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
    Price:
    None
    Category:
    Nucleic acids
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    Structured Review

    Integrated DNA Technologies oligos
    Enhancement of T4 DNA ligase activity by supplemental <t>oligonucleotides.</t> (a) Unsuccessful 4-bp duplex reactions could be salvaged by utilizing a supplementary oligonucleotide, designed to complement the first <t>oligonucleotide-dsDNA</t> 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.
    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 95 stars, based on 10 article reviews
    Price from $9.99 to $1999.99
    oligos - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) 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

    2) 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

    3) 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

    4) 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

    5) 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

    6) 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

    7) 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

    8) 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

    9) 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

    10) 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

    11) 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

    12) 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

    13) 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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    Clone Assay:

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    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
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    Centrifugation:

    Article Title: Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
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    Amplification:

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    Synthesized:

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    Article Title: Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid
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    Autoradiography:

    Article Title: Synthesis and Labeling of RNA In Vitro
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    Blocking Assay:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 200 pmol/µl 2′-O-methyl RNA-DNA chimera (Integrated DNA Technologies, Inc.) RNA substrate from in vitro transcription (Basic Protocol 1) or in vivo purification 10 × buffer for RNase H (see recipe) 2 U/µl RNase H (Amersham) 40 U/µl RNase inhibitor (Thermo Scientific) G50 buffer (see recipe) 10 × buffer for CIP (see recipe) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) 10 × buffer for T4 PNK forward reaction (see recipe) [γ32 P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Bridging DNA oligo (Integrated DNA Technologies, Inc.) 10 × buffer for T4 DNA ligase (see recipe) 5 U/µl T4 DNA ligase (Thermo Scientific) 95°C heat block Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) ..

    Real-time Polymerase Chain Reaction:

    Article Title: Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts *
    Article Snippet: Paragraph title: RNA Isolation, cDNA Synthesis, and Quantitative PCR ... For cDNA synthesis, the RNA pellet was dissolved in diethyl pyrocarbonate (Sigma)-treated water, and 15 μg of RNA was heated at 70 °C for 5 min with oligo(dT) 15-mer primers (Integrated DNA Technologies, Coralville, IA).

    Article Title: Global analysis reveals multiple pathways for unique regulation of mRNA decay in induced pluripotent stem cells
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    Incubation:

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    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
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    Article Title: Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts *
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    Article Title: Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series
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    Activity Assay:

    Article Title: Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series
    Article Snippet: .. RNA-dependent DNA polymerase activity in the presence or absence of NcRTI was quantified on a poly(rA)/oligo(dT)15 substrate in an assay buffer (40-μl volume) containing 2 nM BH10 HIV reverse transcriptase enzyme, 50 mM Tris-HCl (pH 7.8), 60 mM NaCl, 2 mM MgCl2 · 6H2 O, 0.02% 3-[(3-cholamidopropyl)-dimethylamonio]-2-propanesulfonate (CHAPS), 6 mM dithiothreitol (DTT), 2 mM glutathione (GSH, reduced form), 45 nM poly(rA) (GE Healthcare), 4.5 nM oligo(dT15 ) (Integrated DNA Technologies), 1 μM dTTP, 1% dimethyl sulfoxide (DMSO), and 2 mM ATP. .. The reaction plates were incubated for 60 min at 37°C, followed by the addition of 40 μl of the intercalating dye Quant-iT picogreen (Life Technologies; diluted 1:375 from commercial stock solution in 20 mM Tris-HCl–25 mM EDTA, pH 8.0).

    Expressing:

    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
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    Article Title: Conidia but Not Yeast Cells of the Fungal Pathogen Histoplasma capsulatum Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ †
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    Article Title: Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid
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    Modification:

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies). .. The steps involved in the preparation of the vector-insert constructs were modified from the Gibson assembly since standard cloning by restriction digests and ligation was too inefficient to exchange the majority of the old inserts by a synthetic one.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The bridging DNA oligo is purchased by standard order from Integrated DNA Technologies (IDT). .. Except for its specific sequence (depending on the two fragments to be ligated), the oligo is a 30-nucleotide-long DNA oligo without any base or sugar ring modification.

    Hybridization:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: Hybridization with the oligonucleotide probes was done overnight in 6× SSC, 5× Denhardt's solution, and 1% SDS at 43°. .. For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°.

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: The hybridization was carried out by mixing equal amounts (2–10 µl) of 100 µM of two different single-stranded synthetic DNAs in hybridization buffer (50 mM Tris-HCl pH 7.4, 0.1 M NaCl) and incubating them with the following temperature program: 98 °C for 3 min, with a temperature decrease of 1 °C per min, and storage at 8 °C; the hybridization efficiency was monitored on a 10% polyacrylamide gel. .. All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies).

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: In this last phase of the protocol, the 5′ RNA fragment and the radiolabeled 3′ RNA fragment are precisely aligned together through hybridization with a complementary bridging DNA oligonucleotide, and ligated by T4 DNA ligase. .. The bridging DNA oligo is purchased by standard order from Integrated DNA Technologies (IDT).

    Ligation:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA). .. After each ligation step, the ligated products were selected by size fractionation using denaturing PAGE and purified from the gel as above.

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies). .. The steps involved in the preparation of the vector-insert constructs were modified from the Gibson assembly since standard cloning by restriction digests and ligation was too inefficient to exchange the majority of the old inserts by a synthetic one.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: Paragraph title: Two-piece splint ligation to rejoin the 5′ and 3′ RNA fragments ... The bridging DNA oligo is purchased by standard order from Integrated DNA Technologies (IDT).

    Northern Blot:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: Northern blot analysis of the pre-mRNA and spliceosomal snRNAs was conducted with random prime labeled probes (Promega, Madison, WI) prepared from cloned copies of the indicated DNAs that were hybridized and washed under standard conditions ( ). .. For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°.

    Solubility:

    Article Title: Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
    Article Snippet: E72A inactivates the enzyme, while C171A (distal the active site) enhances solubility of the expressed protein. .. The DNA oligo, d(TTTTTTTTCTTTTTT), was synthesized (Integrated DNA Technologies), and mixed with the purified A3A(E72A/C171A) protein at a molar ratio of 2:1.

    Polymerase Chain Reaction:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA). .. The first-strand cDNA was amplified using Taq DNA polymerase (Perkin Elmer) and 3′ PCR FusionB and 5′ PCR FusionA primers .

    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
    Article Snippet: For RT-PCR, 5 µg of total RNA was reverse-transcribed using oligo(dT) (Integrated DNA Technologies Inc., Coralville, IA) and SuperScript II reverse transcriptase (Invitrogen Corp.). .. Specific oligonucleotide primer pairs were incubated with cDNA template for PCR amplification using the Expand High FidelityPLUS PCR System (Roche Applied Science).

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: We used predictions based on type II CRISPR system and Cas9 protein phylogenies ( ; ) to generate crRNA and tracrRNA sequences; the Cdi guide RNA was based on an experimentally validated construct ( ). sgRNA-encoding DNAs were amplified by PCR to add a T7 promoter fragment (TAATACGACTCACTATAGG). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    Reverse Transcription Polymerase Chain Reaction:

    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
    Article Snippet: .. For RT-PCR, 5 µg of total RNA was reverse-transcribed using oligo(dT) (Integrated DNA Technologies Inc., Coralville, IA) and SuperScript II reverse transcriptase (Invitrogen Corp.). .. Specific oligonucleotide primer pairs were incubated with cDNA template for PCR amplification using the Expand High FidelityPLUS PCR System (Roche Applied Science).

    Nucleic Acid Electrophoresis:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°. .. Five units of Escherichia coli RNase H (United States Biochemical, Cleveland) were added and the reaction was incubated at 37° for 60 min prior to gel electrophoresis and Northern analysis.

    Article Title: Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
    Article Snippet: The purity and integrity of A3A(E72A/C171A) was confirmed by SDS–polyacrylamide gel electrophoresis. .. The DNA oligo, d(TTTTTTTTCTTTTTT), was synthesized (Integrated DNA Technologies), and mixed with the purified A3A(E72A/C171A) protein at a molar ratio of 2:1.

    In Vivo:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 200 pmol/µl 2′-O-methyl RNA-DNA chimera (Integrated DNA Technologies, Inc.) RNA substrate from in vitro transcription (Basic Protocol 1) or in vivo purification 10 × buffer for RNase H (see recipe) 2 U/µl RNase H (Amersham) 40 U/µl RNase inhibitor (Thermo Scientific) G50 buffer (see recipe) 10 × buffer for CIP (see recipe) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) 10 × buffer for T4 PNK forward reaction (see recipe) [γ32 P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Bridging DNA oligo (Integrated DNA Technologies, Inc.) 10 × buffer for T4 DNA ligase (see recipe) 5 U/µl T4 DNA ligase (Thermo Scientific) 95°C heat block Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) ..

    Fluorescence:

    Article Title: Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series
    Article Snippet: RNA-dependent DNA polymerase activity in the presence or absence of NcRTI was quantified on a poly(rA)/oligo(dT)15 substrate in an assay buffer (40-μl volume) containing 2 nM BH10 HIV reverse transcriptase enzyme, 50 mM Tris-HCl (pH 7.8), 60 mM NaCl, 2 mM MgCl2 · 6H2 O, 0.02% 3-[(3-cholamidopropyl)-dimethylamonio]-2-propanesulfonate (CHAPS), 6 mM dithiothreitol (DTT), 2 mM glutathione (GSH, reduced form), 45 nM poly(rA) (GE Healthcare), 4.5 nM oligo(dT15 ) (Integrated DNA Technologies), 1 μM dTTP, 1% dimethyl sulfoxide (DMSO), and 2 mM ATP. .. The plate was shaken for 10 s and incubated for 5 min at room temperature, and the fluorescence was measured using a Wallac Victor 2 plate reader (PerkinElmer) with wavelengths specific for fluorescein.

    Isolation:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: The isolated sRNAs were then sequentially ligated to adapters using T4 RNA ligase. .. A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA).

    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
    Article Snippet: Paragraph title: RNA Isolation and RT-PCR ... For RT-PCR, 5 µg of total RNA was reverse-transcribed using oligo(dT) (Integrated DNA Technologies Inc., Coralville, IA) and SuperScript II reverse transcriptase (Invitrogen Corp.).

    Article Title: Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts *
    Article Snippet: Paragraph title: RNA Isolation, cDNA Synthesis, and Quantitative PCR ... For cDNA synthesis, the RNA pellet was dissolved in diethyl pyrocarbonate (Sigma)-treated water, and 15 μg of RNA was heated at 70 °C for 5 min with oligo(dT) 15-mer primers (Integrated DNA Technologies, Coralville, IA).

    Labeling:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: Northern blot analysis of the pre-mRNA and spliceosomal snRNAs was conducted with random prime labeled probes (Promega, Madison, WI) prepared from cloned copies of the indicated DNAs that were hybridized and washed under standard conditions ( ). .. For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°.

    Purification:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: The RNA was purified by two additional PCI extractions followed by ethanol precipitation. .. For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°.

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: Briefly, sRNA fractions of 15 to 40-nt long were purified by size fractionation with 15% polyacrylamide gels (PAGE) containing 8M urea followed by gel elution in 0.3 M NaCl and RNA precipitation. .. A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA).

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 200 pmol/µl 2′-O-methyl RNA-DNA chimera (Integrated DNA Technologies, Inc.) RNA substrate from in vitro transcription (Basic Protocol 1) or in vivo purification 10 × buffer for RNase H (see recipe) 2 U/µl RNase H (Amersham) 40 U/µl RNase inhibitor (Thermo Scientific) G50 buffer (see recipe) 10 × buffer for CIP (see recipe) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) 10 × buffer for T4 PNK forward reaction (see recipe) [γ32 P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Bridging DNA oligo (Integrated DNA Technologies, Inc.) 10 × buffer for T4 DNA ligase (see recipe) 5 U/µl T4 DNA ligase (Thermo Scientific) 95°C heat block Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) ..

    Article Title: Crystal structure of APOBEC3A bound to single-stranded DNA reveals structural basis for cytidine deamination and specificity
    Article Snippet: .. The DNA oligo, d(TTTTTTTTCTTTTTT), was synthesized (Integrated DNA Technologies), and mixed with the purified A3A(E72A/C171A) protein at a molar ratio of 2:1. .. Crystallization and data collection Crystals of the A3A(E72A/C171A)–DNA complex were grown by hanging-drop vapour-diffusion method over a reservoir of 100 mM MOPS (pH 6.5), 50 mM MgCl2 , 50 mM CaCl2 , 23% polyethylene glycol 3,350 and 15% 2-methyl-2,4-pentanediol.

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: The T7 fragment-tagged DNAs were then used as templates for the in vitro transcription of sgRNAs as described The transcribed sgRNAs were purified by denaturing PAGE, ethanol precipitated and folded according to ( ). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    Sequencing:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: Paragraph title: sRNA library construction and sequencing ... A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA).

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: Two bases at positions 2540-2541 of the vector were designed to be different from the original HSI_vector sequence. .. All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies).

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: The bridging DNA oligo is purchased by standard order from Integrated DNA Technologies (IDT). .. Except for its specific sequence (depending on the two fragments to be ligated), the oligo is a 30-nucleotide-long DNA oligo without any base or sugar ring modification.

    Quantitative RT-PCR:

    Article Title: Conidia but Not Yeast Cells of the Fungal Pathogen Histoplasma capsulatum Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ †
    Article Snippet: .. For qRT-PCR, 1 μg of DNase I-treated total macrophage RNA was reverse transcribed with Affinity Script multitemperature reverse transcriptase (Stratagene) and 500 ng oligo(dT19 V) (Integrated DNA Technologies, San Diego, CA) for 2 h. cDNA was diluted 3- to 4-fold with pyrogen-free water. ..

    Article Title: Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid
    Article Snippet: Induction and analyses of gene expression The expression of HMGBs and defense-related maker genes were examined using the real-time RT-PCR (qRT-PCR) technique. .. The first-strand cDNA was synthesized from the total RNA (~2 μg) with Superscript III reverse transcriptase (Invitrogen) and oligo (dT)15 primer (Integrated DNA Technologies).

    Polyacrylamide Gel Electrophoresis:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: Briefly, sRNA fractions of 15 to 40-nt long were purified by size fractionation with 15% polyacrylamide gels (PAGE) containing 8M urea followed by gel elution in 0.3 M NaCl and RNA precipitation. .. A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA).

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: The T7 fragment-tagged DNAs were then used as templates for the in vitro transcription of sgRNAs as described The transcribed sgRNAs were purified by denaturing PAGE, ethanol precipitated and folded according to ( ). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    CRISPR:

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: We used predictions based on type II CRISPR system and Cas9 protein phylogenies ( ; ) to generate crRNA and tracrRNA sequences; the Cdi guide RNA was based on an experimentally validated construct ( ). sgRNA-encoding DNAs were amplified by PCR to add a T7 promoter fragment (TAATACGACTCACTATAGG). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    IA:

    Article Title: p53 Interaction with JMJD3 Results in Its Nuclear Distribution during Mouse Neural Stem Cell Differentiation
    Article Snippet: .. For RT-PCR, 5 µg of total RNA was reverse-transcribed using oligo(dT) (Integrated DNA Technologies Inc., Coralville, IA) and SuperScript II reverse transcriptase (Invitrogen Corp.). .. Specific oligonucleotide primer pairs were incubated with cDNA template for PCR amplification using the Expand High FidelityPLUS PCR System (Roche Applied Science).

    Article Title: Activin/Nodal Signaling Switches the Terminal Fate of Human Embryonic Stem Cell-derived Trophoblasts *
    Article Snippet: .. For cDNA synthesis, the RNA pellet was dissolved in diethyl pyrocarbonate (Sigma)-treated water, and 15 μg of RNA was heated at 70 °C for 5 min with oligo(dT) 15-mer primers (Integrated DNA Technologies, Coralville, IA). .. Moloney murine leukemia virus reverse transcriptase (Invitrogen) and dNTP mix (Invitrogen) were added, and the reaction was carried out for 50 min at 42 °C.

    Plasmid Preparation:

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: Two bases at positions 2540-2541 of the vector were designed to be different from the original HSI_vector sequence. .. All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies).

    Software:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°. .. The membrane transfers were analyzed with a Typhoon 9600 Phosphorimager and ImageQuant 5.2 software (GE Corporation).

    Article Title: Conidia but Not Yeast Cells of the Fungal Pathogen Histoplasma capsulatum Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ Trigger a Type I Interferon Innate Immune Response in Murine Macrophages ▿ †
    Article Snippet: For qRT-PCR, 1 μg of DNase I-treated total macrophage RNA was reverse transcribed with Affinity Script multitemperature reverse transcriptase (Stratagene) and 500 ng oligo(dT19 V) (Integrated DNA Technologies, San Diego, CA) for 2 h. cDNA was diluted 3- to 4-fold with pyrogen-free water. .. Reactions were run on an Mx3000P machine (Stratagene), and MxPro software (Stratagene) was used to determine threshold and threshold cycle ( CT ) values. qRT-PCR data were normalized to hypoxanthine phosphoribosyltransferase 1 (HPRT1) expression using the Pfaffl method ( ).

    SYBR Green Assay:

    Article Title: Activation of Plant Innate Immunity by Extracellular High Mobility Group Box 3 and Its Inhibition by Salicylic Acid
    Article Snippet: The first-strand cDNA was synthesized from the total RNA (~2 μg) with Superscript III reverse transcriptase (Invitrogen) and oligo (dT)15 primer (Integrated DNA Technologies). .. Two μL of 20 times diluted cDNAs were used for quantitative real-time RT PCR (qRT-PCR) with IQ SYBR Green Supermix (Bio-Rad) and gene-specific primer pairs as listed in .

    In Vitro:

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 200 pmol/µl 2′-O-methyl RNA-DNA chimera (Integrated DNA Technologies, Inc.) RNA substrate from in vitro transcription (Basic Protocol 1) or in vivo purification 10 × buffer for RNase H (see recipe) 2 U/µl RNase H (Amersham) 40 U/µl RNase inhibitor (Thermo Scientific) G50 buffer (see recipe) 10 × buffer for CIP (see recipe) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) 10 × buffer for T4 PNK forward reaction (see recipe) [γ32 P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Bridging DNA oligo (Integrated DNA Technologies, Inc.) 10 × buffer for T4 DNA ligase (see recipe) 5 U/µl T4 DNA ligase (Thermo Scientific) 95°C heat block Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) ..

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: The T7 fragment-tagged DNAs were then used as templates for the in vitro transcription of sgRNAs as described The transcribed sgRNAs were purified by denaturing PAGE, ethanol precipitated and folded according to ( ). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    Ethanol Precipitation:

    Article Title: Spp382p Interacts with Multiple Yeast Splicing Factors, Including Possible Regulators of Prp43 DExD/H-Box Protein Function
    Article Snippet: The RNA was purified by two additional PCI extractions followed by ethanol precipitation. .. For RNase H digestion, 1 μm of oligo(dT) 15-mer (Integrated DNA Technologies) or a control oligo (SNR30, 5′-GAAACTGCTCGTAGTCTGACG-3′) was incubated in 1× digestion buffer [20 m m Tris-HCl (pH 7.5), 20 m m KCl, 10 m m MgCl2 , 0.1 m m EDTA, 0.1 m m DTT] at 55° for 10 min and then slowly cooled to 37°.

    Article Title: Synthesis and Labeling of RNA In Vitro
    Article Snippet: .. 200 pmol/µl 2′-O-methyl RNA-DNA chimera (Integrated DNA Technologies, Inc.) RNA substrate from in vitro transcription (Basic Protocol 1) or in vivo purification 10 × buffer for RNase H (see recipe) 2 U/µl RNase H (Amersham) 40 U/µl RNase inhibitor (Thermo Scientific) G50 buffer (see recipe) 10 × buffer for CIP (see recipe) 1 U/µl calf intestine phosphatase (CIP; Thermo Scientific) 10 × buffer for T4 PNK forward reaction (see recipe) [γ32 P]ATP (3000 Ci/mmol, 10 µCi/µl) (PerkinElmer) 10 U/µl T4 polynucleotide kinase (PNK; Thermo Scientific) Bridging DNA oligo (Integrated DNA Technologies, Inc.) 10 × buffer for T4 DNA ligase (see recipe) 5 U/µl T4 DNA ligase (Thermo Scientific) 95°C heat block Additional reagents and equipment for phenol/chloroform/isoamyl alcohol extraction and ethanol precipitation of RNA (Basic Protocol 1, steps 4 to 9), urea-PAGE , autoradiography ( APPENDIX 3A ), and “freeze-thaw” elution/ethanol precipitation (Basic Protocol 1, steps 10 to 13) ..

    Enzymatic Assay:

    Article Title: Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series
    Article Snippet: Paragraph title: HIV RT enzymatic assay. ... RNA-dependent DNA polymerase activity in the presence or absence of NcRTI was quantified on a poly(rA)/oligo(dT)15 substrate in an assay buffer (40-μl volume) containing 2 nM BH10 HIV reverse transcriptase enzyme, 50 mM Tris-HCl (pH 7.8), 60 mM NaCl, 2 mM MgCl2 · 6H2 O, 0.02% 3-[(3-cholamidopropyl)-dimethylamonio]-2-propanesulfonate (CHAPS), 6 mM dithiothreitol (DTT), 2 mM glutathione (GSH, reduced form), 45 nM poly(rA) (GE Healthcare), 4.5 nM oligo(dT15 ) (Integrated DNA Technologies), 1 μM dTTP, 1% dimethyl sulfoxide (DMSO), and 2 mM ATP.

    Produced:

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: Synthetic DNA inserts : We produced six different double-stranded inserts with known DNA lesions by hybridizing synthetic single-stranded DNA fragments ( Supplementary Table S1 ) in different combinations ( A and Supplementary Fig. S2 ). .. All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies).

    Construct:

    Article Title: Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes
    Article Snippet: We used predictions based on type II CRISPR system and Cas9 protein phylogenies ( ; ) to generate crRNA and tracrRNA sequences; the Cdi guide RNA was based on an experimentally validated construct ( ). sgRNA-encoding DNAs were amplified by PCR to add a T7 promoter fragment (TAATACGACTCACTATAGG). .. All other RNA oligos (and all DNA oligos) were synthesized by Integrated DNA Technologies, Inc. (Coralville, Iowa).

    Article Title: Artifactual mutations resulting from DNA lesions limit detection levels in ultrasensitive sequencing applications
    Article Snippet: All inserts were synthesized as Ultramers (which provide a lower synthesis error rate compared with standard oligos, and for which, the synthesis chemistry provides an improved coupling efficiency above 99.5%), except for the strand with 8-oxoG, which was only available as standard DNA Oligo (both from Integrated DNA Technologies). .. Vector-insert (HSI_insert) DNA constructs : Within the HSI_vector, we substituted a 110 bp fragment with the different synthetic inserts ( A and Supplementary Figs S1 and S2 ).

    Fractionation:

    Article Title: High-Throughput Sequencing of RNA Silencing-Associated Small RNAs in Olive (Olea europaea L.)
    Article Snippet: Briefly, sRNA fractions of 15 to 40-nt long were purified by size fractionation with 15% polyacrylamide gels (PAGE) containing 8M urea followed by gel elution in 0.3 M NaCl and RNA precipitation. .. A pre-activated 3′ adenylated oligo ( 5′ rAppCTGTAGGCACCATCAAT3ddC 3′ ) (Integrated DNA technologies) was used as a 3′ adapter to avoid circularization or multimerization of the sRNAs in the pool while the 5′ adapters were chimeric oligonucleotides ( 5′ atcgtAGGCACCUGAUA 3′ and 5′ atcgtAGGCCACUGAUA 3′ ; lower case is DNA, upper case is RNA).

    High Throughput Screening Assay:

    Article Title: Identification and Characterization of a Novel HIV-1 Nucleotide-Competing Reverse Transcriptase Inhibitor Series
    Article Snippet: RNA-dependent DNA polymerase activity in the presence or absence of NcRTI was quantified on a poly(rA)/oligo(dT)15 substrate in an assay buffer (40-μl volume) containing 2 nM BH10 HIV reverse transcriptase enzyme, 50 mM Tris-HCl (pH 7.8), 60 mM NaCl, 2 mM MgCl2 · 6H2 O, 0.02% 3-[(3-cholamidopropyl)-dimethylamonio]-2-propanesulfonate (CHAPS), 6 mM dithiothreitol (DTT), 2 mM glutathione (GSH, reduced form), 45 nM poly(rA) (GE Healthcare), 4.5 nM oligo(dT15 ) (Integrated DNA Technologies), 1 μM dTTP, 1% dimethyl sulfoxide (DMSO), and 2 mM ATP. .. A description of the assay automation for high-throughput screening is provided in the methods provided in the supplemental material.

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    Individually synthesized single stranded DNA sequences that range from 40 to 350 bases providing high fidelity uniformity low error rates and low dropout rates Each pool can be designed with
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    97
    Integrated DNA Technologies synthetic rna oligonucleotide
    Effects of the inclusion of biomolecules on the growth of Mg 2 PPi crystals. (A) Powder XRD patterns and (B, C) Raman spectra of the Mg 2 PPi composites grown in the absence/presence of DNA and/or <t>RNase</t> A (Mg 2 PPi, Mg 2 PPi-R, Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, and Mg 2 PPi/λ DNA) and DNF in the absence/presence of RNase A (DNF and DNF-R). A diffraction pattern of the simulated Mg 2 PPi (Mg 2 P 2 O 7 ·3.5H 2 O) is also shown. The Raman spectra were obtained in (B) dehydrated and (C) hydrated conditions using a 532 nm laser. In (B), the spectra were obtained with a laser power of 13 mW and acquisition time of 5 s and show the average spectra ± s.d. of five different points in the sample area. In (C), the spectra were collected with a laser power of 30 mW and acquisition time of 20 s and show the spectra of one point in the sample area. All Raman spectra are normalized to the area under the curve. (D) UV absorption and (E) CD spectra of AmpDNA, free RNase A (free R), Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, DNF, and DNF-R. (F) Fluorescence intensity (Δ F = F – F 0 , where F and F 0 are the emitted fluorescence of a substrate with and without treatment of RNase-containing samples at λ ex = 490 nm and λ em = 520 nm) as a function of time for four different catalytic systems. The concentration of <t>RNA</t> substrate was 4 μM. (G) Reaction rate against various concentrations of the substrate (0.1–8 μM) for four catalytic systems. The concentration of RNase A in each sample was 0.5 ng mL –1 . Results represent mean ± s.d. for four independent experiments.
    Synthetic Rna Oligonucleotide, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/synthetic rna oligonucleotide/product/Integrated DNA Technologies
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    synthetic rna oligonucleotide - by Bioz Stars, 2020-02
    97/100 stars
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    79
    Integrated DNA Technologies self complementary dna oligonucleotide carrying p53 dna half site
    The A/T dinucleotide pairs at the center of the <t>DNA</t> half-site and their interactions with the wt and rescued <t>p53.</t> ( A ) In type I complex of wt-DNA (PDB ID 2AC0), the A/T base pairs display the Watson–Crick geometry. R273 side chains interact with the DNA phosphate groups (also shown in Figure 5 A). The R248 residues interact occasionally with the backbone phosphates (shown here) or via water molecules (not shown). ( B and C ) In type I complex of R273C/T284R-DNA, the two dinucleotide pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 B). The R248 side chains at the minor groove are occasionally disordered and form water-mediated hydrogen bonds with the N3 atoms of the adenine bases. ( D ) In type II complex of wt-DNA, the A/T base pairs display the Hoogsteen geometry. R273 side chains interact with the phosphate groups. R248 residues do not interact directly with DNA, but rather with the minor-groove hydration shell described previously ( 16 ). Only one dinucleotide pair is shown for type II complexes, as the other pair is related by crystal symmetry. ( E ) In type II complex of R273H/T284R-DNA, the A/T base pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 C). The R248 side chains form direct interactions with the N3 atoms of the adenine bases. ( F ) In type II complex of R273C/S240R-DNA, the A/T base pairs display the Watson–Crick geometry. S240R side chains interact with the backbone phosphate (also shown in Figure 5 D). The R248 side chains form water-mediated interactions with the N3 atoms of the adenine bases.
    Self Complementary Dna Oligonucleotide Carrying P53 Dna Half Site, supplied by Integrated DNA Technologies, used in various techniques. Bioz Stars score: 79/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    self complementary dna oligonucleotide carrying p53 dna half site - by Bioz Stars, 2020-02
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    75
    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: 75/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rna dna oligonucleotides rgrgrgrcrgrgrgrcrcrcrgrcrcrc/product/Integrated DNA Technologies
    Average 75 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    rna dna oligonucleotides rgrgrgrcrgrgrgrcrcrcrgrcrcrc - by Bioz Stars, 2020-02
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    Image Search Results


    Effects of the inclusion of biomolecules on the growth of Mg 2 PPi crystals. (A) Powder XRD patterns and (B, C) Raman spectra of the Mg 2 PPi composites grown in the absence/presence of DNA and/or RNase A (Mg 2 PPi, Mg 2 PPi-R, Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, and Mg 2 PPi/λ DNA) and DNF in the absence/presence of RNase A (DNF and DNF-R). A diffraction pattern of the simulated Mg 2 PPi (Mg 2 P 2 O 7 ·3.5H 2 O) is also shown. The Raman spectra were obtained in (B) dehydrated and (C) hydrated conditions using a 532 nm laser. In (B), the spectra were obtained with a laser power of 13 mW and acquisition time of 5 s and show the average spectra ± s.d. of five different points in the sample area. In (C), the spectra were collected with a laser power of 30 mW and acquisition time of 20 s and show the spectra of one point in the sample area. All Raman spectra are normalized to the area under the curve. (D) UV absorption and (E) CD spectra of AmpDNA, free RNase A (free R), Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, DNF, and DNF-R. (F) Fluorescence intensity (Δ F = F – F 0 , where F and F 0 are the emitted fluorescence of a substrate with and without treatment of RNase-containing samples at λ ex = 490 nm and λ em = 520 nm) as a function of time for four different catalytic systems. The concentration of RNA substrate was 4 μM. (G) Reaction rate against various concentrations of the substrate (0.1–8 μM) for four catalytic systems. The concentration of RNase A in each sample was 0.5 ng mL –1 . Results represent mean ± s.d. for four independent experiments.

    Journal: ACS Nano

    Article Title: Bioinspired Fabrication of DNA–Inorganic Hybrid Composites Using Synthetic DNA

    doi: 10.1021/acsnano.8b06492

    Figure Lengend Snippet: Effects of the inclusion of biomolecules on the growth of Mg 2 PPi crystals. (A) Powder XRD patterns and (B, C) Raman spectra of the Mg 2 PPi composites grown in the absence/presence of DNA and/or RNase A (Mg 2 PPi, Mg 2 PPi-R, Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, and Mg 2 PPi/λ DNA) and DNF in the absence/presence of RNase A (DNF and DNF-R). A diffraction pattern of the simulated Mg 2 PPi (Mg 2 P 2 O 7 ·3.5H 2 O) is also shown. The Raman spectra were obtained in (B) dehydrated and (C) hydrated conditions using a 532 nm laser. In (B), the spectra were obtained with a laser power of 13 mW and acquisition time of 5 s and show the average spectra ± s.d. of five different points in the sample area. In (C), the spectra were collected with a laser power of 30 mW and acquisition time of 20 s and show the spectra of one point in the sample area. All Raman spectra are normalized to the area under the curve. (D) UV absorption and (E) CD spectra of AmpDNA, free RNase A (free R), Mg 2 PPi/AmpDNA, Mg 2 PPi/AmpDNA-R, DNF, and DNF-R. (F) Fluorescence intensity (Δ F = F – F 0 , where F and F 0 are the emitted fluorescence of a substrate with and without treatment of RNase-containing samples at λ ex = 490 nm and λ em = 520 nm) as a function of time for four different catalytic systems. The concentration of RNA substrate was 4 μM. (G) Reaction rate against various concentrations of the substrate (0.1–8 μM) for four catalytic systems. The concentration of RNase A in each sample was 0.5 ng mL –1 . Results represent mean ± s.d. for four independent experiments.

    Article Snippet: RNase Activity Test The ribonucleotic activity of RNase A was evaluated using a synthetic RNA oligonucleotide, which has a fluorescein dye and quencher at both ends (RNase Alert substrate, Integrated DNA Technology).

    Techniques: Fluorescence, Concentration Assay

    The A/T dinucleotide pairs at the center of the DNA half-site and their interactions with the wt and rescued p53. ( A ) In type I complex of wt-DNA (PDB ID 2AC0), the A/T base pairs display the Watson–Crick geometry. R273 side chains interact with the DNA phosphate groups (also shown in Figure 5 A). The R248 residues interact occasionally with the backbone phosphates (shown here) or via water molecules (not shown). ( B and C ) In type I complex of R273C/T284R-DNA, the two dinucleotide pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 B). The R248 side chains at the minor groove are occasionally disordered and form water-mediated hydrogen bonds with the N3 atoms of the adenine bases. ( D ) In type II complex of wt-DNA, the A/T base pairs display the Hoogsteen geometry. R273 side chains interact with the phosphate groups. R248 residues do not interact directly with DNA, but rather with the minor-groove hydration shell described previously ( 16 ). Only one dinucleotide pair is shown for type II complexes, as the other pair is related by crystal symmetry. ( E ) In type II complex of R273H/T284R-DNA, the A/T base pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 C). The R248 side chains form direct interactions with the N3 atoms of the adenine bases. ( F ) In type II complex of R273C/S240R-DNA, the A/T base pairs display the Watson–Crick geometry. S240R side chains interact with the backbone phosphate (also shown in Figure 5 D). The R248 side chains form water-mediated interactions with the N3 atoms of the adenine bases.

    Journal: Nucleic Acids Research

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions

    doi: 10.1093/nar/gkt630

    Figure Lengend Snippet: The A/T dinucleotide pairs at the center of the DNA half-site and their interactions with the wt and rescued p53. ( A ) In type I complex of wt-DNA (PDB ID 2AC0), the A/T base pairs display the Watson–Crick geometry. R273 side chains interact with the DNA phosphate groups (also shown in Figure 5 A). The R248 residues interact occasionally with the backbone phosphates (shown here) or via water molecules (not shown). ( B and C ) In type I complex of R273C/T284R-DNA, the two dinucleotide pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 B). The R248 side chains at the minor groove are occasionally disordered and form water-mediated hydrogen bonds with the N3 atoms of the adenine bases. ( D ) In type II complex of wt-DNA, the A/T base pairs display the Hoogsteen geometry. R273 side chains interact with the phosphate groups. R248 residues do not interact directly with DNA, but rather with the minor-groove hydration shell described previously ( 16 ). Only one dinucleotide pair is shown for type II complexes, as the other pair is related by crystal symmetry. ( E ) In type II complex of R273H/T284R-DNA, the A/T base pairs display the Watson–Crick geometry. T284R side chains interact with the backbone phosphate (also shown in Figure 5 C). The R248 side chains form direct interactions with the N3 atoms of the adenine bases. ( F ) In type II complex of R273C/S240R-DNA, the A/T base pairs display the Watson–Crick geometry. S240R side chains interact with the backbone phosphate (also shown in Figure 5 D). The R248 side chains form water-mediated interactions with the N3 atoms of the adenine bases.

    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.

    Techniques:

    Close-up views of the mutation sites in the rescued p53 proteins bound to DNA, compared with the wt p53-DNA complex. ( A ) Wild-type p53-DNA interface showing the interaction site of R273 (PDB ID 2AC0). ( B–D ) The corresponding sites of the rescued proteins, indicating that both R273H and R273C side chains are too short to interact with the DNA backbone. The corresponding distances to the DNA are shown by gray dashed lines. Alternative hydrogen bonds to the DNA backbone are formed by the second-site suppressor mutations, T284R and S240R, shown by red dashed lines. Black labels denote wt residues. Red and blue labels denote primary and suppressor mutations, respectively.

    Journal: Nucleic Acids Research

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions

    doi: 10.1093/nar/gkt630

    Figure Lengend Snippet: Close-up views of the mutation sites in the rescued p53 proteins bound to DNA, compared with the wt p53-DNA complex. ( A ) Wild-type p53-DNA interface showing the interaction site of R273 (PDB ID 2AC0). ( B–D ) The corresponding sites of the rescued proteins, indicating that both R273H and R273C side chains are too short to interact with the DNA backbone. The corresponding distances to the DNA are shown by gray dashed lines. Alternative hydrogen bonds to the DNA backbone are formed by the second-site suppressor mutations, T284R and S240R, shown by red dashed lines. Black labels denote wt residues. Red and blue labels denote primary and suppressor mutations, respectively.

    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.

    Techniques: Mutagenesis

    The effect of base-pairing geometry on DNA shape in type II complexes. ( A ) Stereo view of the A/T dinucleotide pairs at the center of the DNA half-site, showing the local backbone shift and minor-groove narrowing caused by Hoogsteen base pairs in the wt complex (gray, PDB ID 3IGL) relative to Watson–Crick base pairs of the rescued complex R273H/T284R-DNA (cyan). ( B ) Comparison of three DNA helices bound to the wt and rescued p53. The color code is gray for wt-DNA (PDB ID 3IGL), cyan for R273H/T284R-DNA and green for R273C/S240R-DNA. Also shown is a superposition of the three helices. The continuous 20 bp long helices were obtained by modeling the missing phosphate groups in the full-length binding site (see scheme B in Supplementary Figure S3 ). The overall shape of the three DNA helices is similar except for the constriction shown by the wt DNA at the center of each half site, caused by the Hoogsteen base-pairing geometry (highlighted in red and indicated by arrows). ( C ) Close-up stereo view of the interaction modes of R273, T284R and S240R with their DNA-binding sites, showing the shift in the DNA backbone of the wt complex (gray) relative to those of the rescued complexes (cyan and green). The red arrow points to the position of the oxygen atom interacting with R273. Only one interaction site is shown for each complex. The other three sites are equivalent by symmetry.

    Journal: Nucleic Acids Research

    Article Title: Structural studies of p53 inactivation by DNA-contact mutations and its rescue by suppressor mutations via alternative protein-DNA interactions

    doi: 10.1093/nar/gkt630

    Figure Lengend Snippet: The effect of base-pairing geometry on DNA shape in type II complexes. ( A ) Stereo view of the A/T dinucleotide pairs at the center of the DNA half-site, showing the local backbone shift and minor-groove narrowing caused by Hoogsteen base pairs in the wt complex (gray, PDB ID 3IGL) relative to Watson–Crick base pairs of the rescued complex R273H/T284R-DNA (cyan). ( B ) Comparison of three DNA helices bound to the wt and rescued p53. The color code is gray for wt-DNA (PDB ID 3IGL), cyan for R273H/T284R-DNA and green for R273C/S240R-DNA. Also shown is a superposition of the three helices. The continuous 20 bp long helices were obtained by modeling the missing phosphate groups in the full-length binding site (see scheme B in Supplementary Figure S3 ). The overall shape of the three DNA helices is similar except for the constriction shown by the wt DNA at the center of each half site, caused by the Hoogsteen base-pairing geometry (highlighted in red and indicated by arrows). ( C ) Close-up stereo view of the interaction modes of R273, T284R and S240R with their DNA-binding sites, showing the shift in the DNA backbone of the wt complex (gray) relative to those of the rescued complexes (cyan and green). The red arrow points to the position of the oxygen atom interacting with R273. Only one interaction site is shown for each complex. The other three sites are equivalent by symmetry.

    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.

    Techniques: Binding Assay

    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: