a3h catalyzed deaminations (New England Biolabs)
New England Biolabs is a verified supplier 
New England Biolabs manufactures this product 
88
|
Name:
Uracil DNA Glycosylase UDG
Description:
Uracil DNA Glycosylase UDG 5 000 units
Catalog Number:
m0280l
Price:
301
Size:
5 000 units
Category:
DNA Glycosylases
|
Buy from Supplier |
Structured Review
New England Biolabs
a3h catalyzed deaminations
Uracil DNA Glycosylase UDG 5 000 units
https://www.bioz.com/result/a3h catalyzed deaminations/product/New England Biolabs
Average 88 stars, based on 1348 article reviews
Price from $9.99 to $1999.99
Uracil DNA Glycosylase UDG 5 000 units
https://www.bioz.com/result/a3h catalyzed deaminations/product/New England Biolabs
Average 88 stars, based on 1348 article reviews
Price from $9.99 to $1999.99
a3h catalyzed deaminations - by Bioz Stars,
2020-07
88/100 stars
Related Products / Commonly Used Together
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
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.](https://www.ncbi.nlm.nih.gov/pmc/articles/PMC137176/bin/gkf630f4.jpg "... The released 3 H-labeled material was separated from DNA by a Sephadex G-25 column. The column fractions ...")
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:
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
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
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
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
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
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:
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
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 "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
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
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
6) 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
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
7) 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
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
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
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
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
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
8) 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
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
9) 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
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
10) 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
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
11) 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
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
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
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
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
12) 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.](https://storage.googleapis.com/bioz_article_images/PMC4770248/gkw054fig4.jpg "... cleaves 5′ to the AP site, 3.5 nM EndoIII that incises 3′ to the AP site, or ...")
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
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
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
13) 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.](https://storage.googleapis.com/bioz_article_images/PMC5537341/41598_2017_7378_Fig7_HTML.jpg "... incubated in the presence of either E. coli EndoIII that incises 3′ to the AP site, or ...")
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
14) 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
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
15) 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
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
16) 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
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
17) 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
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
18) 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
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
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
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
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
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
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
19) 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
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
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
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
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
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
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
20) 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
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
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
21) 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
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
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
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:
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
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
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
22) 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
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
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
23) 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
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
24) 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
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
25) 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
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
26) Product Images from "Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries"
Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries
Journal: Methods (San Diego, Calif.)
doi: 10.1016/j.ymeth.2012.08.008
Figure Legend Snippet: Removal of non-recombinant clones by digestion with StuI The phagemid modified with sequences of stop codons and StuI restriction enzyme sites was electroporated into CJ236 bacterial cells for generating uracilated ssDNA, which was annealed to mutagenic oligonucleotides (With NNK codons) and converted to dsDNA in vitro . Heteroduplex dsDNA was electroporated into TG1 cells, and the next day, transformed cells were scraped off the surface of agar plates. Phagemid DNA was then extracted from the scraped cells, digested with StuI, and electroporated back into TG1 cells. Bacterial colonies from electroporation of the synthesized dsDNA and the digested DNA were picked and grown up for phage replication. The following day, a phage ELISA was performed with anti-Flag antibody to identify mutant clones for sequencing analysis. Due to the presence of two amber stop codons encoded by the NNK codons, some recombinant clones appeared negative in the ELISA assay. On the microtiter plate of phage ELISA, the red-circled and green-circled wells correspond to positive and negative controls, respectively.
Techniques Used: Recombinant, Clone Assay, Modification, In Vitro, Transformation Assay, Electroporation, Synthesized, Enzyme-linked Immunosorbent Assay, Mutagenesis, Sequencing
Figure Legend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.
Techniques Used: Mutagenesis, Infection, Purification
27) Product Images from "Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications"
Article Title: Combinatorial Domain Hunting: An effective approach for the identification of soluble protein domains adaptable to high-throughput applications
Journal: Protein Science : A Publication of the Protein Society
doi: 10.1110/ps.062082606
Figure Legend Snippet: Fragment library distribution. ( A ) Fragment size distribution is unbiased. SYBR-Safe stained 1% agarose gel of 144 individual clones, generated by shotgun capture of the fragmentation reaction in the ligase-free cloning vector pCR-Blunt-II TOPO (Invitrogen). Clones were pooled in lots of 12 and miniprepped, and captured DNA inserts were released as EcoRI fragments, with 12 vector-derived bases still attached to each end. The distribution of fragment sizes populates the desired range 0.1–1.0 kb. ( B ) The fragment position is random. Coverage plot of 63 randomly selected and sequenced clones (black lines) from the p85α fragment library, ordered according to their 5′-end ( bottom to top ), arrayed against the 2175-bp sequence of human p85α. Apart from clones beginning at the actual 5′-end of the target gene, the start positions of the fragments are evenly distributed across the target gene, which is fully sampled. Although the sample size is far too small for statistical significance, it is fully consistent with random and unbiased fragmentation. ( C ) As B , but with the data sorted by 3′-end position. ( D ) Histogram of fragment size frequency (N). Fragment sizes are binned in intervals of 200 bp. Although the sample size is too small for statistical significance, the distribution is consistent with the expected Poisson distribution for a random fragmentation process.
Techniques Used: Staining, Agarose Gel Electrophoresis, Clone Assay, Generated, Plasmid Preparation, Polymerase Chain Reaction, Derivative Assay, Sequencing
28) Product Images from "Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism"
Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism
Journal: PLoS ONE
doi: 10.1371/journal.pone.0006201
![Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.](https://storage.googleapis.com/bioz_article_images/PMC2705183/pone.0006201.g004.jpg "... and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography ...")
Figure Legend Snippet: Cleavage reactions with transposase mutants and an abasic substrate. Transpososomes were first assembled in the absence of divalent metal ions. The cleavage reaction was initiated by the addition of MgCl 2 at time zero. Aliquots were withdrawn at the indicated times and the reaction halted by the addition of EDTA and SDS. The products were analyzed on a DNA sequencing gel and recorded and quantified by autoradiography on a phosphoimager. The DNA substrates were labeled at both 5′-ends so that all three phosphoryl transfer reactions could be observed in a single experiment. The steps of the cleavage reaction are shown in panel A of the figure below the gel panel. The flanking DNA is to the left and the transposon arm to the right of the half bracket that indicates the location of the transposon end. The positions of the radioactive labels are indicated by the asterisks. Since the reactions are analyzed on denaturing gels, the unlabeled DNA strands, illustrated in grey, are not detected in the autoradiograms. The identity of each band is indicated to the right of the gel in panel A. Bands I and IV each represent a single product of the reaction as indicated. Bands II and III each represent mixtures of more than one co-migrating product and/or substrate as indicated. A B Cleavage reactions of wild type and abasic DNA substrates. The diagonal slashes indicate regions of the gels that have been removed because they contain no relevant information. Unaltered images of the gels are provided in Figure S1 . The identity of the products are indicated next to each band: Band I is the hairpin intermediate; Band II consists the unreacted substrate plus the top strand of the nicked product; Band III contains the bottom strand of the nicked product and the bottom strand of the cleaved transposon end (the resolved hairpin); Band IV contains the top strand of the cleaved flanking DNA that is released upon hairpin formation. In panel B the substrate has an abasic residue at position +2 of the top strand. This was prepared by incorporating a uracil residue at that position by PCR and subsequently treating the substrate with uracil glycosylase. This approach was preferred over one in which the abasic site could have been incorporated during oligonucleotide synthesis. Tn 10 transposon arms are folded during assembly of the transpososome [32] , [39] , [40] , and the DNA fragments required are too long for convenient oligonucleotide synthesis. C-F Quantification of cleavage intermediates. The respective products are plotted as a percentage of the total substrate in the reaction. The amount of each intermediate present at each time point is indicated by the shading within the column. None of the conditions tested severely inhibit the nicking step of the reaction. Sixty minutes is sufficient time for all of the transpososome complexes present at the start of the reaction to achieve the first nick. The height of the column at the 60 minute time point is therefore equivalent to the efficiency of transposome assembly, which varied over a 3-fold range in the reactions presented in this experiment. Bands I and IV (corresponding to the hairpin and cleaved top strand, respectively) are unique and unambiguous products of the reaction and can be quantified directly from the gel by phosphorimager analysis. Other intermediates and/or substrate comigrate and therefore can not be quantified directly. They were calculated as follows: first strand cleavage (first nick) = Band III - (Band IV - Band I). Hairpin resolution = Band IV - Band I. These calculations rely on equal labeling efficiency at either end of the substrate. To determine the efficiency of labeling an aliquot of the substrate was cleaved into two parts by NdeI, and the ratio of label incorporated at each end of the fragment was determined by phosphoimager analysis. This ratio was used to adjust all quantifications described above.
Techniques Used: DNA Sequencing, Autoradiography, Labeling, Polymerase Chain Reaction, Oligonucleotide Synthesis
29) 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:
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
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
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
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
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
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
30) Product Images from "Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification"
Article Title: Highly Sensitive Detection of Uracil-DNA Glycosylase Activity Based on Self-Initiating Multiple Rolling Circle Amplification
Journal: ACS Omega
doi: 10.1021/acsomega.8b03376
Figure Legend Snippet: Inhibition effect of different concentrations of UGI on the UDG activity. The concentration of UDG was 5 × 10 –4 U/mL. Error bars were obtained from three repetitive measurements.
Techniques Used: Inhibition, Activity Assay, Concentration Assay
31) Product Images from "Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair"
Article Title: Deployment of DNA polymerases beta and lambda in single-nucleotide and multinucleotide pathways of mammalian base excision DNA repair
Journal: DNA repair
doi: 10.1016/j.dnarep.2019.02.001
Figure Legend Snippet: BER activity in MEFs. Linear oligonucleotide duplexes containing a single uracil (A) or F lesion (B) were used as substrates in repair assays with WT and POLB −/− . Where indicated (* time points), after the reaction an aliquot was treated with UDG and Ape1 (U substrate) or Ape1 alone (F-substrate) to cleave any unrepaired DNA. After electrophoresis, the resolved products from the UDG/Ape1-treated treated samples were quantified to determine the fraction of DNA repaired. Positions of the substrate (S), intermediates, and repaired product (P) bands are indicated. For each experiment, a representative gel image is shown. The means ± standard errors are plotted (right; n=3).
Techniques Used: Activity Assay, Electrophoresis
Figure Legend Snippet: Analysis of repaired plasmid DNA recovered after transfection. Recovered DNA (A, lane 2) was confirmed by restriction digestion with AatII and BamHI (A, lane 3) . The pGEM3Zf (+) plasmid was loaded as a marker (A, lane1) . Leftmost lane: a 1-kb ladder. The amount of DNA repaired was estimated by treating the samples with UDG and Ape1 (using amounts sufficient to cleave all unrepaired DNA, as determined by experiments with the unrepaired substrate), and the products were resolved on a 0.8% agarose gel containing ethidium bromide. Analysis of the DNA recovered from two experiments is shown (B, lane 1 and 2) .
Techniques Used: Plasmid Preparation, Transfection, Marker, Agarose Gel Electrophoresis
Figure Legend Snippet: Uracil-repair patch size distribution. ( A ) Quantitative analysis of total repair of the circular substrate using MEF extracts. Reaction mixtures containing 0.1 pmol of circular substrate in the standard in vitro ) and 3.5 μg of cell extract were incubated at 37°C for 60 min, followed by incubation at 65°C for 20 min to inactivate enzymes. The total repaired DNA in each sample was determined by adding UDG and Ape1 to ensure cleavage of any unrepaired DNA. Reaction products were resolved on a 0.8% agarose gel and quantified using ImageJ. ( B .
Techniques Used: In Vitro, Incubation, Agarose Gel Electrophoresis
32) Product Images from "Expression and purification of active mouse and human NEIL3 proteins"
Article Title: Expression and purification of active mouse and human NEIL3 proteins
Journal: Protein Expression and Purification
doi: 10.1016/j.pep.2012.04.022
Figure Legend Snippet: DNA glycosylase/lyase activity of MmuNeil3Δ324 and NEIL3Δ324. Single- and double-stranded substrates containing Tg, Sp, or an AP site (25 nM) were incubated with 25 nM active MmuNeil3Δ324 (lane 4) and NEIL3Δ324 (lane 5),
Techniques Used: Activity Assay, Incubation
33) Product Images from "Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries"
Article Title: Improvements to the Kunkel mutagenesis protocol for constructing primary and secondary phage-display libraries
Journal: Methods (San Diego, Calif.)
doi: 10.1016/j.ymeth.2012.08.008
Figure Legend Snippet: Overview of Kunkel mutagenesis Phagemid DNA is electroporated into the CJ236 strain of E. coli . These cells are then infected with M13-K07 helper virus for amplifying phage particles that yield uracilated (U) single-stranded DNA (ssDNA). The ssDNA is annealed to two phosphorylated mutagenic oligonucleotides, which prime the synthesis of heteroduplex, double-stranded DNA (dsDNA) in the presence of T7 DNA polymerase, T4 DNA ligase, and deoxynucleotides. The resulting dsDNA is purified and electroporated into TG1 cells, where the uracilated parental strand is degraded and the mutant strand is preserved and converted into the replicative form of phagemid DNA.
Techniques Used: Mutagenesis, Infection, Purification
34) Product Images from "DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY"
Article Title: DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY
Journal: DNA repair
doi: 10.1016/j.dnarep.2017.01.002
Figure Legend Snippet: Cisplatin cytotoxicity and effect on glycosylase activity (A) Colony survival assay in MDA-MB-231 cells following UNG and SMUG1 knockdown: shControl (open circles), shUNG (closed triangles), shSMUG1 (closed circles) and shUNG + shSMUG1 (open squares). Results are represented as mean ± SE from 3 independent experiments. Cells were transfected with shRNA directed against UNG and SMUG1. (B) Colony survival assay in MDA-MB-231 cells following MBD4 knockdown with shControl (open circles) and shMBD4 (closed triangles). shRNA transfected cells were treated with increasing doses of cisplatin and cytotoxicity. Results are represented as mean ± SE from 3 independent experiments. (C) In vitro glycosylase assay, DNA (5nM) was incubated with either pure enzyme or HeLa extract. Lane 1, undamaged DNA alone.; lane 2, undamaged DNA treated with UDG and APE1 to generate a 19 mer product; lane 3, undamaged DNA substrate treated with UDG, APE1 and 1 unit of UGI; lane 4, undamaged DNA incubated with HeLa extract; lane 5 reactions in which HeLa extract was preincubated with 1 unit of UGI before adding the undamaged DNA substrate. Lanes 6–10 follow the same set up as lanes 1–5, but with ICL DNA substrate. Both undamaged and ICL substrates contain a central uracil and a 3′ Cy3 label. M is a 21-nt marker.
Techniques Used: Activity Assay, Clonogenic Cell Survival Assay, Multiple Displacement Amplification, Transfection, shRNA, In Vitro, Incubation, Marker
35) Product Images from "A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses"
Article Title: A Single Nucleotide Polymorphism in Human APOBEC3C Enhances Restriction of Lentiviruses
Journal: PLoS Pathogens
doi: 10.1371/journal.ppat.1005865
![I188 is a SNP in APOBEC3C and is the conserved residue in the other six human APOBEC3 paralogs. Alignment of the seven APOBEC3 proteins, using both deaminase domains (N and C terminal) of the double-domain APOBEC3 proteins (11 domains total). The residue homologous to APOBEC3C in the other ten deaminase domains is conserved as an isoleucine, whereas APOBEC3C is the only domain with a serine at that position. However, human APOBEC3C is polymorphic at that position, with an isoleucine at an allele frequency of 2.4% globally[ 29 ].](https://storage.googleapis.com/bioz_article_images/PMC5061367/ppat.1005865.g002.jpg "I188 is a SNP in APOBEC3C and is the conserved residue in the other ...")
Figure Legend Snippet: I188 is a SNP in APOBEC3C and is the conserved residue in the other six human APOBEC3 paralogs. Alignment of the seven APOBEC3 proteins, using both deaminase domains (N and C terminal) of the double-domain APOBEC3 proteins (11 domains total). The residue homologous to APOBEC3C in the other ten deaminase domains is conserved as an isoleucine, whereas APOBEC3C is the only domain with a serine at that position. However, human APOBEC3C is polymorphic at that position, with an isoleucine at an allele frequency of 2.4% globally[ 29 ].
Techniques Used:
Figure Legend Snippet: APOBEC3C is targeted by Vif. (A) Restriction of HIV-1 Δ vif by APOBEC3C S188 (A3C S188), APOBEC3C I188 (A3C I188), and full recovery of infectivity by the presence of HIV-1 (LAI strain) and HIV-2 (ROD strains) vif . 0.4μg of APOBEC3 plasmid was used for each condition, and 0.6μg of each provirus was used. Infectivity of each virus is set to 100% for infection with No APOBEC3 (No A3) present. Error bars represent standard deviation of three independent transfections and infections. Black bars indicate no Vif, orange bars indicate HIV-1 (LAI) Vif, and green bars indicate HIV-2 (ROD) Vif (B) Vif degradation of APOBEC3C S188 and I188 was detected by Western blot analysis. 0.4μg of APOBEC3C and 0.6μg of an HIV-1 provirus (either Δ vif , lane labelled as (-)) or containing HIV-1 vif or containing HIV-2 vif , lanes labelled as HIV-1 or HIV-2) were used to transfect 293T cells and lysates were probed for APOBEC3C expression. Tubulin was used as a loading control.
Techniques Used: Infection, Plasmid Preparation, Standard Deviation, Transfection, Western Blot, Expressing
Figure Legend Snippet: APOBEC3C SNP Isoleucine 188 confers increased antiviral activity. (A) Infectivity of HIV-1 Δ vif in the absence of APOBEC3 (No A3), APOBEC3G (A3G), APOBEC3C S188 (A3C S188) and APOBEC3C I188 (A3C I188). 0.3μg of HA-tagged APOBEC3 was expressed in virus-producing cells, and viruses were collected and use for infection. Infectivity in the absence of APOBEC3 is set to 100%. Error bars indicate the standard deviation of triplicate transfections and infections, and this experiment was repeated four times with similar results. Intracellular expression of APOBEC3 was measured by Western Blot using an anti-HA antibody. A section of the blot was probed with an anti-tubulin antibody as a loading control. (B) Dose-response analysis showing restriction of HIVΔ vif in the presence of two-fold dilutions of transfected APOBEC3C S188, or APOBEC3C plasmids I188 along with Western blot analysis of APOBEC3C S188, and APOBEC3C I188 protein expression during virus production. This experiment was performed three times, and a representative result is shown. (C) Infectivity of Simian Immunodeficiency virus SIVagmΔ vif , in the presence of APOBEC3C S188, APOBEC3C I188. Infectivity is set to 100% for infection with No APOBEC3 present. Error bars indicate the standard deviation of three independent experiments.
Techniques Used: Activity Assay, Infection, Standard Deviation, Transfection, Expressing, Western Blot
![Isoleucine 188 changes to serine in some hominoids but was maintained or reverted back to isoleucine for some human populations. (A) A phylogram of catarrhines, along with an alignment of the C-terminus of APOBEC3C. All old world monkey sequences contained isoleucine at 188 (15 sequences total). (B) Allele frequency of Ile SNP in global human populations, as well as Homo neanderthalensis . (C) Allele frequency of Ile 188 in chimpanzees, bonobos, gorillas, and orangutans (n = 79). Sequences were derived from the Great Ape Genome Project[ 39 ].](https://storage.googleapis.com/bioz_article_images/PMC5061367/ppat.1005865.g008.jpg "... along with an alignment of the C-terminus of APOBEC3C. All old world monkey sequences contained isoleucine at ...")
Figure Legend Snippet: Isoleucine 188 changes to serine in some hominoids but was maintained or reverted back to isoleucine for some human populations. (A) A phylogram of catarrhines, along with an alignment of the C-terminus of APOBEC3C. All old world monkey sequences contained isoleucine at 188 (15 sequences total). (B) Allele frequency of Ile SNP in global human populations, as well as Homo neanderthalensis . (C) Allele frequency of Ile 188 in chimpanzees, bonobos, gorillas, and orangutans (n = 79). Sequences were derived from the Great Ape Genome Project[ 39 ].
Techniques Used: Derivative Assay
Figure Legend Snippet: In vitro characterization of APOBEC3C S188 and I188. (Top) The specific activity of APOBEC3C S188 and I188 was determined by incubating the enzyme with a 118 nt ssDNA substrate with an internal fluorescein label (yellow star) and 2 possible sites for cytidine deamination (marked as “C”). Single deaminations of the 5'C and 3'C are detected as the appearance of fluorescently labeled 100 nt and 81 nt fragments, respectively; double deamination of both C residues on the same molecule results in a 63 nt labeled fragment. Substrate usage is quantified for below each lane of the gels. (Bottom) The substrate usage during a 60 min time course was plotted from three independent experiments (bottom left) and used to calculate the specific activity of the enzymes (bottom right).
Techniques Used: In Vitro, Activity Assay, Labeling
Figure Legend Snippet: Purified APOBEC3C I188 forms dimers in solution. (A) Size exclusion chromatography profiles of the APOBEC3C S188 and I188 from a 10mL G200 Superdex column were used to calculate the oligomerization states of the enzymes. Molecular weights were calculated by comparing to a calibration curve (see inset on right). When APOBEC3C S188 and APOBEC3C I188 were loaded onto the column, APOBEC3C S188 was a monomer in solution (apparent MW 21 kD in peak fraction), whereas APOBEC3C I188 could form dimers (apparent MW 42 kD in peak fraction) in addition to monomers (apparent MW 21 kD in peak fraction). Chromatograms were made using the integrated gel band intensities from three independent experiments of each protein fraction after resolution by SDS-PAGE. A representative Western blot of the size exclusion chromatography fractions is shown. (B) A3C S188 or I188 were incubated in the absence or presence of 10 μM bis(sulfosuccinimidyl)suberate (BS3) crosslinker (indicated as -BS3 or +BS3) and then visualized through SDS-PAGE and Western blotting. The Western blot demonstrates that A3C S188 remained monomeric in the presence of crosslinker, whereas A3C I188 was both monomeric and dimeric in the presence of the crosslinker. Molecular weight standards are indicated.
Techniques Used: Purification, Size-exclusion Chromatography, SDS Page, Western Blot, Incubation, Molecular Weight
![APOBEC3C is rapidly evolving in primates. (A) Twenty-two primate APOBEC3C coding sequences were obtained by PCR or from the NCBI sequence database. A phylogeny of APOBEC3C, indicating the branch analysis results of the positive selection tests. The ratio of rate of nonsynonymous changes (dN) and the rate of synonymous changes (dS) that occurred along each branch are shown above each branch. For dN/dS values = ∞, the total number of non-synonymous changes (N) and synonymous changes (S) are shown (N:S) below the branch. The red asterisks mark the branches where the dN/dS is significantly greater than 1 across the entire gene. (B) Maximum likelihood tests for positive selection, with 2lnl values indicating twice the log difference between the model that allows for positive selection (M8) and the model that does not allow for positive selection (M8a), as well as a P-value to indicate whether the M8 model better fits the data than the M8a model. (C) Sites under positive selection in APOBEC3C are shown in a cartoon diagram, comparing these sites to the Vif binding domain and the cytidine deaminase enzymatic domain (CD). Red triangles depict sites with a posterior probability > 0.99 (red triangle). The structure of APOBEC3C[ 31 ] is represented, with the Vif binding domain[ 31 ] shown in blue. Two of the seven positively selected sites (PP > 0.99) overlap with this domain, are shown with arrows. The cytidine deaminase domain is shown in green.](https://storage.googleapis.com/bioz_article_images/PMC5061367/ppat.1005865.g001.jpg "APOBEC3C is rapidly evolving in primates. (A) Twenty-two primate ...")
Figure Legend Snippet: APOBEC3C is rapidly evolving in primates. (A) Twenty-two primate APOBEC3C coding sequences were obtained by PCR or from the NCBI sequence database. A phylogeny of APOBEC3C, indicating the branch analysis results of the positive selection tests. The ratio of rate of nonsynonymous changes (dN) and the rate of synonymous changes (dS) that occurred along each branch are shown above each branch. For dN/dS values = ∞, the total number of non-synonymous changes (N) and synonymous changes (S) are shown (N:S) below the branch. The red asterisks mark the branches where the dN/dS is significantly greater than 1 across the entire gene. (B) Maximum likelihood tests for positive selection, with 2lnl values indicating twice the log difference between the model that allows for positive selection (M8) and the model that does not allow for positive selection (M8a), as well as a P-value to indicate whether the M8 model better fits the data than the M8a model. (C) Sites under positive selection in APOBEC3C are shown in a cartoon diagram, comparing these sites to the Vif binding domain and the cytidine deaminase enzymatic domain (CD). Red triangles depict sites with a posterior probability > 0.99 (red triangle). The structure of APOBEC3C[ 31 ] is represented, with the Vif binding domain[ 31 ] shown in blue. Two of the seven positively selected sites (PP > 0.99) overlap with this domain, are shown with arrows. The cytidine deaminase domain is shown in green.
Techniques Used: Polymerase Chain Reaction, Sequencing, Selection, Binding Assay
Figure Legend Snippet: A synthetic dimer of APOBEC3C has increased antiviral activity. (A) Cartoon schematic showing the sequence of the double-domain APOBEC3C. Restriction of HIV-1 Δ vif by APOBEC3G(A3G), APOBEC3C S188 (A3C S188), APOBEC3C I188 (A3C I188) and the double-domain APOBEC3C (S188-S188). 0.3μg APOBEC3 was used in this assay. Error bars represent standard deviation of four independent transfections and infections. A Western blot for expression of the APOBEC3 proteins is shown, and is representative of three experiments. (B) Packaging of APOBEC3C variants. Left side: Intracellular expression. Right side: Proteins in the pelleted virions. The intracellular blot was probed with antibody to the HA tag and with antibody to tubulin. The virion pellet blot was probed with antibody to the HA tag and with an antibody to p24gag. A background band in the virion blot with the HA antibody is marked with an asterisk. The single domain APOBEC3C proteins are marked with a solid arrow, while the synthetic double domain APOBEC3C is marked with an open arrow. Relative quantitation of the amounts of APOBEC3C is shown under the panels. An HIV provirus is transfected in each condition; Lanes 1, no APOBEC3; lanes 2, APOBEC3C I188; lanes 3, APOBEC3C S188; lanes 4; the double-domain APOBEC3C (S188-S188).
Techniques Used: Activity Assay, Sequencing, Standard Deviation, Transfection, Western Blot, Expressing, Quantitation Assay
36) Product Images from "Removal of oxygen free-radical-induced 5?,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells"
Article Title: Removal of oxygen free-radical-induced 5?,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision-repair pathway in human cells
Journal: Proceedings of the National Academy of Sciences of the United States of America
doi:
Figure Legend Snippet: Inhibition of primer extension by T7 DNA polymerase and mammalian DNA Pol δ by cyPu. Autoradiograph after denaturing 14% PAGE showing the inhibition of primer extension by 5′ R - and 5′ S -cyclodeoxyadenosine. A 32 P end-labeled primer was annealed to cyPu-containing plasmid DNA predigested with Pvu I and Xho I, then extension reactions were performed with either T7 DNA polymerase or purified recombinant human DNA Pol δ with PCNA. Lanes 1–3, T7 DNA polymerase; Lane 1, control plasmid without cyclo-dA; lane 2, 5′ R -cyclo-dA; lane 3, 5′ S -cyclo-dA. Lanes 4–6, Pol δ/PCNA. Lane 4, control without cyclo-dA; lane 5, 5′ R -cyclo-dA; lane 6, 5′ S -cyclo-dA. M, size markers (3′- 32 P-labeled Msp I digest of pBR322).
Techniques Used: Inhibition, Autoradiography, Polyacrylamide Gel Electrophoresis, Labeling, Plasmid Preparation, Purification, Recombinant
Figure Legend Snippet: Search for a DNA glycosylase or lesion reversal enzyme acting on cyclopurine deoxynucleosides. ( a ) DNA glycosylase assay. Lanes 1 and 2, duplex oligonucleotide containing a uracil instead of cyPu at the same position; lanes 3 and 4, control oligonucleotide without any lesion; lanes 5 and 6, 5′ R -cyclodeoxyadenosine-containing oligonucleotide; lanes 7 and 8, 5′ S -cyclodeoxyadenosine-containing oligonucleotide. These oligonucleotides were incubated with (lanes 2, 4, 6, and 8) or without (lanes 1, 3, 5, and 7) HeLa whole cell extract (WCE) and then treated with 1 M piperidine at 90°C. The arrows mark the positions of intact oligonucleotide (I), and product (P) after DNA glycosylase action followed by piperidine treatment. ( b ) Lesion reversal assay. Lanes 1 and 4, control oligonucleotide without any lesion; lanes 2 and 5, 5′ R -cyclodeoxyadenosine-containing oligonucleotide; lanes 3 and 6, 5′ S -cyclodeoxyadenosine-containing oligonucleotide. The duplex oligonucleotides were incubated with (lanes 4–6) or without (lanes 1–3) HeLa cell extract and then digested with Sau 3AI. The arrows mark the positions of intact oligonucleotide (I), and product of Sau 3AI cleavage (S). Only partial restriction enzyme cleavage of the short control oligonucleotide was observed, even at high Sau 3AI concentrations. ( a and b ) The relevant, lesion-containing strand had been 5′- 32 P-labeled, and 14% polyacrylamide gels are shown.
Techniques Used: Incubation, Labeling
37) Product Images from "DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA"
Article Title: DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkh533
Figure Legend Snippet: DNA glycosylase levels and activities in WCEs of wt and ρ 0 cells are similar. ( a ) Western blot showing αOGG1, βOGG1, PCNA and polymerase β levels in WCEs. ( b ) Representative gel showing substrates and products of OGG1 assay. ( c ) Mean OGG1 activity in wt and ρ 0 WCEs. ( d ) Representative gel showing substrates and products of UDG assay. ( e ) Mean UDG activity in wt and ρ 0 WCEs. Values are means ± S.D. of duplicate measurements on each of two samples. Open bars = wt; filled bars = ρ 0 .
Techniques Used: Western Blot, Activity Assay
38) Product Images from "DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY"
Article Title: DIFFERENTIAL ROLE OF BASE EXCISION REPAIR PROTEINS IN MEDIATING CISPLATIN CYTOTOXICITY
Journal: DNA repair
doi: 10.1016/j.dnarep.2017.01.002
Figure Legend Snippet: Cisplatin cytotoxicity and effect on glycosylase activity (A) Colony survival assay in MDA-MB-231 cells following UNG and SMUG1 knockdown: shControl (open circles), shUNG (closed triangles), shSMUG1 (closed circles) and shUNG + shSMUG1 (open squares). Results are represented as mean ± SE from 3 independent experiments. Cells were transfected with shRNA directed against UNG and SMUG1. (B) Colony survival assay in MDA-MB-231 cells following MBD4 knockdown with shControl (open circles) and shMBD4 (closed triangles). shRNA transfected cells were treated with increasing doses of cisplatin and cytotoxicity. Results are represented as mean ± SE from 3 independent experiments. (C) In vitro glycosylase assay, DNA (5nM) was incubated with either pure enzyme or HeLa extract. Lane 1, undamaged DNA alone.; lane 2, undamaged DNA treated with UDG and APE1 to generate a 19 mer product; lane 3, undamaged DNA substrate treated with UDG, APE1 and 1 unit of UGI; lane 4, undamaged DNA incubated with HeLa extract; lane 5 reactions in which HeLa extract was preincubated with 1 unit of UGI before adding the undamaged DNA substrate. Lanes 6–10 follow the same set up as lanes 1–5, but with ICL DNA substrate. Both undamaged and ICL substrates contain a central uracil and a 3′ Cy3 label. M is a 21-nt marker.
Techniques Used: Activity Assay, Clonogenic Cell Survival Assay, Multiple Displacement Amplification, Transfection, shRNA, In Vitro, Incubation, Marker
39) Product Images from "A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution"
Article Title: A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution
Journal: Nucleic Acids Research
doi: 10.1093/nar/gkw994
Figure Legend Snippet: The mouse H19 DMR region of the genome (chr7:142580029-142580514) from the tissues of the BJF1 and JBF1 was analyzed by the EnIGMA method and is shown as Figure 3 . The C57BL/6 genome in this region has seven CpGs, while JF1 has six CpGs because of the C to T single nucleotide polymorphism in the first CpG. A total of 50 sequences were picked up and summarized. ( A ) Sperm of the C57BL/6. ( B ) BJF1 liver and JBF1 liver. ( C ) ES cells derived from a BJF1 embryo.
Techniques Used: Derivative Assay
40) Product Images from "A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition"
Article Title: A Label-Free Fluorescent Assay for the Rapid and Sensitive Detection of Adenosine Deaminase Activity and Inhibition
Journal: Sensors (Basel, Switzerland)
doi: 10.3390/s18082441
Figure Legend Snippet: Relative fluorescence intensity of the reaction systems upon addition of ADA, hoGG I, UDG, RNase H and λexo. Error bars were estimated from three replicate measurements.
Techniques Used: Fluorescence
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 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 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 Polymerase Chain Reaction:Article Title: Base Flipping in Tn10 Transposition: An Active Flip and Capture Mechanism Article Snippet: .. The PCR product was treated with 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 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 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 |