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  • 91
    New England Biolabs afu udg
    Afu Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 91/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    New England Biolabs udg
    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) <t>Udg</t> activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex <t>DNA</t> containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 635 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs offers antarctic thermolabile udg
    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) <t>Udg</t> activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex <t>DNA</t> containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    Offers Antarctic Thermolabile Udg, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 1x udg buffer
    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) <t>Udg</t> activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex <t>DNA</t> containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    1x Udg Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs 1x udg reaction buffer
    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) <t>Udg</t> activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex <t>DNA</t> containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    1x Udg Reaction Buffer, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    New England Biolabs udg amperase
    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) <t>Udg</t> activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex <t>DNA</t> containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.
    Udg Amperase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

    Journal: Nucleic Acids Research

    Article Title: AP endonuclease independent repair of abasic sites in Schizosaccharomyces pombe

    doi: 10.1093/nar/gkr933

    Figure Lengend Snippet: Tdp1 possesses 3′-α,β-unsaturated aldehyde activity leaving a 3′-P terminus. ( A ) Assay for processing 3′-dRP termini. Ten micrograms total protein extracts from nth1 − (RHP357) and tdp1 − nth1 − (RHP378) cells were analyzed for cleavage of an Nth-nicked ds AP substrate as described in Figure 1 A. The substrate (S; 3′-dRP) and the cleavage product (3′-P) are indicated. Escherichia coli Fpg was used as a positive control for the 3′-P cleavage product. ( B ) Udg activity in the nth1 − and tdp1 − nth1 − extracts. The nth1 − and tdp1 − nth1 − extracts (0.03, 0.06, 0.12, 0.25, 0.5 and 1.0 µg; as in A) were incubated with 10 fmol duplex DNA containing an uracil (opposite C) in reaction buffer for 30 min at 37°C, following incubation with 100 mM NaOH for 10 min at 70°C. The cleavage products were separated on a sequencing gel and visualized by phosphorimaging. The substrate (S) and the cleavage product (P) are indicated. Escherichia coli Udg was used as a positive control.

    Article Snippet: To generate intact AP substrate or a nicked AP substrate with a 3′-dRP terminus, the uracil substrate was pretreated with uracil DNA glycosylase (Udg, NEB) or Udg and Nth (NEB), respectively, for 15 min at 37°C.

    Techniques: Activity Assay, Positive Control, Incubation, Sequencing

    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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

    Techniques: 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.

    Journal: Oncotarget

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

    doi:

    Figure Lengend 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.

    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.

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

    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

    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