the SWR1 footprint in blue on the linker-distal side of the dyad axis. The H2A on the linker-distal face is in yellow. DOI: http://dx.doi.org/10.7554/eLife.06845.008
    Figure Legend Snippet: Position of SWR1 footprint on linker-distal face of nucleosome. The 601 DNA-containing nucleosome structure PDB 3MVD was modeled to highlight the position of the SWR1 footprint in blue on the linker-distal side of the dyad axis. The H2A on the linker-distal face is in yellow. DOI: http://dx.doi.org/10.7554/eLife.06845.008

    Techniques Used:

    SWR1 mediates histone exchange without net change of nucleosome position. ( A ) Left : EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right : SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. ( B ) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. ( C ) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. ( D ) Intensity plots for AA, AZ, and ZZ nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06845.012
    Figure Legend Snippet: SWR1 mediates histone exchange without net change of nucleosome position. ( A ) Left : EMSA (6% native PAGE) shows INO80-mediated nucleosome sliding. An asymmetrically positioned 601 nucleosome with a 43 bp and 0 bp DNA linker was used for the sliding assay. Right : SWR1-mediated incorporation of H2A.Z-H2B dimer (without 3FLAG epitope tag). Incorporation of H2A.Z in nucleosome was confirmed by immunoblotting with anti-H2A.Z antibody. ( B ) Hydroxyl radical footprinting strategy. A canonical nucleosome with 60 bp and 0 bp linker DNA and fluorescence end-label (bottom strand) was used as substrate for histone replacement, followed by hydroxyl radical treatment and separation by 6% native PAGE. ( C ) Recovered DNA from gel slices containing AA, AZ, and ZZ states was analyzed on DNA sequencing gels. ( D ) Intensity plots for AA, AZ, and ZZ nucleosomes. DOI: http://dx.doi.org/10.7554/eLife.06845.012

    Techniques Used: Clear Native PAGE, Footprinting, Fluorescence, DNA Sequencing

    SWR1 binding to nucleosome core particle with gaps on both sides of dyad. Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles. DOI: http://dx.doi.org/10.7554/eLife.06845.010
    Figure Legend Snippet: SWR1 binding to nucleosome core particle with gaps on both sides of dyad. Fluorescently labeled WT (green) and Gap (red) nucleosome core particles (5 nM) were mixed with indicated amounts of SWR1. Free and SWR1-bound nucleosome core particles were resolved on a 1.3% agarose gel. Bottom: binding curves for WT and Gap particles. DOI: http://dx.doi.org/10.7554/eLife.06845.010

    Techniques Used: Binding Assay, Labeling, Agarose Gel Electrophoresis

    SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone. EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles. DOI: http://dx.doi.org/10.7554/eLife.06845.004
    Figure Legend Snippet: SWR1 binding to nucleosome core particles containing H2A or H2A.Z histone. EMSA shows SWR1 binding to Alexa 647-labeled H2A- and H2A.Z-nucleosome core particles (1 nM). Free and bound complexes are resolved on 1.3% agarose gel. Bottom: binding curves for H2A- and H2A.Z-nucleosome core particles. DOI: http://dx.doi.org/10.7554/eLife.06845.004

    Techniques Used: Binding Assay, Labeling, Agarose Gel Electrophoresis

    33) Product Images from "Synthesis, characterization and DNA interaction studies of new triptycene derivatives"

    Article Title: Synthesis, characterization and DNA interaction studies of new triptycene derivatives

    Journal: Beilstein Journal of Organic Chemistry

    doi: 10.3762/bjoc.10.130

    Effect of triptycene derivatives on the restriction endonuclease activity of HindIII and BamHI enzymes on pUC19 plasmid. Agarose gel (top) shows the presence of the different forms of the plasmid following incubation with the compound and the respective
    Figure Legend Snippet: Effect of triptycene derivatives on the restriction endonuclease activity of HindIII and BamHI enzymes on pUC19 plasmid. Agarose gel (top) shows the presence of the different forms of the plasmid following incubation with the compound and the respective

    Techniques Used: Activity Assay, Plasmid Preparation, Agarose Gel Electrophoresis, Incubation

    Nuclease activities of the triptycene derivatives 1–8 . Agarose gel (top) shows results of the incubation of pUC19 plasmid DNA with triptycene derivatives and the bar diagram (bottom) represents the quantitative analysis of the gel bands. This
    Figure Legend Snippet: Nuclease activities of the triptycene derivatives 1–8 . Agarose gel (top) shows results of the incubation of pUC19 plasmid DNA with triptycene derivatives and the bar diagram (bottom) represents the quantitative analysis of the gel bands. This

    Techniques Used: Agarose Gel Electrophoresis, Incubation, Plasmid Preparation

    34) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    35) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    36) Product Images from "Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ"

    Article Title: Mechanism of Error-Free DNA Replication Past Lucidin-Derived DNA Damage by Human DNA Polymerase κ

    Journal: Chemical research in toxicology

    doi: 10.1021/acs.chemrestox.7b00227

    Translesion DNA synthesis by human DNA polymerases κ , η , ι , or Rev1 with unmodified or LdG-containing DNA substrate. Reaction conditions are described in the Materials and Methods section. (A) Primer extension reactions in the presence of all four dNTPs at their physiological concentrations (i.e., 10 μ M for dGTP and 40 μ . Changes in catalytic efficiency relative to a native base pair were calculated from ( k cat / K m,dCTP ) unmodified /( k cat / K m,dCTP ) LdG and indicated as x-fold decrease.
    Figure Legend Snippet: Translesion DNA synthesis by human DNA polymerases κ , η , ι , or Rev1 with unmodified or LdG-containing DNA substrate. Reaction conditions are described in the Materials and Methods section. (A) Primer extension reactions in the presence of all four dNTPs at their physiological concentrations (i.e., 10 μ M for dGTP and 40 μ . Changes in catalytic efficiency relative to a native base pair were calculated from ( k cat / K m,dCTP ) unmodified /( k cat / K m,dCTP ) LdG and indicated as x-fold decrease.

    Techniques Used: DNA Synthesis

    37) Product Images from "Construction of a circular single-stranded DNA template containing a defined lesion"

    Article Title: Construction of a circular single-stranded DNA template containing a defined lesion

    Journal: DNA repair

    doi: 10.1016/j.dnarep.2009.03.006

    ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.
    Figure Legend Snippet: ( A ) Sequence of pSOcpd surrounding the cis-syn CPD (indicated as T-T in bold font). The binding site of the labeled primer M13-TT, is shown as an arrow. ( B ) In vitro DNA replication assay with pSOcpd. The TLS reactions were performed in the presence of pol I (Kf) (lane 1), T7 DNA polymerase (lane 2), pol V (R391) + RecA protein in the absence (lane 3), or presence of the β-clamp and γ-clamp loader complex (lane 4), or human polη (lane 5). The position of the labeled primer (lane 6), is shown on the right of the gel (P), while the local template sequence context and position of the T-T CPD (in bold font) is shown on the left side of the gel.

    Techniques Used: Sequencing, Binding Assay, Labeling, In Vitro

    38) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    39) Product Images from "Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio"

    Article Title: Inactivation of folylpolyglutamate synthetase Met7 results in genome instability driven by an increased dUTP/dTTP ratio

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz1006

    Inactivation of Met7 results in a high dUTP/dTTP ratio that causes increased uracil incorporation into DNA. HPLC quantification of NTP ( A ) and dNTP ( B ) concentrations in cell extracts of indicated strains. Numbers on top in green or red, indicate the fold increase or decrease, respectively, relative to WT levels. In ( B ), cell extracts were (+) or not (−) treated with recombinant human Dut1 prior quantification of dNTPs. ‘nd’ (or ‘not detectable’) indicates dUTP concentrations below our detection limit (≤3 pmol dUTP). ( C ) Schematic diagram illustrating approach used in ( D ) for the detection of incorporated uracil into genomic DNA. ( D ) Genomic DNAs isolated from the indicated strains were (or not) digested with UDG + Ape1 enzymes and subsequently visualized by agarose gel electrophoresis.
    Figure Legend Snippet: Inactivation of Met7 results in a high dUTP/dTTP ratio that causes increased uracil incorporation into DNA. HPLC quantification of NTP ( A ) and dNTP ( B ) concentrations in cell extracts of indicated strains. Numbers on top in green or red, indicate the fold increase or decrease, respectively, relative to WT levels. In ( B ), cell extracts were (+) or not (−) treated with recombinant human Dut1 prior quantification of dNTPs. ‘nd’ (or ‘not detectable’) indicates dUTP concentrations below our detection limit (≤3 pmol dUTP). ( C ) Schematic diagram illustrating approach used in ( D ) for the detection of incorporated uracil into genomic DNA. ( D ) Genomic DNAs isolated from the indicated strains were (or not) digested with UDG + Ape1 enzymes and subsequently visualized by agarose gel electrophoresis.

    Techniques Used: High Performance Liquid Chromatography, Recombinant, Isolation, Agarose Gel Electrophoresis

    40) Product Images from "Genome-wide profiling of adenine base editor specificity by EndoV-seq"

    Article Title: Genome-wide profiling of adenine base editor specificity by EndoV-seq

    Journal: Nature Communications

    doi: 10.1038/s41467-018-07988-z

    Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p
    Figure Legend Snippet: Using EndoV-seq to profile genome-wide off-target deamination by ABE. a Genome-wide cleavage scores (cutoff score of > 2.5) of genomic DNA treated with Cas9 (blue), BE3 (yellow), or ABE7.10 (coral) using human HBG , VEGFA3 , HEK293-2 , or mouse Dmd gRNAs. Untreated genomic DNA (gray) served as controls. Red arrows, on-target sites. b Sequence logos of EndoV-captured (ABE7.10) and Digenome-captured (Cas9 and BE3) off-target (with scores of > 2.5) and on-target sites of the listed gRNAs. Target sequences are shown with PAM in blue. Note: The length of Dmd gRNA is 19-nt. c Venn diagrams that compare Digenome-captured sites for Cas9 and BE3 with EndoV-seq captured sites of ABE7.10 (score of > 0.1 for ABE7.10 and BE3, score of > 2.5 for Cas9) are shown for the target sites listed. d HEK-293T cells were co-transfected with vectors encoding ABE7.10 together with HBG gRNA (that targets both HBG1 and HBG2 ) and VEGFA3 gRNA. At 48 h after transfection, genomic DNA was extracted for PCR amplification and deep sequencing. GFP-transfected cells were used as controls. Error bars represent SEM ( n = 3). Statistical significance was calculated using a two-tailed unpaired t -test (*** p

    Techniques Used: Genome Wide, Sequencing, Transfection, Polymerase Chain Reaction, Amplification, Two Tailed Test

    Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue
    Figure Legend Snippet: Using EndoV-seq to evaluate on-target deamination by ABE. a A flow chart for assessing in vitro ABE off-target effects by EndoV-seq is shown, using sequences from the HEK293-2 site as an example. Genomic DNA is first incubated with recombinant ABE7.10 and the appropriate gRNA and then digested with EndoV, thereby allowing the DNA to be nicked by both nCas9 nickase (black triangle) and EndoV (red triangle, one residue downstream of base I). The cleaved DNA is subsequently fragmented and end repaired for whole-genome sequencing (WGS) with ~30–40 fold coverage. b Genomic DNA of 293T cells was used to PCR amplify regions spanning the HEK293-2 site. The PCR products (100 ng) were incubated with ABE7.10 (300 nM) and HEK293-2 gRNA (900 nM) for 3 h before EndoV (1U) incubation (30 min). The treated products were resolved by agarose gel electrophoresis. Recombinant Cas9 was used as a positive control for DNA cleavage. Molecular weight marker size is in base pairs. Source data are provided as a Source Data file. c Sanger sequencing chromatograms of PCR products amplified from the HEK293-2 gRNA target site using genomic DNA (10 µg) treated with ABE7.10 (300 nM, 8 h) ± EndoV (8U, 3 h). Mock treated genomic DNA served as a control. PAM, blue. Target base A, red and highlighted with red arrow. Peaks on the chromatograph, green for A, red for T, blue for C, and black for G. d PCR products from c were deep sequenced. The frequency of each allele is shown on the right. PAM, blue. Target base A, red. e Alignment of whole-genome sequencing reads of the HEK293-2 gRNA target region as visualized by the Integrative Genomics Viewer (IGV). Target base A, red. PAM, blue

    Techniques Used: Flow Cytometry, In Vitro, Incubation, Recombinant, Sequencing, Polymerase Chain Reaction, Agarose Gel Electrophoresis, Positive Control, Molecular Weight, Marker, Amplification

    Related Articles

    Concentration Assay:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

    Incubation:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

    Formalin-fixed Paraffin-Embedded:

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase
    Article Snippet: .. Treatment of FFPE DNA with uracil-DNA-glycosylase (UDG) To perform the UDG treatment and subsequent PCR/HRM assays without opening of reaction tubes, UDG (0.5 units/reaction, unless specified) and the UDG buffer (New England BioLabs, Ipswich, MA) were directly added to PCR/HRM master mixes. .. The reaction tubes were first incubated at 37°C for 30 minutes for UDG treatment, followed by the standard PCR/HRM assay conditions on the RotorGene Q instrument.

    Polymerase Chain Reaction:

    Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism
    Article Snippet: .. The PCR product was treated with uracil DNA glycosylase (NEB) for 2 h. The abasic site was stabilized by making the solution 100 mM in freshly diluted NaBH4 and incubating on ice for 30 min. .. The DNA was then purified using a MicroSpin G-50 gel filtration device (Amersham Pharmacia).

    Article Title: Dramatic reduction of sequence artefacts from DNA isolated from formalin-fixed cancer biopsies by treatment with uracil-DNA glycosylase
    Article Snippet: .. Treatment of FFPE DNA with uracil-DNA-glycosylase (UDG) To perform the UDG treatment and subsequent PCR/HRM assays without opening of reaction tubes, UDG (0.5 units/reaction, unless specified) and the UDG buffer (New England BioLabs, Ipswich, MA) were directly added to PCR/HRM master mixes. .. The reaction tubes were first incubated at 37°C for 30 minutes for UDG treatment, followed by the standard PCR/HRM assay conditions on the RotorGene Q instrument.

    other:

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity
    Article Snippet: This was then added to 10 μl of master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase, 50 mM Tris (pH 7.4), and 10 mM EDTA, and assayed as described for cell lysates.

    Article Title: Human abasic endonuclease action on multilesion abasic clusters: implications for radiation-induced biological damage
    Article Snippet: The components are annealed, ligated and the uracil residues converted to abasic sites by uracil-DNA glycosylase (UDG).

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient
    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

    Hybridization:

    Article Title: Specificity and Efficiency of the Uracil DNA Glycosylase-Mediated Strand Cleavage Surveyed on Large Sequence Libraries
    Article Snippet: .. Enzyme exposure Microarrays were incubated with 1× UDG Reaction Buffer (20 mM Tris-HCl, 1 mM DTT and 1 mM EDTA pH 8) and 5 units of UDG (E. coli UDG, New England Biolabs, M0280S) in a 300 μl final volume (final enzyme concentration 0.016 U/μl) for either 1 hour or for different time periods ranging from 7 to 120 minutes (7, 15, 30, 60 and 120 min) at 37 °C in a hybridization oven (Boekel Scientific). .. Subsequently, the microarrays were rinsed in deionized water and dried in a microarray centrifuge.

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    New England Biolabs uracil dna glycosylase
    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil <t>DNA</t> <t>glycosylase</t> (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.
    Uracil Dna Glycosylase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs escherichia coli uracil dna glycosylase ung
    N <t>-glycosylase</t> activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from <t>DNA</t> by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.
    Escherichia Coli Uracil Dna Glycosylase Ung, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Journal: PLoS Pathogens

    Article Title: T Cells Contain an RNase-Insensitive Inhibitor of APOBEC3G Deaminase Activity

    doi: 10.1371/journal.ppat.0030135

    Figure Lengend Snippet: A Gel-Based Assay Reveals That Endogenous A3G in T Cell Lines Exhibits Unexpectedly Low Deaminase Activity Compared to Exogenous A3G in Transfected Epithelial-Derived Cell Lines (A) Deaminase activity was measured using an infrared 700 (IR700)–labeled oligo containing the A3G recognition site (CCC) either with or without exogenous recombinant uracil DNA glycosylase (+/- UDG). Oligos were incubated with crude cell lysates containing 10 μg of total cellular protein obtained from H9 cells, H9 cells expressing the HIV genome containing a deletion in Vif (H9-HIV), or from HeLa or 293FT cells transfected with the indicated amounts of A3G plasmid DNA (pA3G). Extent of oligo cleavage (indicating extent of deamination) was determined by gel electrophoresis followed by detection on a LI-COR scanner (top panel), and the percentage of probe cleaved was graphed (second panel). Below, equivalent amounts of cell lysate were analyzed in parallel by western blot (WB) to show A3G protein content. Western blot of calreticulin is shown as a loading control. (B) UDG activity was measured in select lysates from (A) using an IR700-labeled dU-containing oligo in the presence or absence of exogenous UDG (+/- UDG). Results are displayed as in (A) and show that unlike A3G activity shown in (A), UDG activity is similar in all cell lysates analyzed. All assays were performed on RNAse A–treated samples.

    Article Snippet: To each well was added 10 μl of cell lysate in NP40 buffer and 70 μl of a master mix containing 10 pmol Taqman probe, 0.4 units uracil DNA glycosylase (NEB, http://www.neb.com/ ), 50 mM Tris (pH 7.4), and 10 mM EDTA.

    Techniques: Activity Assay, Transfection, Derivative Assay, Labeling, Countercurrent Chromatography, Recombinant, Incubation, Expressing, Plasmid Preparation, Nucleic Acid Electrophoresis, Western Blot

    L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Journal: Nucleic Acids Research

    Article Title: The Leu22Pro tumor-associated variant of DNA polymerase beta is dRP lyase deficient

    doi: 10.1093/nar/gkm1053

    Figure Lengend Snippet: L22P does not support BER.( A ) Reconstituted BER with purified proteins. Lane 1, annealed oligo substrate, treated with uracil DNA glycosylase (UDG); lane 2, UDG-treated substrate incubated with APE1 for 10 min; lane 3, UDG treated substrate incubated with APE1 and T4 DNA ligase for 10 min; lane 4, UDG-treated substrate, incubated with APE1, 400 nM of purified WT pol β and T4 DNA ligase for 10 min; lane 5, UDG-treated substrate, incubated with APE1, 400 nM L22P pol β and T4 DNA ligase for 10 min. ( B ) L22P lacks BER activity even at high concentrations. A reconstituted BER assay was carried with increasing protein concentrations (500–10 000 nM). Lane 1: UDG- and APE1-treated substrate, lanes 2–6: BER assay with WT, lanes 7–11: BER assay with L22P. ( C ) L22P can fill in a single nucleotide gap. A single-nucleotide primer extension assay was carried out in presence of 50 μM dTTP and 10 mM MgCl 2 using 45AG (50 nM) as substrate; 500 nM WT and 5000 nM L22P were used to carry out the reaction at 37°C for 10 min. Reactions were performed in presence (lanes 3 and 6) and absence (lanes 2 and 5) of T4 DNA ligase.

    Article Snippet: Uracil DNA [Glycosylase (UDG) (M0280S), human AP endonuclease I (APE1) (M0282S), terminal transferase (M0252S), T4 PNK (M0201S)] and T4 DNA ligase (M0202S) were purchased from New England Biolabs.

    Techniques: Purification, Incubation, Activity Assay, Primer Extension Assay

    N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Journal: Nucleic Acids Research

    Article Title: Novel repair activities of AlkA (3-methyladenine DNA glycosylase II) and endonuclease VIII for xanthine and oxanine, guanine lesions induced by nitric oxide and nitrous acid

    doi:

    Figure Lengend Snippet: N -glycosylase activity assays of AlkA and Endo VIII for Xan. ( A ) HPLC separation of authentic guanine (G) and Xan. Analysis was performed as described in Materials and Methods. ( B ) HPLC analysis of [ 3 H]Xan released by AlkA. 2.25 pmol of 25XAN/COM25C containing [ 3 H]Xan was incubated with 3 pmol of AlkA at 37°C for 30 min. The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions containing the released 3 H-labeled material were pooled and evaporated. The sample was resuspended in a small volume of water and was subjected to HPLC analysis. HPLC analysis was performed as described in panel (A). ( C ) HPLC analysis of [ 3 H]Xan released by Endo VIII. The experiment was performed in a similar manner using 6 pmol of Endo VIII.

    Article Snippet: Escherichia coli uracil DNA glycosylase (Ung) and exonuclease (Exo) III were purchased from New England Biolabs.

    Techniques: Activity Assay, High Performance Liquid Chromatography, Incubation, Labeling