genomic dna Search Results


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  • 99
    Zymo Research genomic dna gdna
    CtIP and its nuclease activity promote ssDNA break formation. ( A ) <t>DNA</t> breaks were quantified in wild-type and CtIP-depleted U2OS cells by alkaline comet assay. Cells were untreated (DMSO) or exposed to 5 µM CPT for 1 hr, with DRB (20 µM) or aphidicolin (2 µg/mL) pretreatment as indicated. Olive moments were calculated by analyzing at least 100 comets for each sample; error bars represent S.E.M. ( B ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type, CtIP-depleted, XPG-depleted, or CtIP/XPG-depleted U2OS cells as in ( A ). ( C ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type or CtIP-depleted U2OS cells complemented with wild-type eGFP-CtIP or nuclease-deficient NA/HA mutant CtIP as in ( A ). ( D ) A schematic representation of Ligation-mediated (LM)-PCR assay (top). Primer extension with a biotinylated primer (in red) from genomic DNA produces a double-stranded DNA end that is isolated with streptavidin and amplified by ligation-mediated PCR (asymmetric duplex and nested primers are presented in blue and green, respectively). LM-PCR assay measuring DNA breaks on the bottom strand of the RPL13A gene (bottom). DNA single-strand breaks were measured by LM-PCR at the endogenous RPL13A locus in wild-type or CtIP-depleted cells; n = 6, error bars represent S.E.M. * and **** denote p
    Genomic Dna Gdna, supplied by Zymo Research, used in various techniques. Bioz Stars score: 99/100, based on 281 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    New England Biolabs genomic dna
    Isolation of Δ recV mutants. A. PCR confirmation of recV gene disruption using ClosTron. Primers NF1215+NF1356 flanking the ClosTron target site in recV give a 665 bp product in 630Δ erm (WT). After mutagenesis, four erythromycin resistant colonies were tested and a 2839 bp product was amplified, indicative of insertion of the group II intron into recV . These clones were designated Δ recV 1-4. B. Diagrammatic representation of the <t>cwpV</t> <t>DNA</t> switch orientation-specific PCR assay. Primers NF823+NF826 amplify a product from OFF, whilst primers NF823+NF825 amplify a product from ON. C. Analysis of the orientation of the cwpV DNA switch in C. difficile clones by orientation PCR. (i), products for the ON and OFF orientations are amplified from WT. (ii), all four isolated Δ recV mutants contain only the OFF orientation of the cwpV DNA switch, therefore these strains are referred to as 630Δ recV OFF. (iii), complementation of 630Δ recV OFF using a plasmid encoding recV (pRecV+) reconstituted the switching phenotype. (iv), a recV Y176F mutant was unable to complement Δ recV OFF confirming the key role of this tyrosine residue in RecV activity. (v), Δ recV (pRecV+) was serially sub-cultured without thiamphenicol selection to enable curing of the pRecV+ plasmid. Four thiamphenicol sensitive colonies were isolated from which only the ON orientation of the cwpV DNA switch could be amplified. These strains were therefore designated Δ recV ON. D. Colony morphologies of WT and Δ recV OFF are similar, however Δ recV ON exhibit a smaller, smoother-edged colony morphology.
    Genomic Dna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 10746 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore genomic dna
    <t>PCR</t> of genomic <t>DNA</t> and RT-PCR of RNA extracted from larvae and juvenile and mature male and female M. rosenbergii individuals. Genomic-based PCR indicates maleness, where no female specific marker was amplified. RT-PCR was applied for detection of Mr-IAG expression by using Mr-IAG specific primers and β -actin as positive control. (a) For adult male and female individuals, there is a correlation between the absence of the female specific marker and Mr-IAG expression. On the basis of this result, larvae and juveniles were defined as males according to the absence of the genomic sex marker (marked by arrows in Figure 2(b) ). (b) None of the individuals sampled at larval stages (zoeae 3, 6, 9, and 11) expressed Mr-IAG regardless of the presence or absence of the genomic sex marker. At PL 20 and onwards, all individuals identified as males according to absence of the female specific marker also expressed Mr-IAG .
    Genomic Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 10774 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Qiagen genomic dna gdna
    TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic <t>DNA</t> <t>(gDNA)</t> from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P
    Genomic Dna Gdna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 3368 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher genomic dna gdna
    Measurement of P. falciparum rRNA and <t>DNA</t> associated with HUVECs by SYBR Green quantitative PCR. P. falciparum infected RBCs were added to HUVEC cell cultures for the indicated times, as described in Materials and Methods. Following these incubations, RNA and genomic DNA were purified from HUVECS. A. P. falciparum SSU rRNA amounts from HUVECs as measured by reverse-transcription qPCR. No PCR products were detected in RNA samples not treated with reverse-transcriptase (data not shown). Error bars represent standard deviation from three independent replicates. B. The SSU rRNA gene was amplified from P. falciparum genomic DNA <t>(gDNA)</t> from HUVECs and measured by qPCR. P. falciparum RNA and DNA amounts were normalized to human GAPDH RNA and DNA amounts, respectively. As a control, HUVECs were incubated with uninfected RBCs. No P. falciparum -specific PCR products were detected (data not shown). All experiments were done in triplicate.
    Genomic Dna Gdna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1160 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ATCC genomic dna gdna
    For a given <t>DNA</t> input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line <t>gDNA</t> serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.
    Genomic Dna Gdna, supplied by ATCC, used in various techniques. Bioz Stars score: 98/100, based on 57 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    90
    TaKaRa genomic dna gdna eraser
    For a given <t>DNA</t> input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line <t>gDNA</t> serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.
    Genomic Dna Gdna Eraser, supplied by TaKaRa, used in various techniques. Bioz Stars score: 90/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    PerkinElmer genomic dna gdna
    For a given <t>DNA</t> input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line <t>gDNA</t> serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.
    Genomic Dna Gdna, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 97/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genomic dna gdna extraction
    For a given <t>DNA</t> input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line <t>gDNA</t> serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.
    Genomic Dna Gdna Extraction, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genomic dna genomic dna gdna
    Characterization of Disease-Specific iPSCs from Csf2rb −/− Mice (A and B) ESC-like morphology in bright-field images (A) and positive alkaline phosphatase staining of miPAP1 iPSCs (B). Scale bar, 200 μm. (C) NANOG, OCT4, and SOX2 expression by immunofluorescence staining (C; scale bar, 50 μm) as well as by (D) qRT-PCR using murine-specific primers (independent experiments, n = 3, mean ± SD). ns, not significant compared with ESCs, two-way ANOVA. (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker. (F) Representative pictures of miPAP1-derived teratomas containing tissues of all three embryonic germ layers. Scale bar, 50 μm for ectoderm and endoderm; 100 μm for mesoderm. (G) Scheme and gel electrophoresis of PCR on genomic <t>DNA</t> for genotyping miPAP clones.
    Genomic Dna Genomic Dna Gdna, supplied by Millipore, used in various techniques. Bioz Stars score: 93/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Toyobo genomic dna gdna remover kit
    Characterization of Disease-Specific iPSCs from Csf2rb −/− Mice (A and B) ESC-like morphology in bright-field images (A) and positive alkaline phosphatase staining of miPAP1 iPSCs (B). Scale bar, 200 μm. (C) NANOG, OCT4, and SOX2 expression by immunofluorescence staining (C; scale bar, 50 μm) as well as by (D) qRT-PCR using murine-specific primers (independent experiments, n = 3, mean ± SD). ns, not significant compared with ESCs, two-way ANOVA. (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker. (F) Representative pictures of miPAP1-derived teratomas containing tissues of all three embryonic germ layers. Scale bar, 50 μm for ectoderm and endoderm; 100 μm for mesoderm. (G) Scheme and gel electrophoresis of PCR on genomic <t>DNA</t> for genotyping miPAP clones.
    Genomic Dna Gdna Remover Kit, supplied by Toyobo, used in various techniques. Bioz Stars score: 93/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche human genomic dna gdna
    Agarose gel of PCR product and bis-treated PCR product following (A) 15 minutes and (B) 40 minutes of electrophoresis. Size of each PCR product is as indicated. <t>gDNA–untreated</t> genomic <t>DNA;</t> bis gDNA–bis-treated genomic DNA.
    Human Genomic Dna Gdna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega gdna
    c x43 null zebrafish have reduced motile cilia in ECs. a Electropherograms of the target sequences of the cx43 <t>gDNA</t> in WT and cx43 −/− zebrafish. Arrow indicates the deletion of the T nucleotide. b Zebrafish ( n = 71) at 2 months post-fertilization (mpf) from mating of cx43 +/− zebrafish were genotyped for cx43 . c Images of 3 mpf zebrafish with the indicated cx43 genotype. d WT or cx43 −/− embryos at 2 dpf were immunostained with anti-acetylated-α-tubulin antibody, imaged with a confocal microscope and genotyped for cx43 . Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. e Larvae at 8 dpf from mating of cx43 +/− zebrafish were cut into cranial and caudal halves. The cranial half was used for cx43 <t>genotyping,</t> and the caudal half was coronally sectioned at a thickness of 14 μm and then processed for IF staining with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 30 μm. f Quantification of the number of cilia per frame in embryos in e . Mean ± SD. **** P
    Gdna, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 2004 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore genomic dna gdna
    Alignment of vertebrate six6 genes reveals an evolutionary conserved region 3′ of the six6 coding region. PIP analysis was used to compare vertebrate genomic <t>DNA</t> sequences from mammalian (human, mouse), marsupial (tazmanian devil), aves (chicken), reptile (green sea turtle), lobe-finned fish (coelacanth) and fish (medaka). Golden boxes show evolutionarily conserved regions (R); purple boxes indicate exons; white box, intron; dark and light blue lines, 5′ and 3′ flanking genomic regions, respectively. The line at the bottom of the PIP analysis indicates distance in kilobases from the translation start site. The percent identity of X. tropicalis R3 to each species is included. Exon 1 contains 146 bp of 5′ untranslated region, while exon 2 contains 964 bp of 3′ untranslated region, both depicted in light purple.
    Genomic Dna Gdna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 531 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    96
    Thermo Fisher easy dna genomic dna gdna purification kit
    Comparison of <t>DNA</t> extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA <t>gDNA</t> purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).
    Easy Dna Genomic Dna Gdna Purification Kit, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 96/100, based on 8 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Illumina Inc genomic dna gdna
    Sequencing results for Human genomic <t>DNA.</t> Libraries are prepared using 50 ng of input DNA. Similar enzyme concentrations of Tn5-059 and standard Tn5 are used and 2x151 bp sequencing run is performed on a HiSeqX. a Uniformity of Coverage. Sequencing results are down sampled to 20× coverage. Tn5-059 shows improved uniformity over standard Tn5 in NexteraV2 kit. b Normalized GC plot. Grey bar shows schematic GC composition of Human genome. Standard Tn5 has a clear bias towards GC rich regions while undercovers AT rich regions. c AT/GC dropout. Tn5-059 improves AT dropout while adding little on GC dropout
    Genomic Dna Gdna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 136 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Otogenetics Corporation genomic dna gdna
    Sequencing results for Human genomic <t>DNA.</t> Libraries are prepared using 50 ng of input DNA. Similar enzyme concentrations of Tn5-059 and standard Tn5 are used and 2x151 bp sequencing run is performed on a HiSeqX. a Uniformity of Coverage. Sequencing results are down sampled to 20× coverage. Tn5-059 shows improved uniformity over standard Tn5 in NexteraV2 kit. b Normalized GC plot. Grey bar shows schematic GC composition of Human genome. Standard Tn5 has a clear bias towards GC rich regions while undercovers AT rich regions. c AT/GC dropout. Tn5-059 improves AT dropout while adding little on GC dropout
    Genomic Dna Gdna, supplied by Otogenetics Corporation, used in various techniques. Bioz Stars score: 92/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    BioChain Institute genomic dna gdna
    Sequencing results for Human genomic <t>DNA.</t> Libraries are prepared using 50 ng of input DNA. Similar enzyme concentrations of Tn5-059 and standard Tn5 are used and 2x151 bp sequencing run is performed on a HiSeqX. a Uniformity of Coverage. Sequencing results are down sampled to 20× coverage. Tn5-059 shows improved uniformity over standard Tn5 in NexteraV2 kit. b Normalized GC plot. Grey bar shows schematic GC composition of Human genome. Standard Tn5 has a clear bias towards GC rich regions while undercovers AT rich regions. c AT/GC dropout. Tn5-059 improves AT dropout while adding little on GC dropout
    Genomic Dna Gdna, supplied by BioChain Institute, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    CtIP and its nuclease activity promote ssDNA break formation. ( A ) DNA breaks were quantified in wild-type and CtIP-depleted U2OS cells by alkaline comet assay. Cells were untreated (DMSO) or exposed to 5 µM CPT for 1 hr, with DRB (20 µM) or aphidicolin (2 µg/mL) pretreatment as indicated. Olive moments were calculated by analyzing at least 100 comets for each sample; error bars represent S.E.M. ( B ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type, CtIP-depleted, XPG-depleted, or CtIP/XPG-depleted U2OS cells as in ( A ). ( C ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type or CtIP-depleted U2OS cells complemented with wild-type eGFP-CtIP or nuclease-deficient NA/HA mutant CtIP as in ( A ). ( D ) A schematic representation of Ligation-mediated (LM)-PCR assay (top). Primer extension with a biotinylated primer (in red) from genomic DNA produces a double-stranded DNA end that is isolated with streptavidin and amplified by ligation-mediated PCR (asymmetric duplex and nested primers are presented in blue and green, respectively). LM-PCR assay measuring DNA breaks on the bottom strand of the RPL13A gene (bottom). DNA single-strand breaks were measured by LM-PCR at the endogenous RPL13A locus in wild-type or CtIP-depleted cells; n = 6, error bars represent S.E.M. * and **** denote p

    Journal: eLife

    Article Title: Sae2/CtIP prevents R-loop accumulation in eukaryotic cells

    doi: 10.7554/eLife.42733

    Figure Lengend Snippet: CtIP and its nuclease activity promote ssDNA break formation. ( A ) DNA breaks were quantified in wild-type and CtIP-depleted U2OS cells by alkaline comet assay. Cells were untreated (DMSO) or exposed to 5 µM CPT for 1 hr, with DRB (20 µM) or aphidicolin (2 µg/mL) pretreatment as indicated. Olive moments were calculated by analyzing at least 100 comets for each sample; error bars represent S.E.M. ( B ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type, CtIP-depleted, XPG-depleted, or CtIP/XPG-depleted U2OS cells as in ( A ). ( C ) Quantification of ssDNA breaks by alkaline comet assay was performed in wild-type or CtIP-depleted U2OS cells complemented with wild-type eGFP-CtIP or nuclease-deficient NA/HA mutant CtIP as in ( A ). ( D ) A schematic representation of Ligation-mediated (LM)-PCR assay (top). Primer extension with a biotinylated primer (in red) from genomic DNA produces a double-stranded DNA end that is isolated with streptavidin and amplified by ligation-mediated PCR (asymmetric duplex and nested primers are presented in blue and green, respectively). LM-PCR assay measuring DNA breaks on the bottom strand of the RPL13A gene (bottom). DNA single-strand breaks were measured by LM-PCR at the endogenous RPL13A locus in wild-type or CtIP-depleted cells; n = 6, error bars represent S.E.M. * and **** denote p

    Article Snippet: Genomic DNA (gDNA) was purified using genomic DNA preparation kit (Zymo Research Quick-gDNA MiniPrep - Capped column, Genesee Scientific, 11-317AC) and gDNA concentration was determined using Nanodrop.

    Techniques: Activity Assay, Alkaline Single Cell Gel Electrophoresis, Cycling Probe Technology, Mutagenesis, Ligation, Polymerase Chain Reaction, Isolation, Amplification

    Isolation of Δ recV mutants. A. PCR confirmation of recV gene disruption using ClosTron. Primers NF1215+NF1356 flanking the ClosTron target site in recV give a 665 bp product in 630Δ erm (WT). After mutagenesis, four erythromycin resistant colonies were tested and a 2839 bp product was amplified, indicative of insertion of the group II intron into recV . These clones were designated Δ recV 1-4. B. Diagrammatic representation of the cwpV DNA switch orientation-specific PCR assay. Primers NF823+NF826 amplify a product from OFF, whilst primers NF823+NF825 amplify a product from ON. C. Analysis of the orientation of the cwpV DNA switch in C. difficile clones by orientation PCR. (i), products for the ON and OFF orientations are amplified from WT. (ii), all four isolated Δ recV mutants contain only the OFF orientation of the cwpV DNA switch, therefore these strains are referred to as 630Δ recV OFF. (iii), complementation of 630Δ recV OFF using a plasmid encoding recV (pRecV+) reconstituted the switching phenotype. (iv), a recV Y176F mutant was unable to complement Δ recV OFF confirming the key role of this tyrosine residue in RecV activity. (v), Δ recV (pRecV+) was serially sub-cultured without thiamphenicol selection to enable curing of the pRecV+ plasmid. Four thiamphenicol sensitive colonies were isolated from which only the ON orientation of the cwpV DNA switch could be amplified. These strains were therefore designated Δ recV ON. D. Colony morphologies of WT and Δ recV OFF are similar, however Δ recV ON exhibit a smaller, smoother-edged colony morphology.

    Journal: PLoS Pathogens

    Article Title: The Clostridium difficile Cell Wall Protein CwpV is Antigenically Variable between Strains, but Exhibits Conserved Aggregation-Promoting Function

    doi: 10.1371/journal.ppat.1002024

    Figure Lengend Snippet: Isolation of Δ recV mutants. A. PCR confirmation of recV gene disruption using ClosTron. Primers NF1215+NF1356 flanking the ClosTron target site in recV give a 665 bp product in 630Δ erm (WT). After mutagenesis, four erythromycin resistant colonies were tested and a 2839 bp product was amplified, indicative of insertion of the group II intron into recV . These clones were designated Δ recV 1-4. B. Diagrammatic representation of the cwpV DNA switch orientation-specific PCR assay. Primers NF823+NF826 amplify a product from OFF, whilst primers NF823+NF825 amplify a product from ON. C. Analysis of the orientation of the cwpV DNA switch in C. difficile clones by orientation PCR. (i), products for the ON and OFF orientations are amplified from WT. (ii), all four isolated Δ recV mutants contain only the OFF orientation of the cwpV DNA switch, therefore these strains are referred to as 630Δ recV OFF. (iii), complementation of 630Δ recV OFF using a plasmid encoding recV (pRecV+) reconstituted the switching phenotype. (iv), a recV Y176F mutant was unable to complement Δ recV OFF confirming the key role of this tyrosine residue in RecV activity. (v), Δ recV (pRecV+) was serially sub-cultured without thiamphenicol selection to enable curing of the pRecV+ plasmid. Four thiamphenicol sensitive colonies were isolated from which only the ON orientation of the cwpV DNA switch could be amplified. These strains were therefore designated Δ recV ON. D. Colony morphologies of WT and Δ recV OFF are similar, however Δ recV ON exhibit a smaller, smoother-edged colony morphology.

    Article Snippet: To clone the cwpV genes from strains of unknown genomic sequence, genomic DNA was digested overnight at 37°C with Ase I (NEB) and then ligated overnight at 16°C using T4 ligase (NEB).

    Techniques: Isolation, Polymerase Chain Reaction, Mutagenesis, Amplification, Clone Assay, Plasmid Preparation, Activity Assay, Cell Culture, Selection

    Activity of NgTet1 on various DNA substrates a–c , The time courses (lanes 5–13) of the reactions using 32-bp DNA substrates containing 5mC (panel a ), 5hmC (panel b ) or 5caC (panel c ). Lanes 1–4: antibody sensitivity against 10 pmol of control oligonucleotides and 2 fold serial dilutions. Lanes 5–13: the rate of conversion appears to be the fastest for the reaction of 5mC to 5hmC, and decreases with each subsequent reaction: 5mC to 5hmC > 5hmC to 5fC > 5fC to 5caC. d , Activities of NgTet1 (20 µM) on genomic DNA (gDNA) of Hela cells (2.5 µg). After 1 h reaction, 87% of the products are 5caC in gDNA with the remaining being 5fC and 5hmC. The percentages were estimated from integration of the peaks in LC-MS traces. The mean and standard deviation (±s.e.m.) were estimated from three repeated experiments. e , Human thymine DNA glycosylase (TDG) excises 5fC and 5caC (but not 5mC and 5hmC) when paired with a guanine in a CpG sequence (lanes 1–4) (He et al., 2011; Maiti and Drohat, 2011; Hashimoto et al., 2012). After NgTet1 reactions with DNA substrates containing 5mC, 5hmC or 5fC, in the presence of αKG, the product DNA containing 5fC and 5caC becomes a substrate for TDG (lanes 6, 8 and 10), but not with NOG (lanes 5 and 7), again demonstrating the production of 5fC and 5caC by NgTet1. f , Activities of NgTet1 on 56-bp double-stranded (ds) DNA-2 with methylation on both strands (M/M) or single strand (hemi-methylated either on top M/C or bottom C/M strand) or single-stranded (ss) DNA (Reaction time 1 h and ±s.e.m. estimated from three repeats). We note that an in vitro activity of the mouse Tet1 catalytic domain on single-stranded DNA has also been observed (Zhang et al., 2012). g , LC-MS traces of a sample reaction mix on the hemi-methylated 5mCpG dsDNA-1 (top panel), reaction control with no enzyme (middle panel), and the standard deoxyribonucleoside mix (bottom panel). Arrows indicate peaks of 5mC, 5hmC, 5fC and 5caC. Identities of the peaks are confirmed by comparing the retention time with the standard as well as by mass spectrometry. Hashimoto, H., Hong, S., Bhagwat, A. S., Zhang, X. Cheng, X. Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation. Nucleic Acids Res 40 , 10203–10214 (2012). He, Y. F. et al . Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333 , 1303–1307 (2011). Maiti, A. Drohat, A. C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem 286 , 35334–35338 (2011). Zhang, L., Yu, M. He, C. Mouse Tet1 protein can oxidize 5mC to 5hmC and 5caC on single-stranded DNA. Acta Chimica Sinica 70 , 2123–2126 (2012).

    Journal: Nature

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA

    doi: 10.1038/nature12905

    Figure Lengend Snippet: Activity of NgTet1 on various DNA substrates a–c , The time courses (lanes 5–13) of the reactions using 32-bp DNA substrates containing 5mC (panel a ), 5hmC (panel b ) or 5caC (panel c ). Lanes 1–4: antibody sensitivity against 10 pmol of control oligonucleotides and 2 fold serial dilutions. Lanes 5–13: the rate of conversion appears to be the fastest for the reaction of 5mC to 5hmC, and decreases with each subsequent reaction: 5mC to 5hmC > 5hmC to 5fC > 5fC to 5caC. d , Activities of NgTet1 (20 µM) on genomic DNA (gDNA) of Hela cells (2.5 µg). After 1 h reaction, 87% of the products are 5caC in gDNA with the remaining being 5fC and 5hmC. The percentages were estimated from integration of the peaks in LC-MS traces. The mean and standard deviation (±s.e.m.) were estimated from three repeated experiments. e , Human thymine DNA glycosylase (TDG) excises 5fC and 5caC (but not 5mC and 5hmC) when paired with a guanine in a CpG sequence (lanes 1–4) (He et al., 2011; Maiti and Drohat, 2011; Hashimoto et al., 2012). After NgTet1 reactions with DNA substrates containing 5mC, 5hmC or 5fC, in the presence of αKG, the product DNA containing 5fC and 5caC becomes a substrate for TDG (lanes 6, 8 and 10), but not with NOG (lanes 5 and 7), again demonstrating the production of 5fC and 5caC by NgTet1. f , Activities of NgTet1 on 56-bp double-stranded (ds) DNA-2 with methylation on both strands (M/M) or single strand (hemi-methylated either on top M/C or bottom C/M strand) or single-stranded (ss) DNA (Reaction time 1 h and ±s.e.m. estimated from three repeats). We note that an in vitro activity of the mouse Tet1 catalytic domain on single-stranded DNA has also been observed (Zhang et al., 2012). g , LC-MS traces of a sample reaction mix on the hemi-methylated 5mCpG dsDNA-1 (top panel), reaction control with no enzyme (middle panel), and the standard deoxyribonucleoside mix (bottom panel). Arrows indicate peaks of 5mC, 5hmC, 5fC and 5caC. Identities of the peaks are confirmed by comparing the retention time with the standard as well as by mass spectrometry. Hashimoto, H., Hong, S., Bhagwat, A. S., Zhang, X. Cheng, X. Excision of 5-hydroxymethyluracil and 5-carboxylcytosine by the thymine DNA glycosylase domain: its structural basis and implications for active DNA demethylation. Nucleic Acids Res 40 , 10203–10214 (2012). He, Y. F. et al . Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA. Science 333 , 1303–1307 (2011). Maiti, A. Drohat, A. C. Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem 286 , 35334–35338 (2011). Zhang, L., Yu, M. He, C. Mouse Tet1 protein can oxidize 5mC to 5hmC and 5caC on single-stranded DNA. Acta Chimica Sinica 70 , 2123–2126 (2012).

    Article Snippet: For quantitative analyses of various 5mC oxidative species, either the 56-bp hemi-methylated dsDNA-1 ( ) or genomic DNA (gDNA) of Hela cells (NEB #N4006S) ( ) were used as substrates.

    Techniques: Activity Assay, Liquid Chromatography with Mass Spectroscopy, Standard Deviation, Sequencing, Methylation, In Vitro, Mass Spectrometry

    Pairwise comparison of NgTet1 and mammalian Tet1 a , Schematic representation of hTet1 C-terminal catalytic domain. b , Sequence alignment of NgTet1, hTet1 and mTet1. Labels above the sequences indicate that i for intra-molecular polar interaction; s for exposed surface residue; h for hydrophobic core; t for structural turn; α for αKG binding; m for metal ion coordination; P for DNA phosphate interaction; g for DNA base interaction with the orphaned guanine; G for DNA base interaction with the 3’ guanine to 5mC; C for 5mC interaction; a for active site residues (A212 and V293) near the methyl group of 5mC. c , Structure of NgTet1 with arrows indicating the two large insertions of mammalian Tet1. Highlighted is the charge-charge interaction between invariant K86 and E108. d , A kinked helix α4, owing to P172 (conserved among NgTet1, human and mouse Tet1, Tet2 and Tet3) located in the middle. f , Antibody detection of 5hmC in genomic DNA of HEK293T cells (top panel) expressing Flag tagged mouse Tet1 catalytic domain or its internal deletions (bottom panel). Top panel: Lane 1 is the 32-bp oligonucleotide containing a single 5hmC (20 pmol and 2 fold serial dilutions) and lanes 2–7 are the genomic DNA (500 ng and 2 fold serial dilutions). Bottom panel: Lane 1 is the molecular weight marker and Lanes 2 and 7 are the whole cell lysates with approximately equal amount of protein.

    Journal: Nature

    Article Title: Structure of a Naegleria Tet-like dioxygenase in complex with 5-methylcytosine DNA

    doi: 10.1038/nature12905

    Figure Lengend Snippet: Pairwise comparison of NgTet1 and mammalian Tet1 a , Schematic representation of hTet1 C-terminal catalytic domain. b , Sequence alignment of NgTet1, hTet1 and mTet1. Labels above the sequences indicate that i for intra-molecular polar interaction; s for exposed surface residue; h for hydrophobic core; t for structural turn; α for αKG binding; m for metal ion coordination; P for DNA phosphate interaction; g for DNA base interaction with the orphaned guanine; G for DNA base interaction with the 3’ guanine to 5mC; C for 5mC interaction; a for active site residues (A212 and V293) near the methyl group of 5mC. c , Structure of NgTet1 with arrows indicating the two large insertions of mammalian Tet1. Highlighted is the charge-charge interaction between invariant K86 and E108. d , A kinked helix α4, owing to P172 (conserved among NgTet1, human and mouse Tet1, Tet2 and Tet3) located in the middle. f , Antibody detection of 5hmC in genomic DNA of HEK293T cells (top panel) expressing Flag tagged mouse Tet1 catalytic domain or its internal deletions (bottom panel). Top panel: Lane 1 is the 32-bp oligonucleotide containing a single 5hmC (20 pmol and 2 fold serial dilutions) and lanes 2–7 are the genomic DNA (500 ng and 2 fold serial dilutions). Bottom panel: Lane 1 is the molecular weight marker and Lanes 2 and 7 are the whole cell lysates with approximately equal amount of protein.

    Article Snippet: For quantitative analyses of various 5mC oxidative species, either the 56-bp hemi-methylated dsDNA-1 ( ) or genomic DNA (gDNA) of Hela cells (NEB #N4006S) ( ) were used as substrates.

    Techniques: Sequencing, Binding Assay, Expressing, Molecular Weight, Marker

    PCR of genomic DNA and RT-PCR of RNA extracted from larvae and juvenile and mature male and female M. rosenbergii individuals. Genomic-based PCR indicates maleness, where no female specific marker was amplified. RT-PCR was applied for detection of Mr-IAG expression by using Mr-IAG specific primers and β -actin as positive control. (a) For adult male and female individuals, there is a correlation between the absence of the female specific marker and Mr-IAG expression. On the basis of this result, larvae and juveniles were defined as males according to the absence of the genomic sex marker (marked by arrows in Figure 2(b) ). (b) None of the individuals sampled at larval stages (zoeae 3, 6, 9, and 11) expressed Mr-IAG regardless of the presence or absence of the genomic sex marker. At PL 20 and onwards, all individuals identified as males according to absence of the female specific marker also expressed Mr-IAG .

    Journal: ISRN Endocrinology

    Article Title: Expression of an Androgenic Gland-Specific Insulin-Like Peptide during the Course of Prawn Sexual and Morphotypic Differentiation

    doi: 10.5402/2011/476283

    Figure Lengend Snippet: PCR of genomic DNA and RT-PCR of RNA extracted from larvae and juvenile and mature male and female M. rosenbergii individuals. Genomic-based PCR indicates maleness, where no female specific marker was amplified. RT-PCR was applied for detection of Mr-IAG expression by using Mr-IAG specific primers and β -actin as positive control. (a) For adult male and female individuals, there is a correlation between the absence of the female specific marker and Mr-IAG expression. On the basis of this result, larvae and juveniles were defined as males according to the absence of the genomic sex marker (marked by arrows in Figure 2(b) ). (b) None of the individuals sampled at larval stages (zoeae 3, 6, 9, and 11) expressed Mr-IAG regardless of the presence or absence of the genomic sex marker. At PL 20 and onwards, all individuals identified as males according to absence of the female specific marker also expressed Mr-IAG .

    Article Snippet: PCR of Female Specific and Positive Control Sequences PCR was performed with 100 ng genomic DNA, 1 μ M forward primer and 1 μ M reverse primer (primer sequences are available upon request), 12.5 μ L Ready Mix REDTaq (Sigma) and water to a final volume of 25 μ L. The PCR conditions were: 35 cycles of 30 s at 94°C, 30 s at 56°C, and 60 s at 72°C.

    Techniques: Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Expressing, Positive Control

    TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Journal: Disease Models & Mechanisms

    Article Title: Decreased N-TAF1 expression in X-linked dystonia-parkinsonism patient-specific neural stem cells

    doi: 10.1242/dmm.022590

    Figure Lengend Snippet: TAF1 and MTS transcript expression levels in fibroblasts. (A) Genomic DNA (gDNA) from all individuals was PCR amplified with primers flanking the insertion site to confirm the presence of the SVA. Lane 1: 1 kb DNA ladder. Lane 2: no template control (H 2 O). Lanes 3-7: XDP lines (left to right) 32517, 33109, 33363, 33808, 34363. Lanes 8-12: Control lines (left to right) 32643, 33113, 33114, 33809, 33362. The predicted 3229 bp SVA product was present in all XDP samples (upper arrow), whereas controls had a product of ∼599 bp (lower arrow), a difference consistent with the size of the SVA. (B) Quantitative expression analysis of TAF1 transcript fragments in XDP vs control fibroblasts ( n =5 each) based on comparative Ct method. Expression levels were normalized to the mean of housekeeping genes HPRT1 and TFRC . Levels of transcript fragments amplified by primer sets TA02-334, TAF1-3′, TA14-385N and TAF1-3′N were significantly lower in XDP vs control cells, whereas expression of the transcript amplified by TA09-693 was significantly increased in XDP vs control samples. The neural-specific transcript, N-TAF1, amplified by primer set TA14-391, as well as all six transcripts incorporating MTS sequences, were not detected in fibroblasts. Data represent mean fold changes±standard errors, analyzed by Student's t -test. * P

    Article Snippet: Genotyping Genomic DNA (gDNA) was isolated from cells using DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA), as recommended.

    Techniques: Expressing, Polymerase Chain Reaction, Amplification

    Measurement of P. falciparum rRNA and DNA associated with HUVECs by SYBR Green quantitative PCR. P. falciparum infected RBCs were added to HUVEC cell cultures for the indicated times, as described in Materials and Methods. Following these incubations, RNA and genomic DNA were purified from HUVECS. A. P. falciparum SSU rRNA amounts from HUVECs as measured by reverse-transcription qPCR. No PCR products were detected in RNA samples not treated with reverse-transcriptase (data not shown). Error bars represent standard deviation from three independent replicates. B. The SSU rRNA gene was amplified from P. falciparum genomic DNA (gDNA) from HUVECs and measured by qPCR. P. falciparum RNA and DNA amounts were normalized to human GAPDH RNA and DNA amounts, respectively. As a control, HUVECs were incubated with uninfected RBCs. No P. falciparum -specific PCR products were detected (data not shown). All experiments were done in triplicate.

    Journal: The Yale Journal of Biology and Medicine

    Article Title: Association of Plasmodium falciparum with Human Endothelial Cells in vitro

    doi:

    Figure Lengend Snippet: Measurement of P. falciparum rRNA and DNA associated with HUVECs by SYBR Green quantitative PCR. P. falciparum infected RBCs were added to HUVEC cell cultures for the indicated times, as described in Materials and Methods. Following these incubations, RNA and genomic DNA were purified from HUVECS. A. P. falciparum SSU rRNA amounts from HUVECs as measured by reverse-transcription qPCR. No PCR products were detected in RNA samples not treated with reverse-transcriptase (data not shown). Error bars represent standard deviation from three independent replicates. B. The SSU rRNA gene was amplified from P. falciparum genomic DNA (gDNA) from HUVECs and measured by qPCR. P. falciparum RNA and DNA amounts were normalized to human GAPDH RNA and DNA amounts, respectively. As a control, HUVECs were incubated with uninfected RBCs. No P. falciparum -specific PCR products were detected (data not shown). All experiments were done in triplicate.

    Article Snippet: Total RNA and genomic DNA (gDNA) were purified from HUVEC cells using Trizol reagent (Invitrogen) according to the manufacturer’s instructions.

    Techniques: SYBR Green Assay, Real-time Polymerase Chain Reaction, Infection, Purification, Polymerase Chain Reaction, Standard Deviation, Amplification, Incubation

    For a given DNA input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line gDNA serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.

    Journal: Clinical chemistry

    Article Title: Denaturation-enhanced droplet digital PCR for liquid biopsies

    doi: 10.1373/clinchem.2018.293845

    Figure Lengend Snippet: For a given DNA input, the number of positive events using dddPCR is twice that of ddPCR, thus improving the relative standard error in the analysis. (A) Demonstration of the concept. (B) ddPCR and dddPCR for BRAF V600E using 10ng of HTB-19 cell line gDNA serially diluted into wild type DNA (WT). Outer bars represent the standard error of the mean for the replicates; the inner error bar is the 95% confidence interval for the Poisson distribution. Each sample is composed of 4 merged replicates. RSE=relative standard error.

    Article Snippet: Genomic DNA (gDNA) from cell-line MDA-MB-435S (HTB-129D, ATCC) and the Tru-Q1 Reference Standard (HD728, Horizon Discovery) were used as mutated DNA controls for BRAF p.V600E and NRAS p.Q61K.

    Techniques:

    dddPCR allows the analysis of 2 different targets in independent reactions using the same quantity of DNA used for a single ddPCR reaction and producing similar number of WT and MT copies in each reaction. (A) demonstration of the concept. (B) and (C) Analysis of gDNA HD728 serially diluted in WT DNA using 30ng (ddPCR, non-denatured), or split in two 15ng samples (dddPCR, denatured) as DNA input: BRAF V600E and NRAS Q61K screened, respectively. Undiluted gDNA HD728 contains BRAF V600E and NRAS Q61K mutations at 8% and 5% allelic frequencies, respectively. Error bars represent the 95% confidence interval for the Poisson distribution. The dddPCR results represent merged wells for 2 replicates. The concentration of WT and MT copies obtained were similar for both dddPCR and ddPCR (for WT, P = 0.49, 0.71 and 0.69 for 1:5, 1:20 and 1:50 dilutions, respectively. For MT, P = is 0.79, 0.34 and 0.19, respectively. The differences are not significant).

    Journal: Clinical chemistry

    Article Title: Denaturation-enhanced droplet digital PCR for liquid biopsies

    doi: 10.1373/clinchem.2018.293845

    Figure Lengend Snippet: dddPCR allows the analysis of 2 different targets in independent reactions using the same quantity of DNA used for a single ddPCR reaction and producing similar number of WT and MT copies in each reaction. (A) demonstration of the concept. (B) and (C) Analysis of gDNA HD728 serially diluted in WT DNA using 30ng (ddPCR, non-denatured), or split in two 15ng samples (dddPCR, denatured) as DNA input: BRAF V600E and NRAS Q61K screened, respectively. Undiluted gDNA HD728 contains BRAF V600E and NRAS Q61K mutations at 8% and 5% allelic frequencies, respectively. Error bars represent the 95% confidence interval for the Poisson distribution. The dddPCR results represent merged wells for 2 replicates. The concentration of WT and MT copies obtained were similar for both dddPCR and ddPCR (for WT, P = 0.49, 0.71 and 0.69 for 1:5, 1:20 and 1:50 dilutions, respectively. For MT, P = is 0.79, 0.34 and 0.19, respectively. The differences are not significant).

    Article Snippet: Genomic DNA (gDNA) from cell-line MDA-MB-435S (HTB-129D, ATCC) and the Tru-Q1 Reference Standard (HD728, Horizon Discovery) were used as mutated DNA controls for BRAF p.V600E and NRAS p.Q61K.

    Techniques: Concentration Assay

    Characterization of Disease-Specific iPSCs from Csf2rb −/− Mice (A and B) ESC-like morphology in bright-field images (A) and positive alkaline phosphatase staining of miPAP1 iPSCs (B). Scale bar, 200 μm. (C) NANOG, OCT4, and SOX2 expression by immunofluorescence staining (C; scale bar, 50 μm) as well as by (D) qRT-PCR using murine-specific primers (independent experiments, n = 3, mean ± SD). ns, not significant compared with ESCs, two-way ANOVA. (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker. (F) Representative pictures of miPAP1-derived teratomas containing tissues of all three embryonic germ layers. Scale bar, 50 μm for ectoderm and endoderm; 100 μm for mesoderm. (G) Scheme and gel electrophoresis of PCR on genomic DNA for genotyping miPAP clones.

    Journal: Stem Cell Reports

    Article Title: Murine iPSC-Derived Macrophages as a Tool for Disease Modeling of Hereditary Pulmonary Alveolar Proteinosis due to Csf2rb Deficiency

    doi: 10.1016/j.stemcr.2016.06.011

    Figure Lengend Snippet: Characterization of Disease-Specific iPSCs from Csf2rb −/− Mice (A and B) ESC-like morphology in bright-field images (A) and positive alkaline phosphatase staining of miPAP1 iPSCs (B). Scale bar, 200 μm. (C) NANOG, OCT4, and SOX2 expression by immunofluorescence staining (C; scale bar, 50 μm) as well as by (D) qRT-PCR using murine-specific primers (independent experiments, n = 3, mean ± SD). ns, not significant compared with ESCs, two-way ANOVA. (E) Representative flow cytometry plot revealing expression of the SSEA-1 surface marker. (F) Representative pictures of miPAP1-derived teratomas containing tissues of all three embryonic germ layers. Scale bar, 50 μm for ectoderm and endoderm; 100 μm for mesoderm. (G) Scheme and gel electrophoresis of PCR on genomic DNA for genotyping miPAP clones.

    Article Snippet: PCR on Genomic DNA Genomic DNA (gDNA) was isolated from tissues or iPSCs using the Mammalian Genomic DNA Miniprep Kit (Sigma-Aldrich) according to the manufacturer's instructions.

    Techniques: Mouse Assay, Staining, Expressing, Immunofluorescence, Quantitative RT-PCR, Flow Cytometry, Cytometry, Marker, Derivative Assay, Nucleic Acid Electrophoresis, Polymerase Chain Reaction, Clone Assay

    Agarose gel of PCR product and bis-treated PCR product following (A) 15 minutes and (B) 40 minutes of electrophoresis. Size of each PCR product is as indicated. gDNA–untreated genomic DNA; bis gDNA–bis-treated genomic DNA.

    Journal: PLoS ONE

    Article Title: Circulating cell-free DNA from plasma undergoes less fragmentation during bisulfite treatment than genomic DNA due to low molecular weight

    doi: 10.1371/journal.pone.0224338

    Figure Lengend Snippet: Agarose gel of PCR product and bis-treated PCR product following (A) 15 minutes and (B) 40 minutes of electrophoresis. Size of each PCR product is as indicated. gDNA–untreated genomic DNA; bis gDNA–bis-treated genomic DNA.

    Article Snippet: Human genomic DNA (gDNA) purchased from Roche (Cat #11691112001) and stored at 4°C as specified by the manufacturer was used as the high molecular weight DNA sample.

    Techniques: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Electrophoresis

    Agarose gel of cirDNA and bis-treated cirDNA following (A) 15 minutes and (B) 40 minutes of electrophoresis. The amount of cirDNA in each lane corresponds to the total cirDNA extracted from the plasma volumes indicated. gDNA–untreated genomic DNA.

    Journal: PLoS ONE

    Article Title: Circulating cell-free DNA from plasma undergoes less fragmentation during bisulfite treatment than genomic DNA due to low molecular weight

    doi: 10.1371/journal.pone.0224338

    Figure Lengend Snippet: Agarose gel of cirDNA and bis-treated cirDNA following (A) 15 minutes and (B) 40 minutes of electrophoresis. The amount of cirDNA in each lane corresponds to the total cirDNA extracted from the plasma volumes indicated. gDNA–untreated genomic DNA.

    Article Snippet: Human genomic DNA (gDNA) purchased from Roche (Cat #11691112001) and stored at 4°C as specified by the manufacturer was used as the high molecular weight DNA sample.

    Techniques: Agarose Gel Electrophoresis, Electrophoresis

    c x43 null zebrafish have reduced motile cilia in ECs. a Electropherograms of the target sequences of the cx43 gDNA in WT and cx43 −/− zebrafish. Arrow indicates the deletion of the T nucleotide. b Zebrafish ( n = 71) at 2 months post-fertilization (mpf) from mating of cx43 +/− zebrafish were genotyped for cx43 . c Images of 3 mpf zebrafish with the indicated cx43 genotype. d WT or cx43 −/− embryos at 2 dpf were immunostained with anti-acetylated-α-tubulin antibody, imaged with a confocal microscope and genotyped for cx43 . Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. e Larvae at 8 dpf from mating of cx43 +/− zebrafish were cut into cranial and caudal halves. The cranial half was used for cx43 genotyping, and the caudal half was coronally sectioned at a thickness of 14 μm and then processed for IF staining with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 30 μm. f Quantification of the number of cilia per frame in embryos in e . Mean ± SD. **** P

    Journal: Nature Communications

    Article Title: Wnt-PLC-IP3-Connexin-Ca2+ axis maintains ependymal motile cilia in zebrafish spinal cord

    doi: 10.1038/s41467-020-15248-2

    Figure Lengend Snippet: c x43 null zebrafish have reduced motile cilia in ECs. a Electropherograms of the target sequences of the cx43 gDNA in WT and cx43 −/− zebrafish. Arrow indicates the deletion of the T nucleotide. b Zebrafish ( n = 71) at 2 months post-fertilization (mpf) from mating of cx43 +/− zebrafish were genotyped for cx43 . c Images of 3 mpf zebrafish with the indicated cx43 genotype. d WT or cx43 −/− embryos at 2 dpf were immunostained with anti-acetylated-α-tubulin antibody, imaged with a confocal microscope and genotyped for cx43 . Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 20 μm. e Larvae at 8 dpf from mating of cx43 +/− zebrafish were cut into cranial and caudal halves. The cranial half was used for cx43 genotyping, and the caudal half was coronally sectioned at a thickness of 14 μm and then processed for IF staining with anti-acetylated-α-tubulin antibody. Arrowheads represent motile cilia. Dorsal view anterior to the left. Scale bar = 30 μm. f Quantification of the number of cilia per frame in embryos in e . Mean ± SD. **** P

    Article Snippet: For mice genotyping, gDNA was extracted from mice ear punches using a Wizard Genomic DNA Purification Kit as per the manufacturer’s instructions (Promega).

    Techniques: Microscopy, Staining

    Alignment of vertebrate six6 genes reveals an evolutionary conserved region 3′ of the six6 coding region. PIP analysis was used to compare vertebrate genomic DNA sequences from mammalian (human, mouse), marsupial (tazmanian devil), aves (chicken), reptile (green sea turtle), lobe-finned fish (coelacanth) and fish (medaka). Golden boxes show evolutionarily conserved regions (R); purple boxes indicate exons; white box, intron; dark and light blue lines, 5′ and 3′ flanking genomic regions, respectively. The line at the bottom of the PIP analysis indicates distance in kilobases from the translation start site. The percent identity of X. tropicalis R3 to each species is included. Exon 1 contains 146 bp of 5′ untranslated region, while exon 2 contains 964 bp of 3′ untranslated region, both depicted in light purple.

    Journal: Developmental biology

    Article Title: Distinct cis-acting regions control six6 expression during eye field and optic cup stages of eye formation

    doi: 10.1016/j.ydbio.2017.04.003

    Figure Lengend Snippet: Alignment of vertebrate six6 genes reveals an evolutionary conserved region 3′ of the six6 coding region. PIP analysis was used to compare vertebrate genomic DNA sequences from mammalian (human, mouse), marsupial (tazmanian devil), aves (chicken), reptile (green sea turtle), lobe-finned fish (coelacanth) and fish (medaka). Golden boxes show evolutionarily conserved regions (R); purple boxes indicate exons; white box, intron; dark and light blue lines, 5′ and 3′ flanking genomic regions, respectively. The line at the bottom of the PIP analysis indicates distance in kilobases from the translation start site. The percent identity of X. tropicalis R3 to each species is included. Exon 1 contains 146 bp of 5′ untranslated region, while exon 2 contains 964 bp of 3′ untranslated region, both depicted in light purple.

    Article Snippet: Genomic DNA (gDNA) was isolated from tails by adding 250μl of lysis buffer [20μg/μl Proteinase K (cat# P2308; Sigma-Aldrich); 10 mM Tris, pH 8.0; 100 mM NaCl; 10 mM EDTA, pH 8.0; 0.5 % SDS] and incubating samples at 60 C overnight.

    Techniques: Fluorescence In Situ Hybridization

    Comparison of DNA extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA gDNA purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).

    Journal: mSystems

    Article Title: Impact of Sample Type and DNA Isolation Procedure on Genomic Inference of Microbiome Composition

    doi: 10.1128/mSystems.00095-16

    Figure Lengend Snippet: Comparison of DNA extraction methods. (A) Experimental design. Human feces, pig feces, and hospital sewage were extracted using seven different DNA extraction methods ( Table 1 ): InnuPure C16, MagNA Pure LC DNA isolation kit III, Easy-DNA gDNA purification kit, MP FastDNA Spin kit, PowerSoil DNA isolation kit, QIAamp DNA stool minikit, and QIAamp DNA stool minikit plus bead beating (for details, see Materials and Methods). DNA concentration, purity, and stability were examined, and microbial community composition was determined using 16S rRNA gene profiling and metagenomics (selected samples). (B) DNA from each method was dissolved in 100 µl solution, and DNA concentrations were determined using Qubit dsDNA BR assay kit measurements. Values represent averages from duplicate or triplicate DNA extractions (see also Table S1A in the supplemental material). (C) Ecological richness (Chao 1) and diversity (Shannon index) were determined based on contingency tables from 16S rRNA gene profiling and metagenomic sequencing data at OTU and species levels, respectively (see also Table S1B ).

    Article Snippet: In a first step, seven DNA isolation procedures were examined, namely, InnuPure C16 from Analytic Jena AG (InnuPure), MagNA Pure LC DNA isolation kit III from Roche (MagNA Pure), Easy-DNA genomic DNA (gDNA) purification kit from Invitrogen (Easy-DNA), MP FastDNA Spin kit from MP Biomedicals (FastDNA), PowerSoil DNA isolation kit from MoBio (PowerSoil.HMP), QIAamp DNA stool minikit from Qiagen (QIAStool), and QIAamp DNA stool minikit plus bead beating from Qiagen (QIAStool+BB) ( and details below).

    Techniques: DNA Extraction, Purification, Concentration Assay, Sequencing

    Sequencing results for Human genomic DNA. Libraries are prepared using 50 ng of input DNA. Similar enzyme concentrations of Tn5-059 and standard Tn5 are used and 2x151 bp sequencing run is performed on a HiSeqX. a Uniformity of Coverage. Sequencing results are down sampled to 20× coverage. Tn5-059 shows improved uniformity over standard Tn5 in NexteraV2 kit. b Normalized GC plot. Grey bar shows schematic GC composition of Human genome. Standard Tn5 has a clear bias towards GC rich regions while undercovers AT rich regions. c AT/GC dropout. Tn5-059 improves AT dropout while adding little on GC dropout

    Journal: BMC Biotechnology

    Article Title: Improved genome sequencing using an engineered transposase

    doi: 10.1186/s12896-016-0326-1

    Figure Lengend Snippet: Sequencing results for Human genomic DNA. Libraries are prepared using 50 ng of input DNA. Similar enzyme concentrations of Tn5-059 and standard Tn5 are used and 2x151 bp sequencing run is performed on a HiSeqX. a Uniformity of Coverage. Sequencing results are down sampled to 20× coverage. Tn5-059 shows improved uniformity over standard Tn5 in NexteraV2 kit. b Normalized GC plot. Grey bar shows schematic GC composition of Human genome. Standard Tn5 has a clear bias towards GC rich regions while undercovers AT rich regions. c AT/GC dropout. Tn5-059 improves AT dropout while adding little on GC dropout

    Article Snippet: In short, 25 ng B. cereus genomic DNA (gDNA) was tagmented by various concentrations of mutants or standard Tn5 from Illumina Nextera kit as a control.

    Techniques: Sequencing

    Bias plots , showing percentage of observed bases at each position (or sequencing cycle). Plots show insertion bias for standard Tn5 in NexteraV2 kit and two Tn5 mutants from sequencing E. coli genomic DNA. The intensities after position (cycle) 20 are the base composition of E. coli genomic DNA. a Standard Tn5 in NexteraV2 kit. Notice the symmetry in the plot centered at position 5, between positions 1 through 9 b Tn5 mutant, notice the change in the insertion bias at positions 3 through 7 and position 12 c Tn5 mutant, notice the change in the insertion bias at positions 3 through 7, as well as higher G bias at position 1

    Journal: BMC Biotechnology

    Article Title: Improved genome sequencing using an engineered transposase

    doi: 10.1186/s12896-016-0326-1

    Figure Lengend Snippet: Bias plots , showing percentage of observed bases at each position (or sequencing cycle). Plots show insertion bias for standard Tn5 in NexteraV2 kit and two Tn5 mutants from sequencing E. coli genomic DNA. The intensities after position (cycle) 20 are the base composition of E. coli genomic DNA. a Standard Tn5 in NexteraV2 kit. Notice the symmetry in the plot centered at position 5, between positions 1 through 9 b Tn5 mutant, notice the change in the insertion bias at positions 3 through 7 and position 12 c Tn5 mutant, notice the change in the insertion bias at positions 3 through 7, as well as higher G bias at position 1

    Article Snippet: In short, 25 ng B. cereus genomic DNA (gDNA) was tagmented by various concentrations of mutants or standard Tn5 from Illumina Nextera kit as a control.

    Techniques: Sequencing, Mutagenesis

    Sequencing results for B. cereus genomic DNA. Libraries are prepared using 50 ng of input DNA. a Uniformity of Coverage. Sequencing results are down sampled to 24× coverage. Tn5-059 shows improved uniformity when compared to standard Tn5. b Normalized GC plot . Grey bar shows schematic GC composition of B. cereus genome. Tn5-059 has a more uniform coverage with less AT dropout. c AT/GC dropout percentages. Both enzyme have no GC dropout while Tn5-059 shows significant improvement in AT dropout

    Journal: BMC Biotechnology

    Article Title: Improved genome sequencing using an engineered transposase

    doi: 10.1186/s12896-016-0326-1

    Figure Lengend Snippet: Sequencing results for B. cereus genomic DNA. Libraries are prepared using 50 ng of input DNA. a Uniformity of Coverage. Sequencing results are down sampled to 24× coverage. Tn5-059 shows improved uniformity when compared to standard Tn5. b Normalized GC plot . Grey bar shows schematic GC composition of B. cereus genome. Tn5-059 has a more uniform coverage with less AT dropout. c AT/GC dropout percentages. Both enzyme have no GC dropout while Tn5-059 shows significant improvement in AT dropout

    Article Snippet: In short, 25 ng B. cereus genomic DNA (gDNA) was tagmented by various concentrations of mutants or standard Tn5 from Illumina Nextera kit as a control.

    Techniques: Sequencing

    Percentage of observed bases at each cycle using standard Tn5 in NexteraV2 kit ( a ), or Tn5-059 ( b ) for sequencing B. cereus genomic DNA. In particular, the two plots differ at positions 3, 4, 6, 7, 11, 13 and 14. For Tn5-059, the bias within positions 10–15 is much closer to the overall genome composition

    Journal: BMC Biotechnology

    Article Title: Improved genome sequencing using an engineered transposase

    doi: 10.1186/s12896-016-0326-1

    Figure Lengend Snippet: Percentage of observed bases at each cycle using standard Tn5 in NexteraV2 kit ( a ), or Tn5-059 ( b ) for sequencing B. cereus genomic DNA. In particular, the two plots differ at positions 3, 4, 6, 7, 11, 13 and 14. For Tn5-059, the bias within positions 10–15 is much closer to the overall genome composition

    Article Snippet: In short, 25 ng B. cereus genomic DNA (gDNA) was tagmented by various concentrations of mutants or standard Tn5 from Illumina Nextera kit as a control.

    Techniques: Sequencing