oligos  (Integrated DNA Technologies)


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    Name:
    DNA oligo
    Description:
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    Catalog Number:
    do-577595
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    Category:
    Nucleic acids
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    Structured Review

    Integrated DNA Technologies oligos
    Source strain multiplex genome engineering and expected metabolic phenotypes of derived lysates post-reduction. A. Strain construction course by <t>MAGE</t> cycling culminating with the 6xHis-4 containing all 4 tags. Each arrow designates the strain being taken through the MAGE process with the <t>oligos</t> used to transform each strain above the arrow. B. MASC-PCR results for additive mutations using primers specifically designed for the 6xHis-taggged version of the gene. C. Expected metabolic phenotypes present in WT and engineered lysate proteomes after the reduction of lysates derived from all generated strains.
    Single stranded pooled or duplexed DNA synthesized to customer specifications Sspecialized platforms with industry leading synthesis capabilities deliver the purest primers for PCR dual labelled probes for qPCR indexed adapters and fusion primers for sequencing and a variety of advanced and custom products
    https://www.bioz.com/result/oligos/product/Integrated DNA Technologies
    Average 99 stars, based on 95 article reviews
    Price from $9.99 to $1999.99
    oligos - by Bioz Stars, 2020-08
    99/100 stars

    Images

    1) Product Images from "A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production"

    Article Title: A Lysate Proteome Engineering Strategy for Enhancing Cell-Free Metabolite Production

    Journal: bioRxiv

    doi: 10.1101/2020.04.05.026393

    Source strain multiplex genome engineering and expected metabolic phenotypes of derived lysates post-reduction. A. Strain construction course by MAGE cycling culminating with the 6xHis-4 containing all 4 tags. Each arrow designates the strain being taken through the MAGE process with the oligos used to transform each strain above the arrow. B. MASC-PCR results for additive mutations using primers specifically designed for the 6xHis-taggged version of the gene. C. Expected metabolic phenotypes present in WT and engineered lysate proteomes after the reduction of lysates derived from all generated strains.
    Figure Legend Snippet: Source strain multiplex genome engineering and expected metabolic phenotypes of derived lysates post-reduction. A. Strain construction course by MAGE cycling culminating with the 6xHis-4 containing all 4 tags. Each arrow designates the strain being taken through the MAGE process with the oligos used to transform each strain above the arrow. B. MASC-PCR results for additive mutations using primers specifically designed for the 6xHis-taggged version of the gene. C. Expected metabolic phenotypes present in WT and engineered lysate proteomes after the reduction of lysates derived from all generated strains.

    Techniques Used: Multiplex Assay, Derivative Assay, Polymerase Chain Reaction, Generated

    2) Product Images from "Efficient assembly of very short oligonucleotides using T4 DNA Ligase"

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Journal: BMC Research Notes

    doi: 10.1186/1756-0500-3-291

    Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.
    Figure Legend Snippet: Enhancement of T4 DNA ligase activity by supplemental oligonucleotides. (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first oligonucleotide-dsDNA duplex but is unphosphorylated to prevent ligation of itself. Two hour ligation of the 4-bp reaction at 16°C supplemented with 3.33 μM of the hexamer, shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (b) Ligation reaction of an octamer supplemented with a second octamer in which one is used for ligation and the other is used to extend the duplex. A two hour ligation at 16°C of serial concentrations of the octamer with 3.33 μM of the supplementary octamer shows significant ligation (■) compared to reactions without the supplemental octamer (◆). (c) Unsuccessful 3-bp duplex reactions could be salvaged by utilizing a supplementary hexamer that hybridized at all six positions. A two hour ligation of the 3-bp reaction at 16°C with 3.33 μM supplementary hexamer shows successful ligation (■) while reactions without the supplementary hexamer show no activity (◆). (d) Ligation using a hexamer pair at 4°C for 16 hours shows limited improvement (■) compared to the unsupplemented (◆) control.

    Techniques Used: Activity Assay, Ligation

    Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).
    Figure Legend Snippet: Evaluation of minimal oligonucleotide substrate requirements for T4 DNA ligase. (a) Schematic diagram of an immobilized DNA strand used in ligation assays and DNA construction. M-270 Dynabeads (Invitrogen) are attached through a streptavidin-biotin linkage to the 5' end of a double stranded DNA. The free end is designed with a variable 5' overhang, complementary to labeled oligonucleotides used in ligation. An additional BbsI restriction site and a forward primer site are included in the case of DNA construction. (b) Increasing concentrations of 5'-phosphorylated, 3'-fluorescently labeled oligonucleotide are ligated to 5 pmoles of immobilized dsDNA with a complementary overhang. Reactions were performed for one hour at 16°C and washed with TE to remove unligated substrate. Successful ligation kinetics are observed at the 5-bp duplex length (▲), but no significant ligation occurs at lengths of 4-bp (■) or 3-bp (◆).

    Techniques Used: Ligation, Labeling

    3) Product Images from "False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine"

    Article Title: False positives and false negatives measure less than 0.001% in labeling ssDNA with osmium tetroxide 2,2’-bipyridine

    Journal: Beilstein Journal of Nanotechnology

    doi: 10.3762/bjnano.7.135

    HPLC profiles of weeks-old-aged osmylated oligos. C* is an abbreviation for C(OsBp) and T* is an abbreviation for T(OsBp). A 10 T(OsBp)A 9 was stable over weeks at room temperature, in contrast to A 10 C(OsBp)A 9 that over time shifts materials to a new, unidentified, product (see major peak with the longest rt in (a) and (c)). Shaded areas correspond to degradation products of the C-oligo and stable osmylation products of the T-oligo. Smaller peaks outside the shaded region correspond to osmylation products of the impurities. The figure illustrates that all the products formed by aging of C(OsBp) carry the OsBp moiety. This is evidenced by comparing the values of R = 0.11 for the C-oligo (peak area in (a) divided by the peak area in (c)) with the value R = 0.11 for the T-oligo (peak area in (b) divided by the peak area in (d)); R = 0.01 for DNA that is an order of magnitude lower compared to the reported values here.
    Figure Legend Snippet: HPLC profiles of weeks-old-aged osmylated oligos. C* is an abbreviation for C(OsBp) and T* is an abbreviation for T(OsBp). A 10 T(OsBp)A 9 was stable over weeks at room temperature, in contrast to A 10 C(OsBp)A 9 that over time shifts materials to a new, unidentified, product (see major peak with the longest rt in (a) and (c)). Shaded areas correspond to degradation products of the C-oligo and stable osmylation products of the T-oligo. Smaller peaks outside the shaded region correspond to osmylation products of the impurities. The figure illustrates that all the products formed by aging of C(OsBp) carry the OsBp moiety. This is evidenced by comparing the values of R = 0.11 for the C-oligo (peak area in (a) divided by the peak area in (c)) with the value R = 0.11 for the T-oligo (peak area in (b) divided by the peak area in (d)); R = 0.01 for DNA that is an order of magnitude lower compared to the reported values here.

    Techniques Used: High Performance Liquid Chromatography

    Direct comparison of the kinetics of a 20 nucleotide long deoxyoligo (triangles, PCR primer “16S RNA for”, for simplicity “20-mer”, sequence given in Table 1 ) and the circular 7249 nucleotide long ssDNA M13mp18 (circles, for simplicity “ssDNA”, see Table 1 ) as obtained by CE (see Experimental). The data show kinetically comparable reactivity of these two dramatically different materials under three different osmylation conditions, i.e. in 3.4 mM, or in 8.5 mM, or in 13.6 mM OsBp (solid symbols). Dotted lines indicate “theoretical” R 2 for 100% osmylation, as estimated from equation R = 2.01 × (C + T) tot / N tot , obtained earlier using a training set of oligos/DNA [ 31 ]. Theoretical R 2 values at 1.01 and 1.09 and observed R 2 values at 1.05 and 1.18, respectively for “16S RNA for” and ssM13mp18. R 2 values are R (312/272) upon protocol B osmylation that yields practically 100% T + C labeling (see Experimental).
    Figure Legend Snippet: Direct comparison of the kinetics of a 20 nucleotide long deoxyoligo (triangles, PCR primer “16S RNA for”, for simplicity “20-mer”, sequence given in Table 1 ) and the circular 7249 nucleotide long ssDNA M13mp18 (circles, for simplicity “ssDNA”, see Table 1 ) as obtained by CE (see Experimental). The data show kinetically comparable reactivity of these two dramatically different materials under three different osmylation conditions, i.e. in 3.4 mM, or in 8.5 mM, or in 13.6 mM OsBp (solid symbols). Dotted lines indicate “theoretical” R 2 for 100% osmylation, as estimated from equation R = 2.01 × (C + T) tot / N tot , obtained earlier using a training set of oligos/DNA [ 31 ]. Theoretical R 2 values at 1.01 and 1.09 and observed R 2 values at 1.05 and 1.18, respectively for “16S RNA for” and ssM13mp18. R 2 values are R (312/272) upon protocol B osmylation that yields practically 100% T + C labeling (see Experimental).

    Techniques Used: Polymerase Chain Reaction, Sequencing, Labeling

    4) Product Images from "Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents"

    Article Title: Fluorophore-labelled RNA aptamers to common protein tags as super-resolution imaging reagents

    Journal: bioRxiv

    doi: 10.1101/2020.02.27.968578

    Additional unique advantages of aptamer labeling. (a) Two-rounds of sequential binding of Alexa 647 labeled AP3 on GFP beads using RNase treatment demonstrating aptamers as multiplexing reagents. (b) Left: structure of modified AP3 with 60nt linker at its 3’end that is hybridized to bind three labeled ssDNA oligos. Right: brightness comparison of beads with directly labeled AP3 and the 3X modified version (equal concentrations of AP3 were used).
    Figure Legend Snippet: Additional unique advantages of aptamer labeling. (a) Two-rounds of sequential binding of Alexa 647 labeled AP3 on GFP beads using RNase treatment demonstrating aptamers as multiplexing reagents. (b) Left: structure of modified AP3 with 60nt linker at its 3’end that is hybridized to bind three labeled ssDNA oligos. Right: brightness comparison of beads with directly labeled AP3 and the 3X modified version (equal concentrations of AP3 were used).

    Techniques Used: Labeling, Binding Assay, Multiplexing, Modification

    5) Product Images from "Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis"

    Article Title: Rapid and dynamic nucleic acid hybridization enables enzymatic oligonucleotide synthesis by cyclic reversible termination: A novel mechanism for enzymatic DNA synthesis

    Journal: bioRxiv

    doi: 10.1101/561092

    Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.
    Figure Legend Snippet: Dynamic hybridization of DNA enables EOS. (a) Extension of single-stranded DNA by DNA polymerases and reverse transcriptases. Denaturing PAGE analysis of a 20 base single-stranded sequence, self-priming oligo-1 aka SPO-1, using different enzymes and dGTP. Despite a maximum of two bases of hybridization, some enzymes are able to extend this solid-phase oligonucleotide. None of these enzymes have previously been reported to have nucleotidyl transferase activity on single-stranded DNA. (b) Sequence-specific extension of single-stranded DNA. Duplase extension of four different 20 base oligos proceeds in a sequence-specific manner, and only two bases of hybridization are required for extension. * indicates unextended control. Extension with non-templated bases can be explained by misincorporation that occurs at long reaction times . (c) Extension of solid-phase oligos through intermolecular reactions. Magnetic beads were conjugated with either a 20 base poly-T oligo as illustrated in i, or with that oligo plus a 30 base poly-T oligo with an internal 5’-CAA-3’ sequence as illustrated in ii . Beads were extended using Duplase-3 and Cy5-ddGTP (* indicates unextended control during this step), after which, all samples were labeled with TdT and fluorescein-12-ddUTP. Oligos appear blue if extended with Duplase-3 and green if extended wtih TdT.

    Techniques Used: Hybridization, Polyacrylamide Gel Electrophoresis, Sequencing, Activity Assay, Magnetic Beads, Labeling

    6) Product Images from "Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing"

    Article Title: Identification of MiR-21-5p as a Functional Regulator of Mesothelin Expression Using MicroRNA Capture Affinity Coupled with Next Generation Sequencing

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0170999

    Test for capture efficiency and specificity of MSLN_1 (A), MSLN_2 (B), and MSLN_3 (C) oligos. QRT-PCR was used to calculate MSLN and off-target mRNA enrichment for each tested oligo in Mero-14 samples. Relative mRNA expression was quantified using the 2 -ΔΔCt method comparing MSLN-captured vs scrambled-captured samples. The punctual P-value according to Dunnett’s test calculated within the ANOVA model is also reported. The columns represent mean values, the bars show standard error of the mean (SEM) of three independent experiments. Ns = not statistically significant.
    Figure Legend Snippet: Test for capture efficiency and specificity of MSLN_1 (A), MSLN_2 (B), and MSLN_3 (C) oligos. QRT-PCR was used to calculate MSLN and off-target mRNA enrichment for each tested oligo in Mero-14 samples. Relative mRNA expression was quantified using the 2 -ΔΔCt method comparing MSLN-captured vs scrambled-captured samples. The punctual P-value according to Dunnett’s test calculated within the ANOVA model is also reported. The columns represent mean values, the bars show standard error of the mean (SEM) of three independent experiments. Ns = not statistically significant.

    Techniques Used: Quantitative RT-PCR, Expressing

    7) Product Images from "Oligonucleotide Recombination in Gram-Negative Bacteria"

    Article Title: Oligonucleotide Recombination in Gram-Negative Bacteria

    Journal: Molecular Microbiology

    doi: 10.1111/j.1365-2958.2009.06976.x

    Effect of DNA concentration on recombination frequency. Recombination frequencies are the average of at least three independent transformations and are shown with error bars indicating standard deviation. (A) P. syringae cells were transformed with different amounts of oligo oSWC1255, which encodes a 4 base change that directs the rpsL K43R plus a silent marker, which is used to confirm that the mutation is derived from the oligo. (B) The recombination rate was determined for P. syringae cells transformed with the rpsL K43R encoding oligo (oSWC1255) in the presence or absence of carrier oligo. Carrier oligos oSWC1515 and oSWC1449 have sequence homology to the leading or lagging strands, respectively, within gene PSPTO_5020 in the P. syringae sequence. The PSPTO_5020 gene was chosen because it and rpsL are equidistant from the origin of replication on opposite arms of the chromosome. Carrier oligos random A (oSWC1447) and Random B (oSWC1448) are complementary oligos. They were designed by generating a random sequence of 84 nt for Random A and then creating its complement Random B.
    Figure Legend Snippet: Effect of DNA concentration on recombination frequency. Recombination frequencies are the average of at least three independent transformations and are shown with error bars indicating standard deviation. (A) P. syringae cells were transformed with different amounts of oligo oSWC1255, which encodes a 4 base change that directs the rpsL K43R plus a silent marker, which is used to confirm that the mutation is derived from the oligo. (B) The recombination rate was determined for P. syringae cells transformed with the rpsL K43R encoding oligo (oSWC1255) in the presence or absence of carrier oligo. Carrier oligos oSWC1515 and oSWC1449 have sequence homology to the leading or lagging strands, respectively, within gene PSPTO_5020 in the P. syringae sequence. The PSPTO_5020 gene was chosen because it and rpsL are equidistant from the origin of replication on opposite arms of the chromosome. Carrier oligos random A (oSWC1447) and Random B (oSWC1448) are complementary oligos. They were designed by generating a random sequence of 84 nt for Random A and then creating its complement Random B.

    Techniques Used: Concentration Assay, Standard Deviation, Transformation Assay, Marker, Mutagenesis, Derivative Assay, Sequencing

    8) Product Images from "Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition"

    Article Title: Rapid Generation of MicroRNA Sponges for MicroRNA Inhibition

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0029275

    Confirmation of miR-19 binding to miR-19 sponge constructs. ( A ) Luciferase reporter assays in HEK293 cells reveal that repression of Renilla activity is more prominent in reporter vectors that contain perfect MBS sequences as compared to reporter vectors that encode bulged MBS sequences and is in both cases dependent on the number of MBS. ( B ) Release of miR-19 specific repression of Renilla luciferase activity by anti-miR-19a/b oligos confirms that miR-19 binds to the miR-19 MBS sequences. No release of luciferase activity is observed with a control anti-miR-16 oligo. Open bars: Mock, grey bars: miR-16 inhibitor and black bar: miR-19 inhibitor mix. For each graph the number of MBS per reporter vector is indicated on the x-axis and on the y-axis the ratio of Renilla (R) over Firefly (F) luciferase is depicted. * = p-value
    Figure Legend Snippet: Confirmation of miR-19 binding to miR-19 sponge constructs. ( A ) Luciferase reporter assays in HEK293 cells reveal that repression of Renilla activity is more prominent in reporter vectors that contain perfect MBS sequences as compared to reporter vectors that encode bulged MBS sequences and is in both cases dependent on the number of MBS. ( B ) Release of miR-19 specific repression of Renilla luciferase activity by anti-miR-19a/b oligos confirms that miR-19 binds to the miR-19 MBS sequences. No release of luciferase activity is observed with a control anti-miR-16 oligo. Open bars: Mock, grey bars: miR-16 inhibitor and black bar: miR-19 inhibitor mix. For each graph the number of MBS per reporter vector is indicated on the x-axis and on the y-axis the ratio of Renilla (R) over Firefly (F) luciferase is depicted. * = p-value

    Techniques Used: Binding Assay, Construct, Luciferase, Activity Assay, Plasmid Preparation

    9) Product Images from "Synthetic Zinc Finger Nuclease Design and Rapid Assembly"

    Article Title: Synthetic Zinc Finger Nuclease Design and Rapid Assembly

    Journal: Human Gene Therapy

    doi: 10.1089/hum.2011.072

    CoDA-syn DNA assembly. (A) Overlapping extension PCR. The left (fragment 1) or right (fragment 4) ZFN arrays containing eight overlapping oligonucleotides were mixed together for use in an overlapping extension PCR to generate dsDNA. (B) Expression vector
    Figure Legend Snippet: CoDA-syn DNA assembly. (A) Overlapping extension PCR. The left (fragment 1) or right (fragment 4) ZFN arrays containing eight overlapping oligonucleotides were mixed together for use in an overlapping extension PCR to generate dsDNA. (B) Expression vector

    Techniques Used: Polymerase Chain Reaction, Expressing, Plasmid Preparation

    10) Product Images from "Improved bacterial recombineering by parallelized protein discovery"

    Article Title: Improved bacterial recombineering by parallelized protein discovery

    Journal: bioRxiv

    doi: 10.1101/2020.01.14.906594

    Recombineering in Gammaproteobacteria. (A) Recombineering experiments were run with Redβ, PapRecT, and CspRecT expressed off of the pORTMAGE311B backbone, or with a pBBR1 origin in the case of P. aeruginosa . Editing efficiency was measured by colony counts on selective vs. non-selective plates (n=3; see methods). Vector optimization resulted in improved efficiency of PapRecT in P. aeruginosa (see Figure S7 ) (B) Diagram of a simple multi-drug resistance experiment in P. aeruginosa harboring an optimized PapRecT plasmid expression system, pORTMAGE-Pa1. In a single round of MAGE, a pool of five oligos was used to incorporate genetic modifications that would provide resistance to STR, RIF, and CIP (n=3). These populations were then selected by plating on all combinations of 1-, 2-, or 3-antibiotic agarose plates and compared with a non-selective control. (C) Observed efficiencies were calculated by comparing colony counts on selective vs. non-selective plates. Expected efficiencies for multi-locus events were calculated as the product of all relevant single-locus efficiencies.
    Figure Legend Snippet: Recombineering in Gammaproteobacteria. (A) Recombineering experiments were run with Redβ, PapRecT, and CspRecT expressed off of the pORTMAGE311B backbone, or with a pBBR1 origin in the case of P. aeruginosa . Editing efficiency was measured by colony counts on selective vs. non-selective plates (n=3; see methods). Vector optimization resulted in improved efficiency of PapRecT in P. aeruginosa (see Figure S7 ) (B) Diagram of a simple multi-drug resistance experiment in P. aeruginosa harboring an optimized PapRecT plasmid expression system, pORTMAGE-Pa1. In a single round of MAGE, a pool of five oligos was used to incorporate genetic modifications that would provide resistance to STR, RIF, and CIP (n=3). These populations were then selected by plating on all combinations of 1-, 2-, or 3-antibiotic agarose plates and compared with a non-selective control. (C) Observed efficiencies were calculated by comparing colony counts on selective vs. non-selective plates. Expected efficiencies for multi-locus events were calculated as the product of all relevant single-locus efficiencies.

    Techniques Used: Plasmid Preparation, Expressing

    11) Product Images from "Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection"

    Article Title: Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks455

    Mechanism for MAGE AR with CoS. The dividing chromosome is schematized, with integration of a mutagenic oligo into the genome at a replication fork [adapted from Costantino and Court ( 4 )]. An oligo electroporated into the cell is bound by multiple copies of the λ -bacteriophage β protein and anneals to the lagging strand during DNA replication. When multiple oligos are incorporated into nearby sites (black and gray rectangles), they are predicted to co-segregate at high frequency, often inherited by the same daughter cell. Thus, a permissive replication fork seems to be a limiting factor in MAGE. Using one of these modifications to change the function of a selectable gene allows selection to remove unmodified cells.
    Figure Legend Snippet: Mechanism for MAGE AR with CoS. The dividing chromosome is schematized, with integration of a mutagenic oligo into the genome at a replication fork [adapted from Costantino and Court ( 4 )]. An oligo electroporated into the cell is bound by multiple copies of the λ -bacteriophage β protein and anneals to the lagging strand during DNA replication. When multiple oligos are incorporated into nearby sites (black and gray rectangles), they are predicted to co-segregate at high frequency, often inherited by the same daughter cell. Thus, a permissive replication fork seems to be a limiting factor in MAGE. Using one of these modifications to change the function of a selectable gene allows selection to remove unmodified cells.

    Techniques Used: Selection

    12) Product Images from "Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation"

    Article Title: Tracking replication enzymology in vivo by genome-wide mapping of ribonucleotide incorporation

    Journal: Nature structural & molecular biology

    doi: 10.1038/nsmb.2957

    Mapping ribonucleotides by HydEn-Seq (a) HydEn-Seq protocol. The procedure was performed as described in Methods, using the oligonucleotides listed in Supplementary Table 1 . (b) Alkaline agarose gel electrophoresis. The analysis was performed as previously described 18 . Genomic DNA samples from the indicated yeast strains ( lanes 1–10 ) were treated with alkali, separated by 1% alkaline agarose gel electrophoresis, and imaged after staining with SYBR Gold. Migration positions of two DNA size standards are indicated. (c) Densitometry scans of the gel image in (b). The Y-axis is scaled to maximum intensity for each pair of lanes. (d) Mean HydEn-seq end counts per haploid genome (see N ends calculation in Methods; error bars represent ranges of two to four independent measurements).
    Figure Legend Snippet: Mapping ribonucleotides by HydEn-Seq (a) HydEn-Seq protocol. The procedure was performed as described in Methods, using the oligonucleotides listed in Supplementary Table 1 . (b) Alkaline agarose gel electrophoresis. The analysis was performed as previously described 18 . Genomic DNA samples from the indicated yeast strains ( lanes 1–10 ) were treated with alkali, separated by 1% alkaline agarose gel electrophoresis, and imaged after staining with SYBR Gold. Migration positions of two DNA size standards are indicated. (c) Densitometry scans of the gel image in (b). The Y-axis is scaled to maximum intensity for each pair of lanes. (d) Mean HydEn-seq end counts per haploid genome (see N ends calculation in Methods; error bars represent ranges of two to four independent measurements).

    Techniques Used: Agarose Gel Electrophoresis, Staining, Migration

    13) Product Images from "Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer"

    Article Title: Runx2 contributes to murine Col10a1 gene regulation through direct interaction with its cis-enhancer

    Journal: Journal of Bone and Mineral Research

    doi: 10.1002/jbmr.504

    Putative Runx2 sites within 3′prime of the 150-bp Col10a1 promoter. ( A ) Positions of 11 consecutive short DNA oligos (25 bases each, SP1-11 ) and three long DNA oligos (∼40 bases each, LP1-3 ) within the 150-bp Col10a1 distal promoter (−4296 to −4147 bp) were as indicated (see also Table 1 ). The previously reported two AP-1 sites (16) and the putative tandem-repeat Runx2 binding sites (−4187 to −4176 bp) were underlined. SP = short probe; LP = long probe. ( B ) EMSA assay showed that SP9 forms specific binding complexes with hypertrophic MCT cell nuclear extracts (lane 9, arrow). Weak signal was also observed with SP11 (lane 11). Similar migration pattern but stronger signal intensity was observed with LP3 (lane LP3 , arrows). The sequence of LP3 (−4201 to −4163 bp) and SP9 (−4296 to −4274 bp) was shown in ( A ) and in Table 1 . Bottom signals correspond to free probes. ( C ) Forward oligo sequences of LP3 and SP9 are shown. The putative Runx2 core binding sites (underlined) as well as mutations inside (“CA”) or outside (“GC”, or “CC”) of the core binding sequence are as highlighted (bold and italic). “GATCC” and “A” are BamHI and BglII adaptor sequence. WT = wild type; MI = mutated inside; MO = mutated outside. ( D ) EMSA assays with probes LP3 , SP9 , and their mutant forms of oligomers were performed. Probes LP3 and SP9 form similar binding complexes with hypertrophic MCT cell nuclear extracts as seen in ( B ) (WT, lane 1). When the putative Runx2 core binding sites were mutated, no DNA/protein complexes were observed (MI, lane 1). However, mutations outside of the core sequence did not abolish the DNA/protein complexes (MO, lane 1). No binding complexes formed when competitive DNA oligos without biotin modification were used (lane 2). Bottom signals show free probes.
    Figure Legend Snippet: Putative Runx2 sites within 3′prime of the 150-bp Col10a1 promoter. ( A ) Positions of 11 consecutive short DNA oligos (25 bases each, SP1-11 ) and three long DNA oligos (∼40 bases each, LP1-3 ) within the 150-bp Col10a1 distal promoter (−4296 to −4147 bp) were as indicated (see also Table 1 ). The previously reported two AP-1 sites (16) and the putative tandem-repeat Runx2 binding sites (−4187 to −4176 bp) were underlined. SP = short probe; LP = long probe. ( B ) EMSA assay showed that SP9 forms specific binding complexes with hypertrophic MCT cell nuclear extracts (lane 9, arrow). Weak signal was also observed with SP11 (lane 11). Similar migration pattern but stronger signal intensity was observed with LP3 (lane LP3 , arrows). The sequence of LP3 (−4201 to −4163 bp) and SP9 (−4296 to −4274 bp) was shown in ( A ) and in Table 1 . Bottom signals correspond to free probes. ( C ) Forward oligo sequences of LP3 and SP9 are shown. The putative Runx2 core binding sites (underlined) as well as mutations inside (“CA”) or outside (“GC”, or “CC”) of the core binding sequence are as highlighted (bold and italic). “GATCC” and “A” are BamHI and BglII adaptor sequence. WT = wild type; MI = mutated inside; MO = mutated outside. ( D ) EMSA assays with probes LP3 , SP9 , and their mutant forms of oligomers were performed. Probes LP3 and SP9 form similar binding complexes with hypertrophic MCT cell nuclear extracts as seen in ( B ) (WT, lane 1). When the putative Runx2 core binding sites were mutated, no DNA/protein complexes were observed (MI, lane 1). However, mutations outside of the core sequence did not abolish the DNA/protein complexes (MO, lane 1). No binding complexes formed when competitive DNA oligos without biotin modification were used (lane 2). Bottom signals show free probes.

    Techniques Used: Binding Assay, Migration, Sequencing, Mutagenesis, Modification

    Runx2 binds to the putative tandem-repeat Runx2 binding sites. ( A ) Candidate EMSA assay using Runx2 antibody showed that the signal intensity of the two major DNA/protein complexes decreased when Runx2 antibody was used (lanes “R2Ab,” dashed arrows) compared to the ones using only biotin-labeled probes LP3 and SP9 (lanes “Bio,” black arrows). No binding complexes formed using competitive DNA oligos (“Comp” lanes). Bottom signals show free probe LP3 (left). Free probe SP9 runs out of the gel (right). Bio = biotin-labeled probes; Comp = probes without biotin modification; R2Ab = Runx2 antibody. ( B ) Candidate EMSA assay was also performed using a diluted Runx2 antibody series. Signal intensity decreased with 200 ng of Runx2 antibody (“200” lanes). Meanwhile, 500 ng of Runx2 antibody significantly inhibited, whereas 1000ng of Runx2 antibody completely abolished, formation of the DNA/protein complexes. Bottom signals show free probes LP3 and SP9 . ( C ). ChIP experiment was performed using MCT cells and Runx2 antibody (R2Ab). Position of the primers flanking the enhancer and control sequence was illustrated (top, arrows). Semiquantitative PCR showed clear amplicon of the target enhancer sequence precipitated by Runx2 antibody but only faint band by control IgG (bottom, left), whereas the control sequence was barely detectable from DNA samples that use either Runx2 antibody or control IgG (bottom, right). Both enhancer and control sequences were amplified from the input samples. ( D ) Real-time PCR was performed using the same input sample and DNAs precipitated with Runx2 antibody or control IgG. The enhancer sequence was significantly enriched (7.44-fold, p = 0.009) by Runx2 antibody compared to DNA precipitated by control IgG, whereas no enrichment of control sequence was obtained ( p = 0.878) by Runx2 antibody.
    Figure Legend Snippet: Runx2 binds to the putative tandem-repeat Runx2 binding sites. ( A ) Candidate EMSA assay using Runx2 antibody showed that the signal intensity of the two major DNA/protein complexes decreased when Runx2 antibody was used (lanes “R2Ab,” dashed arrows) compared to the ones using only biotin-labeled probes LP3 and SP9 (lanes “Bio,” black arrows). No binding complexes formed using competitive DNA oligos (“Comp” lanes). Bottom signals show free probe LP3 (left). Free probe SP9 runs out of the gel (right). Bio = biotin-labeled probes; Comp = probes without biotin modification; R2Ab = Runx2 antibody. ( B ) Candidate EMSA assay was also performed using a diluted Runx2 antibody series. Signal intensity decreased with 200 ng of Runx2 antibody (“200” lanes). Meanwhile, 500 ng of Runx2 antibody significantly inhibited, whereas 1000ng of Runx2 antibody completely abolished, formation of the DNA/protein complexes. Bottom signals show free probes LP3 and SP9 . ( C ). ChIP experiment was performed using MCT cells and Runx2 antibody (R2Ab). Position of the primers flanking the enhancer and control sequence was illustrated (top, arrows). Semiquantitative PCR showed clear amplicon of the target enhancer sequence precipitated by Runx2 antibody but only faint band by control IgG (bottom, left), whereas the control sequence was barely detectable from DNA samples that use either Runx2 antibody or control IgG (bottom, right). Both enhancer and control sequences were amplified from the input samples. ( D ) Real-time PCR was performed using the same input sample and DNAs precipitated with Runx2 antibody or control IgG. The enhancer sequence was significantly enriched (7.44-fold, p = 0.009) by Runx2 antibody compared to DNA precipitated by control IgG, whereas no enrichment of control sequence was obtained ( p = 0.878) by Runx2 antibody.

    Techniques Used: Binding Assay, Labeling, Modification, Chromatin Immunoprecipitation, Sequencing, Polymerase Chain Reaction, Amplification, Real-time Polymerase Chain Reaction

    14) Product Images from "Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection"

    Article Title: Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks455

    Mechanism for MAGE AR with CoS. The dividing chromosome is schematized, with integration of a mutagenic oligo into the genome at a replication fork [adapted from Costantino and Court ( 4 )]. An oligo electroporated into the cell is bound by multiple copies of the λ -bacteriophage β protein and anneals to the lagging strand during DNA replication. When multiple oligos are incorporated into nearby sites (black and gray rectangles), they are predicted to co-segregate at high frequency, often inherited by the same daughter cell. Thus, a permissive replication fork seems to be a limiting factor in MAGE. Using one of these modifications to change the function of a selectable gene allows selection to remove unmodified cells.
    Figure Legend Snippet: Mechanism for MAGE AR with CoS. The dividing chromosome is schematized, with integration of a mutagenic oligo into the genome at a replication fork [adapted from Costantino and Court ( 4 )]. An oligo electroporated into the cell is bound by multiple copies of the λ -bacteriophage β protein and anneals to the lagging strand during DNA replication. When multiple oligos are incorporated into nearby sites (black and gray rectangles), they are predicted to co-segregate at high frequency, often inherited by the same daughter cell. Thus, a permissive replication fork seems to be a limiting factor in MAGE. Using one of these modifications to change the function of a selectable gene allows selection to remove unmodified cells.

    Techniques Used: Selection

    15) Product Images from "OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes"

    Article Title: OligoMiner provides a rapid, flexible environment for the design of genome-scale oligonucleotide in situ hybridization probes

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

    doi: 10.1073/pnas.1714530115

    OligoMiner enables highly efficient FISH. ( A and B ) Representative single-channel minimum-maximum (min-max) contrasted image ( Left ) and two-color image with manual contrast adjustment ( Right ) ( A ) and signal number quantification ( B ) of 3D FISH experiment performed with a probe set consisting of 4,776 UM oligos targeting 817 kb at Xq28 in human XX 2N WI-38 fibroblasts. ( C and D ) Representative single-channel min-max contrasted image ( C , Left ) and two-color contrast-adjusted ( C , Right ) and signal number quantification ( D ) of 3D FISH experiment performed with a probe set consisting of 3,678 LDM oligos targeting 1,035 kb at 19p13.2 in human XY 2N PGP-1 fibroblasts. ( E ) Quantification of background-subtracted SNR for the Xq28 and 19p13.2 probes. ( F ) Three-color 3D FISH experiment performed using ATTO 488-labeled “X.1” (green), ATTO 565-labeled “X.2” (magenta), and Alexa Fluor 647-labeled “X.3” UM probe sets targeting adjacent regions on Xq28 in WI-38 fibroblasts. ( G and H ) Two-color metaphase FISH experiment performed using ATTO 488-labeled “X.1” (green) and Alexa Fluor 647-labeled “X.2” (magenta) UM probe sets targeting adjacent regions on Xq28 on XX 46N ( G ) and XY 46N ( H ) chromosome spreads. ( I and J ) Two-color metaphase FISH experiment performed using Alexa Fluor 647-labeled “19.1” (green) and Cy3B-labeled “19.2” (magenta) LDM probe sets targeting adjacent regions on 19p13.2 on XX 46N ( I ) and XY 46N ( J ) chromosome spreads. All images in are maximum-intensity projections in Z . DNA is stained with DAPI (blue) in multichannel images. In G – J , the multicolor images of the full spread and single-channel images ( Inset ) are min-max contrasted and the multichannel images ( Inset ) have manual contrast adjustments. (Scale bars: 10 µm; G – J , Inset , 1 µm.) For each image, the minimum and maximum pixel intensity value used to set the display scale is indicated in the lower left.
    Figure Legend Snippet: OligoMiner enables highly efficient FISH. ( A and B ) Representative single-channel minimum-maximum (min-max) contrasted image ( Left ) and two-color image with manual contrast adjustment ( Right ) ( A ) and signal number quantification ( B ) of 3D FISH experiment performed with a probe set consisting of 4,776 UM oligos targeting 817 kb at Xq28 in human XX 2N WI-38 fibroblasts. ( C and D ) Representative single-channel min-max contrasted image ( C , Left ) and two-color contrast-adjusted ( C , Right ) and signal number quantification ( D ) of 3D FISH experiment performed with a probe set consisting of 3,678 LDM oligos targeting 1,035 kb at 19p13.2 in human XY 2N PGP-1 fibroblasts. ( E ) Quantification of background-subtracted SNR for the Xq28 and 19p13.2 probes. ( F ) Three-color 3D FISH experiment performed using ATTO 488-labeled “X.1” (green), ATTO 565-labeled “X.2” (magenta), and Alexa Fluor 647-labeled “X.3” UM probe sets targeting adjacent regions on Xq28 in WI-38 fibroblasts. ( G and H ) Two-color metaphase FISH experiment performed using ATTO 488-labeled “X.1” (green) and Alexa Fluor 647-labeled “X.2” (magenta) UM probe sets targeting adjacent regions on Xq28 on XX 46N ( G ) and XY 46N ( H ) chromosome spreads. ( I and J ) Two-color metaphase FISH experiment performed using Alexa Fluor 647-labeled “19.1” (green) and Cy3B-labeled “19.2” (magenta) LDM probe sets targeting adjacent regions on 19p13.2 on XX 46N ( I ) and XY 46N ( J ) chromosome spreads. All images in are maximum-intensity projections in Z . DNA is stained with DAPI (blue) in multichannel images. In G – J , the multicolor images of the full spread and single-channel images ( Inset ) are min-max contrasted and the multichannel images ( Inset ) have manual contrast adjustments. (Scale bars: 10 µm; G – J , Inset , 1 µm.) For each image, the minimum and maximum pixel intensity value used to set the display scale is indicated in the lower left.

    Techniques Used: Fluorescence In Situ Hybridization, Labeling, Staining

    Single-molecule superresolution imaging of OligoMiner oligos. ( A and B ) Diffraction-limited ( A ) and superresolved STORM ( B ) images of a probe set consisting of 3,678 LDM oligos targeting 1,035 kb at 19p13.2 in human XY 2N PGP-1 fibroblasts. ( C and D ) Diffraction-limited ( C ) and superresolved STORM ( D ) images of a probe set consisting of 104 LDM oligos targeting 20 kb at 19p13.2 in PGP-1 fibroblasts. ( E and F ) Diffraction-limited ( E ) and superresolved DNA-PAINT ( F ) images of a probe set consisting of 4,776 UM oligos targeting 817 kb at Xq28 in human XY 2N MRC-5 fibroblasts. ( G and H ) Diffraction-limited ( G ) and superresolved DNA-PAINT ( H ) images of a probe set consisting of 176 LDM oligos targeting 11 kb of the Xist RNA in human XX 2N WI-38 fibroblasts. ( i – viii ) Normalized single-molecule counts along the indicated 1D line traces (blue bars) and one- or two-component Gaussian fits to the underlying data (black lines). Superresolution data are presented using a “hot” color map in which single-molecule localization density scales from black (lowest) to red to yellow to white (highest). (Scale bars: 500 nm.) The minimum and maximum values of detected photons per square nanometer used to set the display scale is shown to right of each superresolution image, and the SD of the Gaussian blur used in the construction of each superresolution image is denoted in the top right corner.
    Figure Legend Snippet: Single-molecule superresolution imaging of OligoMiner oligos. ( A and B ) Diffraction-limited ( A ) and superresolved STORM ( B ) images of a probe set consisting of 3,678 LDM oligos targeting 1,035 kb at 19p13.2 in human XY 2N PGP-1 fibroblasts. ( C and D ) Diffraction-limited ( C ) and superresolved STORM ( D ) images of a probe set consisting of 104 LDM oligos targeting 20 kb at 19p13.2 in PGP-1 fibroblasts. ( E and F ) Diffraction-limited ( E ) and superresolved DNA-PAINT ( F ) images of a probe set consisting of 4,776 UM oligos targeting 817 kb at Xq28 in human XY 2N MRC-5 fibroblasts. ( G and H ) Diffraction-limited ( G ) and superresolved DNA-PAINT ( H ) images of a probe set consisting of 176 LDM oligos targeting 11 kb of the Xist RNA in human XX 2N WI-38 fibroblasts. ( i – viii ) Normalized single-molecule counts along the indicated 1D line traces (blue bars) and one- or two-component Gaussian fits to the underlying data (black lines). Superresolution data are presented using a “hot” color map in which single-molecule localization density scales from black (lowest) to red to yellow to white (highest). (Scale bars: 500 nm.) The minimum and maximum values of detected photons per square nanometer used to set the display scale is shown to right of each superresolution image, and the SD of the Gaussian blur used in the construction of each superresolution image is denoted in the top right corner.

    Techniques Used: Imaging

    16) Product Images from "Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq"

    Article Title: Analyzing tumor heterogeneity and driver genes in single myeloid leukemia cells with SBCapSeq

    Journal: Nature biotechnology

    doi: 10.1038/nbt.3637

    The SBCapSeq method for sequencing transposon insertions sites from tumors. ( a ) Capture hybridization probes (orange bars) were designed to hybridize to the first 120 nucleotides from the 5′end of the IRDRL transposon inverted repeat and the last 120 nucleotides from the 3′end of the IRDRR of the SB transposon. Capture probes contain biotin moieties that allow for capture by streptavidin beads. Blocking oligos (blue bars) were designed from vector sequence to minimize capture of unmobilized transposon remaining at the donor site where the transposon concatamer first integrated. Blocking oligos contain a bulky adduct (yellow circle) that prevents isolation by bead capture. IRDRL, inverted repeat direct repeat left; IRDRR, inverted repeat direct repeat right; SA, splice acceptor; MSCV, murine stem cell virus minimal promoter; SD, splice donor; En2-SA, engrailed 2 splice acceptor. ( b ) Liquid capture hybridization performed on DNA libraries prepared from sheared genomic tumor DNA enriched for DNA fragments containing transposon sequences. Fragments are isolated and sequenced on the Ion Torrent platform. A custom SBCapSeq bioinformatics pipeline then maps SB insertions to the mouse genome. ( c ) Sequence analysis of nine independent SB capture hybridization reactions from a single ML tumor library to assess reproducibility of the method for detecting reads (left) or fragments (right) with SB insertions in genes.
    Figure Legend Snippet: The SBCapSeq method for sequencing transposon insertions sites from tumors. ( a ) Capture hybridization probes (orange bars) were designed to hybridize to the first 120 nucleotides from the 5′end of the IRDRL transposon inverted repeat and the last 120 nucleotides from the 3′end of the IRDRR of the SB transposon. Capture probes contain biotin moieties that allow for capture by streptavidin beads. Blocking oligos (blue bars) were designed from vector sequence to minimize capture of unmobilized transposon remaining at the donor site where the transposon concatamer first integrated. Blocking oligos contain a bulky adduct (yellow circle) that prevents isolation by bead capture. IRDRL, inverted repeat direct repeat left; IRDRR, inverted repeat direct repeat right; SA, splice acceptor; MSCV, murine stem cell virus minimal promoter; SD, splice donor; En2-SA, engrailed 2 splice acceptor. ( b ) Liquid capture hybridization performed on DNA libraries prepared from sheared genomic tumor DNA enriched for DNA fragments containing transposon sequences. Fragments are isolated and sequenced on the Ion Torrent platform. A custom SBCapSeq bioinformatics pipeline then maps SB insertions to the mouse genome. ( c ) Sequence analysis of nine independent SB capture hybridization reactions from a single ML tumor library to assess reproducibility of the method for detecting reads (left) or fragments (right) with SB insertions in genes.

    Techniques Used: Sequencing, Hybridization, Blocking Assay, Plasmid Preparation, Isolation

    17) Product Images from "Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology"

    Article Title: Efficient mouse genome engineering by CRISPR-EZ (CRISPR RNP Electroporation of Zygotes) technology

    Journal: Nature protocols

    doi: 10.1038/nprot.2018.012

    Overview of CRISPR-EZ technology and workflow. (a) An illustration of the most successful CRISPR-EZ editing strategies. A single sgRNA can be used to create a small indel via NHEJ repair or in conjunction with a ssODNs to create a precision mutation or a small insertion by HDR. Multiple sgRNAs can be used to engineer a genomic deletion by NHEJ repair. Design sgRNAs, HDR donor oligos, and editing validation assays prior to CRISPR-EZ experiments. (b) A graphic overview of CRISPR-EZ workflow. Day 1–3: ~4 week-old females are superovulated, first by PMSG injection, 46–48 hours later by hCG injection, before being housed with stud males for breeding. In parallel, sgRNAs are in vitro transcribed and purified. Day 4: Pronuclear stage embryos are collected and processed for electroporation, while Cas9/sgRNA complexes are assembled in vitro . Embryos (harvested at 0.5 dpc), Cas9/sgRNA RNPs, and optional ssODNs are combined in an electroporation cuvette and subjected to a series of electrical pulses. We recommend ex vivo validation of sgRNA editing efficiency in cultured morulae or blastocysts before generating edited mice. With a validated sgRNA design, electroporated embryos can be transferred to the oviduct of 0.5 dpc, pseudopregnant mothers to generate genetically engineered mice, which are then genotyped to confirm editing efficiency.
    Figure Legend Snippet: Overview of CRISPR-EZ technology and workflow. (a) An illustration of the most successful CRISPR-EZ editing strategies. A single sgRNA can be used to create a small indel via NHEJ repair or in conjunction with a ssODNs to create a precision mutation or a small insertion by HDR. Multiple sgRNAs can be used to engineer a genomic deletion by NHEJ repair. Design sgRNAs, HDR donor oligos, and editing validation assays prior to CRISPR-EZ experiments. (b) A graphic overview of CRISPR-EZ workflow. Day 1–3: ~4 week-old females are superovulated, first by PMSG injection, 46–48 hours later by hCG injection, before being housed with stud males for breeding. In parallel, sgRNAs are in vitro transcribed and purified. Day 4: Pronuclear stage embryos are collected and processed for electroporation, while Cas9/sgRNA complexes are assembled in vitro . Embryos (harvested at 0.5 dpc), Cas9/sgRNA RNPs, and optional ssODNs are combined in an electroporation cuvette and subjected to a series of electrical pulses. We recommend ex vivo validation of sgRNA editing efficiency in cultured morulae or blastocysts before generating edited mice. With a validated sgRNA design, electroporated embryos can be transferred to the oviduct of 0.5 dpc, pseudopregnant mothers to generate genetically engineered mice, which are then genotyped to confirm editing efficiency.

    Techniques Used: CRISPR, Non-Homologous End Joining, Mutagenesis, Injection, In Vitro, Purification, Electroporation, Ex Vivo, Cell Culture, Mouse Assay

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    Article Title: Mechanism of HIV-1 RNA Dimerization in the Central Region of the Genome and Significance for Viral Evolution *
    Article Snippet: .. DNA oligonucleotides and the HPLC-purified RNA strand used for CD spectra analyses were purchased from Integrated DNA Technologies, Inc. (Coralville, IA). ..

    Article Title: Versatile kit of robust nanoshapes self-assembling from RNA and DNA modules
    Article Snippet: .. Materials HPLC or gel-purified RNA and DNA oligonucleotides were purchased from Integrated DNA Technologies. ..

    Clone Assay:

    Article Title: Hyperosmotic stress memory in Arabidopsis is mediated by distinct epigenetically labile sites in the genome and is restricted in the male germline by DNA glycosylase activity
    Article Snippet: .. A pair of guide RNAs was selected using the CRISPR-PLANT tool , the corresponding DNA oligonucleotides (Integrated DNA Technologies) were cloned into pEN-Chimera using BbsI and BsmBI to generate plasmids pEN-CNI1.1. .. Constructs were transferred into pDE-CAS9 plasmid by Gateway cloning (Invitrogen) and transformed by floral dipping ( ).

    Ion Exchange Chromatography:

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions
    Article Snippet: .. A self-complementary DNA oligonucleotide carrying p53 DNA half-site of the sequence 5′-c GGGCATGCCC g-3′ (consensus sequence underlined) was purchased after standard desalting and lyophilization from IDT (Integrated DNA Technologies, Israel) and purified by ion-exchange chromatography. ..

    Synthesized:

    Article Title: Efficient assembly of very short oligonucleotides using T4 DNA Ligase
    Article Snippet: .. Preparation of immobilized dsDNA All oligos, including those 5'-biotinylated, 3'-FAM6, and 5'-phosphorylated were synthesized by Integrated DNA Technologies (IDT Inc., IA, USA). .. Immobilized double stranded DNA preparation involved purification of strepdavidin coated magnetic beads, binding of the biotinylated top strand, and then annealing of the complementary bottom strand.

    Purification:

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions
    Article Snippet: .. A self-complementary DNA oligonucleotide carrying p53 DNA half-site of the sequence 5′-c GGGCATGCCC g-3′ (consensus sequence underlined) was purchased after standard desalting and lyophilization from IDT (Integrated DNA Technologies, Israel) and purified by ion-exchange chromatography. ..

    IA:

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    other:

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    Sequencing:

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions
    Article Snippet: .. A self-complementary DNA oligonucleotide carrying p53 DNA half-site of the sequence 5′-c GGGCATGCCC g-3′ (consensus sequence underlined) was purchased after standard desalting and lyophilization from IDT (Integrated DNA Technologies, Israel) and purified by ion-exchange chromatography. ..

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    Integrated DNA Technologies gene specific qrt pcr primers
    Validation of microarray data using <t>qRT-PCR</t> during fibre development stages (0, 5, 10, 15 and 20 dpa) in the fl mutant. Y -axis represents the log2 fold change values at various stages in the fl mutant as compared to their respective stages in WT.
    Gene Specific Qrt Pcr Primers, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 89/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrated DNA Technologies rna dna oligonucleotides rgrgrgrcrgrgrgrcrcrcrgrcrcrc
    Crystal structures of Mss116p D2 bound to A-form duplexes a , The 14-bp self-complementary GC-rich <t>RNA</t> duplex substrate. b, The 14-bp GC-rich chimeric <t>RNA-DNA</t> duplex substrate. c-e , Orthogonal views of the D2-dsRNA complex colored as in Fig. 1a and Fig. 2a. Helix α 14 of D2, which contains motif IVa, faces the major groove of the dsRNA, and α 18 and α 20 of the CTE face the minor groove of the dsRNA. f-h , Orthogonal views of the D2-dsRNA-DNA complex, colored as in Fig. 1a and Fig. 2b, in which D2 is bound to two stacked 14-bp chimeric RNA-DNA duplexes.
    Rna Dna Oligonucleotides Rgrgrgrcrgrgrgrcrcrcrgrcrcrc, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Integrated DNA Technologies microsatellite primers flanking keap1
    Somatic Alterations in <t>KEAP1</t> in Lung Cancer (A) LOH at the 19p13.1–13.3 region. A heat map depicts <t>microsatellite-based</t> LOH at 19p13.1–19p13.3 in 181 lung cancer cell lines. Tumor-derived cell lines that were non-informative for this region were not included. Microsatellite markers showing heterozygous typings are depicted in red, markers demonstrating homozygous typing are in green, and non-informative markers in black. Each vertical column represents one cell line. ET, endocrine tumors; NS, no subtype specified; SCC, small cell carcinoma. (B) Sequence analysis of KEAP1 mutations in lung cancer. Part a shows the H838 cell line showing C–A substitution (G–T, plus strand), resulting in a termination codon. Wild-type sequence is from BEAS2B. A wild-type allele was not detected in H838. Part b shows a 18-bp deletion in one allele but not in the other allele in the PF DNA sample from PF-8. Part c shows tumor PT-23, showing A–T substitution in one allele but not in the other allele. Part d shows a 2-bp deletion in the fourth exon of KEAP1 that was detected in one allele of PT-17. Samples showing deletion mutations were confirmed by subcloning and sequencing. (C) LOH at the KEAP1 locus in human primary lung tumors. Summary of LOH patterns of 39 NSCLC tumors. Retained microsatellites are indicated in red, markers demonstrating allelic loss in green, markers showing genomic instability in white, and non-informative markers in black. KEAP-UM1 (CA 17 ) is present upstream of the KEAP1 locus, and KEAP-DM1 (CA 21 ) is present downstream of the KEAP1 locus.
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    Integrated DNA Technologies elk1 oligonucleotides
    Functional analysis of the KATNB1 promoter region. ( A ) The analyzed KATNB1 promoter region is 1000- bp (F1) excluding 5′-UTR. The CpG island is 332 nucleotides in length and positioned between -1884 and -1553. The optimal promoter region showing the highest promoter activity is 518- bp in length and positioned between -2013 and -1496 (F2). Location of <t>Elk1</t> consensus sequence (CTTCCTCTT) is indicated in the figure ( B ) Luminometric analysis of promoter constructs. SH-SY5Y cells were co-transfected with promoter constructs and pRL-TK Renilla luciferase vector. Results were calculated as fold activation relative to empty pGL3-basic vector. Error bars represent ± SD. Asterisk symbol (*) indicates p-value
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    Validation of microarray data using qRT-PCR during fibre development stages (0, 5, 10, 15 and 20 dpa) in the fl mutant. Y -axis represents the log2 fold change values at various stages in the fl mutant as compared to their respective stages in WT.

    Journal: BMC Genomics

    Article Title: Functional genomics of fuzzless-lintless mutant of Gossypium hirsutum L. cv. MCU5 reveal key genes and pathways involved in cotton fibre initiation and elongation

    doi: 10.1186/1471-2164-13-624

    Figure Lengend Snippet: Validation of microarray data using qRT-PCR during fibre development stages (0, 5, 10, 15 and 20 dpa) in the fl mutant. Y -axis represents the log2 fold change values at various stages in the fl mutant as compared to their respective stages in WT.

    Article Snippet: Gene specific qRT-PCR primers were designed using PrimerQuest software ( http://eu.idtdna.com ) and the list of primers are presented in Additional file .

    Techniques: Microarray, Quantitative RT-PCR, Mutagenesis

    Crystal structures of Mss116p D2 bound to A-form duplexes a , The 14-bp self-complementary GC-rich RNA duplex substrate. b, The 14-bp GC-rich chimeric RNA-DNA duplex substrate. c-e , Orthogonal views of the D2-dsRNA complex colored as in Fig. 1a and Fig. 2a. Helix α 14 of D2, which contains motif IVa, faces the major groove of the dsRNA, and α 18 and α 20 of the CTE face the minor groove of the dsRNA. f-h , Orthogonal views of the D2-dsRNA-DNA complex, colored as in Fig. 1a and Fig. 2b, in which D2 is bound to two stacked 14-bp chimeric RNA-DNA duplexes.

    Journal: Nature

    Article Title: Structural basis for RNA duplex recognition and unwinding by the DEAD-box helicase Mss116p

    doi: 10.1038/nature11402

    Figure Lengend Snippet: Crystal structures of Mss116p D2 bound to A-form duplexes a , The 14-bp self-complementary GC-rich RNA duplex substrate. b, The 14-bp GC-rich chimeric RNA-DNA duplex substrate. c-e , Orthogonal views of the D2-dsRNA complex colored as in Fig. 1a and Fig. 2a. Helix α 14 of D2, which contains motif IVa, faces the major groove of the dsRNA, and α 18 and α 20 of the CTE face the minor groove of the dsRNA. f-h , Orthogonal views of the D2-dsRNA-DNA complex, colored as in Fig. 1a and Fig. 2b, in which D2 is bound to two stacked 14-bp chimeric RNA-DNA duplexes.

    Article Snippet: Oligonucleotides The RNA and RNA-DNA oligonucleotides rGrGrGrCrGrGrGrCrCrCrGrCrCrC and rGrGrGrCrGrGrGdCdCdCdGdCdCdC (Integrated DNA Technologies) were annealed to form 14-bp RNA or chimeric RNA-DNA duplexes by heating 6 mM solutions in 100 mM potassium acetate, 30 mM HEPES (pH 7.5) at 94°C for 1 min and then slowly cooling to room temperature over 1 h.

    Techniques:

    Interactions between Mss116p D2 and duplex RNA a , Schematic of RNA-protein interactions observed in the D2-dsRNA structure. The dsRNA interacts with conserved DEAD-box motifs IV-Va of D2 (green) and the CTE of D2 (orange). The box indicates that the interaction is maintained in closed-state Mss116p 10 . RNA bases are numbered according to their position relative to ssRNA in closed-state Mss116p (see Supplementary Fig. 4d ). Similar nucleic acid-protein interactions were observed in the D2-dsRNA-DNA structure ( Supplementary Fig. 5 ). b , Interactions between strand 1 (yellow) of the duplex RNA and D2 (green). c , Interactions between duplex RNA and the CTE of D2 (orange).

    Journal: Nature

    Article Title: Structural basis for RNA duplex recognition and unwinding by the DEAD-box helicase Mss116p

    doi: 10.1038/nature11402

    Figure Lengend Snippet: Interactions between Mss116p D2 and duplex RNA a , Schematic of RNA-protein interactions observed in the D2-dsRNA structure. The dsRNA interacts with conserved DEAD-box motifs IV-Va of D2 (green) and the CTE of D2 (orange). The box indicates that the interaction is maintained in closed-state Mss116p 10 . RNA bases are numbered according to their position relative to ssRNA in closed-state Mss116p (see Supplementary Fig. 4d ). Similar nucleic acid-protein interactions were observed in the D2-dsRNA-DNA structure ( Supplementary Fig. 5 ). b , Interactions between strand 1 (yellow) of the duplex RNA and D2 (green). c , Interactions between duplex RNA and the CTE of D2 (orange).

    Article Snippet: Oligonucleotides The RNA and RNA-DNA oligonucleotides rGrGrGrCrGrGrGrCrCrCrGrCrCrC and rGrGrGrCrGrGrGdCdCdCdGdCdCdC (Integrated DNA Technologies) were annealed to form 14-bp RNA or chimeric RNA-DNA duplexes by heating 6 mM solutions in 100 mM potassium acetate, 30 mM HEPES (pH 7.5) at 94°C for 1 min and then slowly cooling to room temperature over 1 h.

    Techniques:

    Somatic Alterations in KEAP1 in Lung Cancer (A) LOH at the 19p13.1–13.3 region. A heat map depicts microsatellite-based LOH at 19p13.1–19p13.3 in 181 lung cancer cell lines. Tumor-derived cell lines that were non-informative for this region were not included. Microsatellite markers showing heterozygous typings are depicted in red, markers demonstrating homozygous typing are in green, and non-informative markers in black. Each vertical column represents one cell line. ET, endocrine tumors; NS, no subtype specified; SCC, small cell carcinoma. (B) Sequence analysis of KEAP1 mutations in lung cancer. Part a shows the H838 cell line showing C–A substitution (G–T, plus strand), resulting in a termination codon. Wild-type sequence is from BEAS2B. A wild-type allele was not detected in H838. Part b shows a 18-bp deletion in one allele but not in the other allele in the PF DNA sample from PF-8. Part c shows tumor PT-23, showing A–T substitution in one allele but not in the other allele. Part d shows a 2-bp deletion in the fourth exon of KEAP1 that was detected in one allele of PT-17. Samples showing deletion mutations were confirmed by subcloning and sequencing. (C) LOH at the KEAP1 locus in human primary lung tumors. Summary of LOH patterns of 39 NSCLC tumors. Retained microsatellites are indicated in red, markers demonstrating allelic loss in green, markers showing genomic instability in white, and non-informative markers in black. KEAP-UM1 (CA 17 ) is present upstream of the KEAP1 locus, and KEAP-DM1 (CA 21 ) is present downstream of the KEAP1 locus.

    Journal: PLoS Medicine

    Article Title: Dysfunctional KEAP1-NRF2 Interaction in Non-Small-Cell Lung Cancer

    doi: 10.1371/journal.pmed.0030420

    Figure Lengend Snippet: Somatic Alterations in KEAP1 in Lung Cancer (A) LOH at the 19p13.1–13.3 region. A heat map depicts microsatellite-based LOH at 19p13.1–19p13.3 in 181 lung cancer cell lines. Tumor-derived cell lines that were non-informative for this region were not included. Microsatellite markers showing heterozygous typings are depicted in red, markers demonstrating homozygous typing are in green, and non-informative markers in black. Each vertical column represents one cell line. ET, endocrine tumors; NS, no subtype specified; SCC, small cell carcinoma. (B) Sequence analysis of KEAP1 mutations in lung cancer. Part a shows the H838 cell line showing C–A substitution (G–T, plus strand), resulting in a termination codon. Wild-type sequence is from BEAS2B. A wild-type allele was not detected in H838. Part b shows a 18-bp deletion in one allele but not in the other allele in the PF DNA sample from PF-8. Part c shows tumor PT-23, showing A–T substitution in one allele but not in the other allele. Part d shows a 2-bp deletion in the fourth exon of KEAP1 that was detected in one allele of PT-17. Samples showing deletion mutations were confirmed by subcloning and sequencing. (C) LOH at the KEAP1 locus in human primary lung tumors. Summary of LOH patterns of 39 NSCLC tumors. Retained microsatellites are indicated in red, markers demonstrating allelic loss in green, markers showing genomic instability in white, and non-informative markers in black. KEAP-UM1 (CA 17 ) is present upstream of the KEAP1 locus, and KEAP-DM1 (CA 21 ) is present downstream of the KEAP1 locus.

    Article Snippet: Two pairs of fluorescently labeled microsatellite primers flanking KEAP1 (KEAP-UM1 [CA17 ], present upstream of the KEAP1 locus, and KEAP-DM1 [CA21 ], present downstream of the KEAP1 locus) were designed using Primer3 software and synthesized by Integrated DNA Technologies.

    Techniques: Derivative Assay, Sequencing, Subcloning

    Functional analysis of the KATNB1 promoter region. ( A ) The analyzed KATNB1 promoter region is 1000- bp (F1) excluding 5′-UTR. The CpG island is 332 nucleotides in length and positioned between -1884 and -1553. The optimal promoter region showing the highest promoter activity is 518- bp in length and positioned between -2013 and -1496 (F2). Location of Elk1 consensus sequence (CTTCCTCTT) is indicated in the figure ( B ) Luminometric analysis of promoter constructs. SH-SY5Y cells were co-transfected with promoter constructs and pRL-TK Renilla luciferase vector. Results were calculated as fold activation relative to empty pGL3-basic vector. Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: Functional analysis of the KATNB1 promoter region. ( A ) The analyzed KATNB1 promoter region is 1000- bp (F1) excluding 5′-UTR. The CpG island is 332 nucleotides in length and positioned between -1884 and -1553. The optimal promoter region showing the highest promoter activity is 518- bp in length and positioned between -2013 and -1496 (F2). Location of Elk1 consensus sequence (CTTCCTCTT) is indicated in the figure ( B ) Luminometric analysis of promoter constructs. SH-SY5Y cells were co-transfected with promoter constructs and pRL-TK Renilla luciferase vector. Results were calculated as fold activation relative to empty pGL3-basic vector. Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Functional Assay, Activity Assay, Sequencing, Construct, Transfection, Luciferase, Plasmid Preparation, Activation Assay

    Elk1 activates KATNB1 promoter and increases katanin-p80 mRNA. ( A ) Forced luciferase experiment to compare effects of Elk1 on KATNB1 optimal promoter activation. SH-SY5Y cells were co-transfected with (I) F1 construct and either pCMV-empty vector (F1) or pCMV6-Elk1 (F1+Elk1) and (II) F2 construct and either control (F2) or Elk1 (F2+Elk1). (III) Normalization of data is shown in I and II. Promoter activity of F1 and F2 constructs transfected with Elk1 containing vector were normalized to F1 and F2 constructs transfected with control vector, respectively. ( B ) Western blot image represents the total protein lysate obtained either from Elk1 transfected (overexpression) or naive SH-SY5Y cells (control). Cells were immunolabeled for Elk 1 and β-actin. ( C ) Relative mRNA quantification of katanin-p80 mRNA level in Elk1 overexpressing SH-SY5Y cells (Elk1). Results were calculated by normalizing the Ct values of the KATNB1 gene to β-actin as internal control. Results represent ΔΔCt values which gives mRNA fold change relative to naive cells (control). Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: Elk1 activates KATNB1 promoter and increases katanin-p80 mRNA. ( A ) Forced luciferase experiment to compare effects of Elk1 on KATNB1 optimal promoter activation. SH-SY5Y cells were co-transfected with (I) F1 construct and either pCMV-empty vector (F1) or pCMV6-Elk1 (F1+Elk1) and (II) F2 construct and either control (F2) or Elk1 (F2+Elk1). (III) Normalization of data is shown in I and II. Promoter activity of F1 and F2 constructs transfected with Elk1 containing vector were normalized to F1 and F2 constructs transfected with control vector, respectively. ( B ) Western blot image represents the total protein lysate obtained either from Elk1 transfected (overexpression) or naive SH-SY5Y cells (control). Cells were immunolabeled for Elk 1 and β-actin. ( C ) Relative mRNA quantification of katanin-p80 mRNA level in Elk1 overexpressing SH-SY5Y cells (Elk1). Results were calculated by normalizing the Ct values of the KATNB1 gene to β-actin as internal control. Results represent ΔΔCt values which gives mRNA fold change relative to naive cells (control). Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Luciferase, Activation Assay, Transfection, Construct, Plasmid Preparation, Activity Assay, Western Blot, Over Expression, Immunolabeling

    Confirmation of Elk1 binding to the corresponding site on the KATNB1 promoter by EMSA. ( A ) Extract free biotin-11-UTP labeled Elk1 oligonucleotides (WT, wild type; Mut, mutated) were illustrated in lanes 1 and 3, respectively. SH-SY5Y cell extract was added to the reaction mixture and incubated with WT and Mut oligonucleotides to form nucleotide-protein complex (lane 2 and lane 4, respectively). 1000-fold excess of unlabeled WT competitor Elk1 oligonucleotide was added to the binding reaction mixture (lane 5). Arrow shows band of SH-SY5Y cell extract-WT oligonucleotide complex (lane 2). Lane 1 and 3 represent only WT and Mut oligonucleotides, respectively that were loaded as controls. ( B ) Super-shift assay confirmed the specificity of Elk1 binding on the KATNB1 promoter. WT Elk1 oligonucleotide was incubated with recombinant Elk1- db protein either alone (lane 1) or in the presence of His-tag antibody (lane 2). Mut Elk1 oligonucleotides were also incubated with recombinant Elk1- db protein either alone (lane 3) or in the presence of His-tag antibody (lane 4). Arrowhead shows band of Elk1- db-WT oligonucleotide complex and the arrow shows the band of Elk1- db-WT oligonucleotide-His-tag antibody complex. ( C ) SH-SY5Y cell extract was added to the reaction mixture and incubated with WT oligonucleotides in the presence or absence of 1000 fold excess of unlabeled Mut competitor oligonucleotides to form nucleotide-protein complex (lane 1 and lane 2, respectively). Lane 3 shows Elk1- db-WT oligonucleotide complex. Arrows show bands of SH-SY5Y cell extract-WT oligonucleotide complexes and arrowheads indicate 95 amino acids splice variant of Elk1.

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: Confirmation of Elk1 binding to the corresponding site on the KATNB1 promoter by EMSA. ( A ) Extract free biotin-11-UTP labeled Elk1 oligonucleotides (WT, wild type; Mut, mutated) were illustrated in lanes 1 and 3, respectively. SH-SY5Y cell extract was added to the reaction mixture and incubated with WT and Mut oligonucleotides to form nucleotide-protein complex (lane 2 and lane 4, respectively). 1000-fold excess of unlabeled WT competitor Elk1 oligonucleotide was added to the binding reaction mixture (lane 5). Arrow shows band of SH-SY5Y cell extract-WT oligonucleotide complex (lane 2). Lane 1 and 3 represent only WT and Mut oligonucleotides, respectively that were loaded as controls. ( B ) Super-shift assay confirmed the specificity of Elk1 binding on the KATNB1 promoter. WT Elk1 oligonucleotide was incubated with recombinant Elk1- db protein either alone (lane 1) or in the presence of His-tag antibody (lane 2). Mut Elk1 oligonucleotides were also incubated with recombinant Elk1- db protein either alone (lane 3) or in the presence of His-tag antibody (lane 4). Arrowhead shows band of Elk1- db-WT oligonucleotide complex and the arrow shows the band of Elk1- db-WT oligonucleotide-His-tag antibody complex. ( C ) SH-SY5Y cell extract was added to the reaction mixture and incubated with WT oligonucleotides in the presence or absence of 1000 fold excess of unlabeled Mut competitor oligonucleotides to form nucleotide-protein complex (lane 1 and lane 2, respectively). Lane 3 shows Elk1- db-WT oligonucleotide complex. Arrows show bands of SH-SY5Y cell extract-WT oligonucleotide complexes and arrowheads indicate 95 amino acids splice variant of Elk1.

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Binding Assay, Labeling, Incubation, Super-Shift Assay, Recombinant, Variant Assay

    Putative Elk1 transcription factor binding site on the KATNB1 promoter. Forward primer binding sites for each construct are indicated as F1, F2, F3, F4, F5 and F6. Elk1 binding site represented as bold. Underlined and grey highlighted region show the probe selected for EMSA assay, the dark grey highlighted region represents serum response element (SRE) to which serum response factor (SRF) binds and GC boxes are shown in frames. The largest frame represents CpG island. CpG dinucleotides are shown with arrowheads.

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: Putative Elk1 transcription factor binding site on the KATNB1 promoter. Forward primer binding sites for each construct are indicated as F1, F2, F3, F4, F5 and F6. Elk1 binding site represented as bold. Underlined and grey highlighted region show the probe selected for EMSA assay, the dark grey highlighted region represents serum response element (SRE) to which serum response factor (SRF) binds and GC boxes are shown in frames. The largest frame represents CpG island. CpG dinucleotides are shown with arrowheads.

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Binding Assay, Construct

    KCl treatment increases Elk1 SUMOylation and decreases katanin-p60 expression. ( A ) The effect of KCl treatment on Elk1 mediated regulation of KATNB1 promoter. SH-SY5Y cells were co-transfected with F2 construct and either pCMV-empty vector (F2), or pCMV6-Elk1 (F2+Elk1). 24 h post-transfection, group of the Elk1 transfected cells were incubated in medium containing 50 µM KCl for 1 h (F2+Elk1+KCl). After 48 h following transfection, luminometric analysis was performed and fold activities were calculated relative to empty pGL3-basic vector. ( B ) Western blot analysis of katanin-p80 in SH-SY5Y cells. We performed analysis with both pCMV6-Elk1 transfected cells which were either untreated (Elk1) or treated with KCl (Elk1+KCl) and naive cells which were either untreated (control) or treated with KCl (control+KCl). Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: KCl treatment increases Elk1 SUMOylation and decreases katanin-p60 expression. ( A ) The effect of KCl treatment on Elk1 mediated regulation of KATNB1 promoter. SH-SY5Y cells were co-transfected with F2 construct and either pCMV-empty vector (F2), or pCMV6-Elk1 (F2+Elk1). 24 h post-transfection, group of the Elk1 transfected cells were incubated in medium containing 50 µM KCl for 1 h (F2+Elk1+KCl). After 48 h following transfection, luminometric analysis was performed and fold activities were calculated relative to empty pGL3-basic vector. ( B ) Western blot analysis of katanin-p80 in SH-SY5Y cells. We performed analysis with both pCMV6-Elk1 transfected cells which were either untreated (Elk1) or treated with KCl (Elk1+KCl) and naive cells which were either untreated (control) or treated with KCl (control+KCl). Error bars represent ± SD. Asterisk symbol (*) indicates p-value

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Expressing, Transfection, Construct, Plasmid Preparation, Incubation, Western Blot

    KCl stimulated Elk1 SUMOylation reduces endogenous katanin-p60 protein level in SH-SY5Y cells. ( A ) Immunocytochemistry staining of endogenous katanin-p 80 to reveal the effect of KCl treatment on the Elk1 mediated expression of KATNB1 gene in SH-SY5Y cells. Control represents naive SH-SY5Y cells that were neither transfected nor KCl treated; Elk1 represents SH-SY5Y cells that were pCMV6-Elk1 transfected and finally Elk1+KCl represents SH-SY5Y cells that were both pCMV6-Elk1 transfected and KCl treated. Green indicates katanin-p80 staining, while blue is for nuclear DAPI staining. ( B ) Integrated pixel analysis for katanin-p80 expression in panel A.

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: KCl stimulated Elk1 SUMOylation reduces endogenous katanin-p60 protein level in SH-SY5Y cells. ( A ) Immunocytochemistry staining of endogenous katanin-p 80 to reveal the effect of KCl treatment on the Elk1 mediated expression of KATNB1 gene in SH-SY5Y cells. Control represents naive SH-SY5Y cells that were neither transfected nor KCl treated; Elk1 represents SH-SY5Y cells that were pCMV6-Elk1 transfected and finally Elk1+KCl represents SH-SY5Y cells that were both pCMV6-Elk1 transfected and KCl treated. Green indicates katanin-p80 staining, while blue is for nuclear DAPI staining. ( B ) Integrated pixel analysis for katanin-p80 expression in panel A.

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Immunocytochemistry, Staining, Expressing, Transfection

    Schematic illustration of human katanin-p80 and Elk1 proteins. ( A ) Katanin-p80 has WD40 domain which negatively regulates microtubule severing activity of katanin-p60, and procon80 domain which is required for interaction with katanin-p60 and includes microtubule binding domain (dark grey). ( B ) The A domain (or ETS domain) is responsible for binding to DNA. The B domain is involved in interaction of Elk1 with SRF. The R domain including the lysine residues of Elk1 associated with SUMO modification that is important for repressive activity of Elk1. The D domain is docking site for MAP kinases. The C domain (or transactivation domain) contains serine residues at positions 383 and 389 which are phosphorylated by MAP kinases. Illustrations were performed by the use of DOG 1.0 and Adobe Photoshop CS5 softwares.

    Journal: PLoS ONE

    Article Title: Katanin-p80 Gene Promoter Characterization and Regulation via Elk1

    doi: 10.1371/journal.pone.0069423

    Figure Lengend Snippet: Schematic illustration of human katanin-p80 and Elk1 proteins. ( A ) Katanin-p80 has WD40 domain which negatively regulates microtubule severing activity of katanin-p60, and procon80 domain which is required for interaction with katanin-p60 and includes microtubule binding domain (dark grey). ( B ) The A domain (or ETS domain) is responsible for binding to DNA. The B domain is involved in interaction of Elk1 with SRF. The R domain including the lysine residues of Elk1 associated with SUMO modification that is important for repressive activity of Elk1. The D domain is docking site for MAP kinases. The C domain (or transactivation domain) contains serine residues at positions 383 and 389 which are phosphorylated by MAP kinases. Illustrations were performed by the use of DOG 1.0 and Adobe Photoshop CS5 softwares.

    Article Snippet: Elk1 and mutated Elk1 oligonucleotides were synthesized by Integrated DNA Technologies, Inc. (Munich, Germany).

    Techniques: Activity Assay, Binding Assay, Modification