dna polymerase i Search Results


  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 99
    New England Biolabs klenow fragment
    <t>UL30</t> inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM <t>Klenow</t> Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.
    Klenow Fragment, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 8089 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/New England Biolabs
    Average 99 stars, based on 8089 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher dna polymerase i
    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear <t>DNA</t> using DNA <t>polymerase</t> I and Ampligase. This process substantially improved the complexity of the library during sequencing.
    Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2890 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase i/product/Thermo Fisher
    Average 99 stars, based on 2890 article reviews
    Price from $9.99 to $1999.99
    dna polymerase i - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs dna polymerase
    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed <t>DNA</t> sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and <t>dNTP.</t> The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.
    Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5092 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/New England Biolabs
    Average 99 stars, based on 5092 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    Thermo Fisher dna polymerase
    Knockdown of cyclin D1, CDK4, or Rb disrupts Stat3 and C/EBPβ binding and simultaneously induces C/EBPα binding to the miR-21 and miR-181b promoters in sepsis Gr1 + CD11b + MDSCs Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice and transfected with cyclin D1-, CDK4-, Rb-specific or control siRNA for 36 hr. ( A ) Knockdown of cyclin D1 or CDK4 inhibits Rb phosphorylation. Levels of Rb and p-Rb in cell lysate were determined by immunoblot. Cells were fixed, lyzed, and chromatin immunoprecipitation (ChIP) was performed with antibody specific to p-Stat3, C/EBPβ or C/EBPα, as described in Fig. 2 . ( B and D ) the recovered, ChIP <t>DNA</t> was amplified by semi-quantitative <t>PCR</t> using primers that flank the Stat3 and C/EBP binding sites in the miR-21 and miR-181b promoters. An IgG-immunoprecipitated samples is shown as a negative control. The results are representative of three experiments. ( C and E ) the recovered DNA was amplified by real-time PCR. Samples values were normalized to the “input” DNA values and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. of three experiments. (F) Levels of c-Myc protein after the Rb knockdown in sepsis Gr1 + CD11b + cells isolated from the bone marrow. The results are representative of two experiments.
    Dna Polymerase, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 4605 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/Thermo Fisher
    Average 99 stars, based on 4605 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    TaKaRa dna polymerase
    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was <t>PCR</t> amplified from genomic <t>DNA</t> of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.
    Dna Polymerase, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 2424 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/TaKaRa
    Average 99 stars, based on 2424 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    99
    New England Biolabs klenow dna polymerase
    The principle of PE-PCR for bacterial <t>DNA</t> amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and <t>Klenow</t> DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).
    Klenow Dna Polymerase, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 1415 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow dna polymerase/product/New England Biolabs
    Average 99 stars, based on 1415 article reviews
    Price from $9.99 to $1999.99
    klenow dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    93
    Promega klenow fragment
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Klenow Fragment, supplied by Promega, used in various techniques. Bioz Stars score: 93/100, based on 1150 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/Promega
    Average 93 stars, based on 1150 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    93
    Illumina Inc dna polymerase i
    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier <t>RNA</t> added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and <t>Klenow</t> fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).
    Dna Polymerase I, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 93/100, based on 965 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase i/product/Illumina Inc
    Average 93 stars, based on 965 article reviews
    Price from $9.99 to $1999.99
    dna polymerase i - by Bioz Stars, 2020-09
    93/100 stars
      Buy from Supplier

    99
    Thermo Fisher escherichia coli dna polymerase i
    Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli <t>DNA</t> polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.
    Escherichia Coli Dna Polymerase I, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 177 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/escherichia coli dna polymerase i/product/Thermo Fisher
    Average 99 stars, based on 177 article reviews
    Price from $9.99 to $1999.99
    escherichia coli dna polymerase i - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Boehringer Mannheim klenow fragment
    The effect of Ku on the 3′→5′ exonuclease activity of WRN, <t>Klenow</t> and exo <t>III</t> on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.
    Klenow Fragment, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 155 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/Boehringer Mannheim
    Average 92 stars, based on 155 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    99
    Millipore dna polymerase
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Dna Polymerase, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 456 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dna polymerase/product/Millipore
    Average 99 stars, based on 456 article reviews
    Price from $9.99 to $1999.99
    dna polymerase - by Bioz Stars, 2020-09
    99/100 stars
      Buy from Supplier

    92
    Enzymatics klenow fragment
    PFGE analysis to show the relatedness of E. pyrifoliae strains <t>Ep2/97</t> and Ep4/97. Genomic <t>DNA</t> of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.
    Klenow Fragment, supplied by Enzymatics, used in various techniques. Bioz Stars score: 92/100, based on 112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/klenow fragment/product/Enzymatics
    Average 92 stars, based on 112 article reviews
    Price from $9.99 to $1999.99
    klenow fragment - by Bioz Stars, 2020-09
    92/100 stars
      Buy from Supplier

    Image Search Results


    UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: UL30 inhibits the minicircle replication in the absence of UL42 Reactions contained helicase, polymerase(s), DNA MC70-2 (A) and were quenched after 30 minutes. (B) Lanes 1–6 contained 100 nM Klenow Fragment and increasing concentrations of UL30 (0, 10, 50, 100, 150 or 200 nM). Lanes 7–12 contained 100 nM UL30 and increasing concentrations of Klenow Fragment (0, 10, 50, 100, 150 or 200 nM). DNA products were separated using 1.5% alkaline agarose gel electrophoresis. (C) Amount of dNTPs incorporated was measured using ImageQuant.

    Article Snippet: Both polymerases could replace UL30-UL42, with Klenow Fragment generating products ~1 kB long.

    Techniques: Agarose Gel Electrophoresis

    Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Journal: Biochemistry

    Article Title: Protein Displacement by Herpes Helicase-Primase and the Key Role of UL42 During Helicase-Coupled DNA Synthesis by the Herpes Polymerase

    doi: 10.1021/acs.biochem.6b01128

    Figure Lengend Snippet: Non-cognate polymerases can replace UL30-UL42 during minicircle replication Either Klenow Fragment or T4 DNA Polymerase were titrated into assays containing DNA MC70 (A) and 100 nM UL5-UL8-UL52. (B) DNA products were separated with 1.5% alkaline agarose gel electrophoresis.

    Article Snippet: Both polymerases could replace UL30-UL42, with Klenow Fragment generating products ~1 kB long.

    Techniques: Agarose Gel Electrophoresis

    Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Mechanism for the generation of InDels using TRIAD. (A) Generation of single, double and triple triplet nucleotide deletions. Step 1. Two MlyI recognition sites (5’GAGTC(N) 5 ↓) are positioned at each end of TransDel, 1 bp away from the site of transposon insertion. Transposition with TransDel results in the duplication of 5 bp (N 4 N 5 N 6 N 7 N 8 ) of the target DNA at the insertion point. TransDel carries a selection marker (resistance gene against chloramphenicol; CamR) enabling the recovery of in vitro transposition products after transformation into E. coli . Step 2. MlyI digestion removes TransDel together with 8 bp of the target DNA (4 bp at each end), leaving blunt ends and resulting in the removal of a contiguous 3 bp sequence from the target DNA (N 5 N 6 N 7 ). Step 3a. Self-ligation reforms the target DNA minus 3 bp, as previously described 11 . Step 3b. Alternatively, blunt-ended cassettes Del2 or Del3 are ligated into the gap left upon TransDel removal for the generation of 6 and 9 bp deletions, respectively. Both Del2 and Del3 also contain two MlyI recognition sites advantageously positioned towards the ends of the cassettes. These cassettes also contain a different marker than TransDel (resistance gene against kanamycin; KanR) to avoid cross-contamination. Step 4b. MlyI digestion removes Del2 and Del3 together with respectively 3 and 6 additional bp of the original target DNA. In the case of Del2, MlyI digestion results in the removal of a 3 bp sequence (N 2 N 3 N 4 ) on one side of the cassette. In the case of Del3, MlyI digestion results in the removal of two 3 bp sequence (N 2 N 3 N 4 ) on both side of the cassette (N 2 N 3 N 4 and N 8 N 9 N 10 ). Step 5b. Self-ligation reforms the target DNA minus 6 or 9 bp. (B) Generation of single, double and triple randomized triplet nucleotide insertions. Step 1. TransDel is an asymmetric transposon with MlyI at one end and NotI at the other end. Both recognition sites are positioned 1bp away from TransIns insertion site. Upon transposition, 5 bp (N 1 N 2 N 3 N 4 N 5 ) of the target DNA are duplicated at the insertion point of TransIns. Step 2. Double digestion with NotI and MlyI results in the removal of TransIns. Digestion with MlyI removes TransIns with 4 bp (N 1 N 2 N 3 N 4 ) of the duplicated sequence at the transposon insertion site. Digestion with NotI leaves a 5’, 4-base cohesive overhang. Step 3. DNA cassettes Ins1, Ins2 and Ins3 (Ins1/2/3) carrying complementary ends are ligated in the NotI/MlyI digested TransIns insertion site. Ins1, Ins2 and Ins3 carry respectively 1, 2 and 3 randomized bp triplets at their blunt-ended extremities ([NNN] 1,2 or 3 ; indicated in purple). Ins1/2/3 contain two AcuI recognition sites (5’CTGAAG(16/14)) strategically positioned towards their ends. One site is located so that AcuI will cleave at the point where the target DNA joins Ins1/2/3. The other site is positioned so that AcuI will cut inside Ins1/2/3 to leave the randomized triplet(s) with the target DNA. Step 4. Digestion with AcuI removes Ins1/2/3 leaving 3’, 2-base overhangs with the target DNA ( i.e. , 5’N 5 T on one end and 5’TC on the end carrying the randomized triplet(s)). Digestion with the Large Klenow fragment generates blunt ends by removing the overhangs. This step also enables to discard the extra nucleotide (N 5 ) from the sequence duplicated during the transposition. Step 5. Self-ligation reforms the target DNA with one, two or three randomized nucleotide triplets.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Selection, Marker, In Vitro, Transformation Assay, Sequencing, Ligation

    Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Journal: bioRxiv

    Article Title: Access to unexplored regions of sequence space in directed enzyme evolution via insertion/deletion mutagenesis

    doi: 10.1101/790014

    Figure Lengend Snippet: Schematic outline of TRIAD. (A) Generation of deletion libraries. Step 1 : The TransDel insertion library is generated by in vitro transposition of the engineered transposon TransDel into the target sequence. Step 2 : Mly I digestion removes TransDel together with 3 bp of the original target sequence and generate a single break per variant. Step 3a : self-ligation results in the reformation of the target sequence minus 3 bp, yielding a library of single variants with a deletion of one triplet 11 . Step 3b : DNA cassettes dubbed Del2 and Del3 are then inserted between the break in the target sequence to generate Del2 and Del3 insertion libraries. Step 4b : Mly I digestion removes Del2 and Del3 together with 3 and 6 additional bp of the original target sequence, respectively. Step 5b : self-ligation results in the reformation of the target sequence minus 6 and 9 bp, yielding libraries of single variants with a deletion of 2 and 3 triplets, respectively. Deletions are indicated by red vertical lines. (B) Generation of insertion libraries. Step 1: The TransIns insertion library is generated by in vitro transposition of the engineered transposon TransIns into the target sequence. Step 2: digestion by Not I and Mly I removes TransIns. Step 3: DNA cassettes dubbed Ins1, Ins 3 and Ins3 (with respectively 1, 2 and 3 randomized NNN triplets at one of their extremities; indicated by purple triangles) are then inserted between the break in the target sequence to generate the corresponding Ins1, Ins2 and Ins3 insertion libraries. Step 4: Acu I digestion and 3’-end digestion by the Klenow fragment remove the cassettes, leaving the randomized triplet(s) in the original target sequence. Step 5: Self-ligation results in the reformation of the target sequence plus 3, 6 and 9 random bp, yielding libraries of single variants with an insertion of 1, 2 and 3 triplets, respectively.

    Article Snippet: DNA Polymerase I, Large (Klenow) Fragment was purchased from New England Biolabs.

    Techniques: Generated, In Vitro, Sequencing, Variant Assay, Ligation

    Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Journal: Nucleic Acids Research

    Article Title: Triazole linking for preparation of a next-generation sequencing library from single-stranded DNA

    doi: 10.1093/nar/gky452

    Figure Lengend Snippet: Enzymatic synthesis of DNA complementary to triazole-containing DNA. ( A ) Chemical structures and sequences of the template DNA. TpT contains no triazole linking (left), whereas TzT and TzU contain a single triazole linking (middle and right). Note that the triazole linkage is followed by thymidine and uridine (i.e., ribonucleoside) in TzT and TzU, respectively. Schematic representation of the primer extension assay (bottom). ( B ) Primer extension by the Klenow fragment exo (−).

    Article Snippet: The reaction was supplemented with 1 μl (20 units) of Exonuclease I (New England BioLabs) and incubated at 37°C for 15 min. Then, the reaction was supplemented with 1 μl (50 units) of Klenow fragment exo (−) (New England BioLabs) and further incubated for 15 min at 37°C.

    Techniques: Primer Extension Assay

    Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Journal: Nature biotechnology

    Article Title: Massively parallel polymerase cloning and genome sequencing of single cells using nanoliter microwells

    doi: 10.1038/nbt.2720

    Figure Lengend Snippet: Microwell displacement amplification system. (a) Each slide contains 16 arrays of 255 microwells each. Cells, lysis solution, denaturing buffer, neutralization buffer and MDA master mix were each added to the microwells with a single pipette pump. Amplicon growth was then visualized with a fluorescent microscope using a real-time MDA system. Microwells showing increasing fluorescence over time were positive amplicons. The amplicons were extracted with fine glass pipettes attached to a micromanipulation system. (b) Scanning electron microscopy of a single E. coli cell displayed at different magnifications. This particular well contains only one cell, and most wells observed also contained no more than one cell. (c) A custom microscope incubation chamber was used for real time MDA. The chamber was temperature and humidity controlled to mitigate evaporation of reagents. Additionally, it prevented contamination during amplicon extraction by self-containing the micromanipulation system. An image of the entire microwell array is also shown, as well as a micropipette probing a well. (d ) Complex three-dimensional MDA amplicons were reduced to linear DNA using DNA polymerase I and Ampligase. This process substantially improved the complexity of the library during sequencing.

    Article Snippet: 10 U of DNA Polymerase I (Invitrogen, Carlsbad, CA) was added to the denatured amplicons along with 250 nanograms of unmodified random hexamer primer, 1 mM dNTPs, 1x Ampligase buffer (Epicentre, Madison, Wi), and 1x NEB buffer 2 (NEB, Cambridge, MA).

    Techniques: Amplification, Lysis, Neutralization, Multiple Displacement Amplification, Transferring, Microscopy, Fluorescence, Micromanipulation, Electron Microscopy, Incubation, Evaporation, Sequencing

    Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

    Journal: Nucleic Acids Research

    Article Title: Motif programming: a microgene-based method for creating synthetic proteins containing multiple functional motifs

    doi: 10.1093/nar/gkm017

    Figure Lengend Snippet: Schematic diagram of a motif-mixing protocol used in this study. Initially, we designed DNA sequences for microgenes core that each encode a peptide motif to be mixed in their first reading frames, after which sense and antisense MPR primers were synthesized based on these microgenes core . These primers share 3′ sequences that enable base-pair formation between the sense and antisense primers, but contain mismatched bases at their 3′-OH ends (shown by red letters with dots). In the polymerization step, motifs can be embedded either in the sense or antisense primer. In the figure, motifs A and B are embedded in the sense primers, producing primers A S and B S , while motifs C and D are in the antisense primers, producing primers C AS and D AS . The thermal cycle reaction is carried out in the presence of these MPR primers, a thermostable, a DNA polymerase and dNTP. The resultant high molecular weight DNAs are combinatorial polymers of multiple microgenes created by stochastic base paring of the MPR primers. In some clones, nucleotide insertions or deletions allow frame shift mutations (denoted by FS), so that peptide sequences encoded by the second and third reading frames appear in the translated products.

    Article Snippet: For the experiments schematically depicted in , 0.4 µM sense- and antisense MPR primers, four dNTP (0.35 mM each) and 2.6 units of 3′–5′ exo + Vent DNA polymerase (New England Biolabs) were mixed in reaction buffer (10 mM KCl, 10 mM (NH4 )2 SO4 , 20 mM Tris-HCl, 2 mM MgSO4 , 0.1% TritonX-100, pH 8.8).

    Techniques: Synthesized, Molecular Weight, Clone Assay

    (A) RCA detection using 20 pmol of circularizable probe and 10 11 artificial DNA and RNA template. Six of seven probes were shown to detect their corresponding artificial template (Table ), the negative signal (OCP-E) could be due to

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification

    doi: 10.1128/JCM.43.5.2339-2344.2005

    Figure Lengend Snippet: (A) RCA detection using 20 pmol of circularizable probe and 10 11 artificial DNA and RNA template. Six of seven probes were shown to detect their corresponding artificial template (Table ), the negative signal (OCP-E) could be due to

    Article Snippet: Two DNA polymerases were chosen to perform the RCA: Vent (exo-) DNA polymerase and Bst DNA polymerase (New England Biolabs).

    Techniques:

    Pictorial representation of the RCA method. (A) Padlock probe containing target-complementary segment hybridization to a target DNA or RNA sequence. (B) The padlock probe can be circularized by DNA ligase. (C) Ligated probe and binding of complementary

    Journal: Journal of Clinical Microbiology

    Article Title: Rapid and Sensitive Detection of Severe Acute Respiratory Syndrome Coronavirus by Rolling Circle Amplification

    doi: 10.1128/JCM.43.5.2339-2344.2005

    Figure Lengend Snippet: Pictorial representation of the RCA method. (A) Padlock probe containing target-complementary segment hybridization to a target DNA or RNA sequence. (B) The padlock probe can be circularized by DNA ligase. (C) Ligated probe and binding of complementary

    Article Snippet: Two DNA polymerases were chosen to perform the RCA: Vent (exo-) DNA polymerase and Bst DNA polymerase (New England Biolabs).

    Techniques: Hybridization, Sequencing, Binding Assay

    DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]

    Journal: International Journal of Molecular Sciences

    Article Title: DNA Break Mapping Reveals Topoisomerase II Activity Genome-Wide

    doi: 10.3390/ijms150713111

    Figure Lengend Snippet: DSBs ( a ) and SSBs ( b – d ) generated in presence of Top2 ( a ); ETO ( b ); Top1 ( c ); and DNA-damaging agents that modify the DNA termini ( d ). Red arrow represents successful nick translation. Stop sign represents unsuccessful nick translation. Nick translation by DNA polymerase I necessitates a 3'-OH, which is not reconstituted in case of Top1 cleavage or when the DNA termini is damaged (shown by asterisk). In these cases the principal enzymes involved in processing and repair of the ends are listed below the black arrow. TDP1, tyrosyl-DNA phosphodiesterase 1, PNKP, polynucleotide kinase 3'-phosphatase, APE1, AP endonuclease I [ 17 ]

    Article Snippet: 500 μg of DNA was incubated for 40 s at 16 °C with a mixture of 200 μM of dATP, dGTP, dCTP and 20 μM of digoxigenin-11-dUTP (Roche), 117 μM of ddATP, ddGTP, ddCTP (Roche) and 1000 units of Escherichia coli DNA Polymerase I (New England Biolabs, Ipswich, MA, USA).

    Techniques: Generated, Nick Translation

    EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: EM of the complexes formed of 7kMk with pUC19/ Bam HI DNA. Complexes were obtained after incubation in the presence of 1 M K-Glu at 70°C at a R w of 0 ( A ), 1.5 ( B – E ) and 10 ( F – J ). Arrows indicate DNA loops and bends. The scale bar represents 200 nm.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation

    EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: EMSA of 7kMk–DNA complexes. Bam HI linearized pUC19 DNA (150 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. The R w (protein:DNA weight ratio) values of the samples loaded in lanes 1–7 were 0.5, 1, 1.5, 2, 3, 10 and 0, respectively. M, 1 kb DNA ladder (Gibco BRL). Electrophoresis was performed in 1.5% agarose at 1.5 V/cm for 16 h in TBE buffer containing 100 mM Na-Glu.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation, Electrophoresis

    DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.

    Journal: Nucleic Acids Research

    Article Title: Identification, cloning and characterization of a new DNA-binding protein from the hyperthermophilic methanogen Methanopyrus kandleri

    doi:

    Figure Lengend Snippet: DNA topology assay of 7kMk protein. Relaxed pUC19 (300 ng) was incubated with various concentrations of 7kMk in GB buffer at 70°C for 30 min. 7kMk was added at a R w of 0 (lane 10), 0.4 (lane 3), 0.8 (lanes 4 and 11), 1.6 (lanes 5 and 12), 3.3 (lanes 6 and 13) 5 (lane 7), 6.7 (lanes 8 and 14) and 12 (lane 9). The resulting complexes were digested with topoisomerase V (100 ng) at the same temperature for 15 min (lanes 3–10) or 1 h (lanes 11–14). Lanes M, 1 and 2 were a 1 kb DNA ladder (Gibco BRL), negatively supercoiled pUC19 DNA isolated from E.coli and relaxed pUC19 DNA, respectively. The reaction products were analyzed by 1.5% agarose gel electrophoresis in TBE buffer ( A ) or TBE buffer containing 1 µg/ml chloroquine ( B ) at 1.5 V/cm for 16 h, stained with ethidium bromide and digitized.

    Article Snippet: ThermoFidelase I was from Fidelity Systems (USA), while pUC19 DNA, restriction endonucleases and the Klenow fragment of E.coli DNA polymerase I were purchased from New England Biolabs (USA).

    Techniques: Incubation, Isolation, Agarose Gel Electrophoresis, Staining

    Knockdown of cyclin D1, CDK4, or Rb disrupts Stat3 and C/EBPβ binding and simultaneously induces C/EBPα binding to the miR-21 and miR-181b promoters in sepsis Gr1 + CD11b + MDSCs Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice and transfected with cyclin D1-, CDK4-, Rb-specific or control siRNA for 36 hr. ( A ) Knockdown of cyclin D1 or CDK4 inhibits Rb phosphorylation. Levels of Rb and p-Rb in cell lysate were determined by immunoblot. Cells were fixed, lyzed, and chromatin immunoprecipitation (ChIP) was performed with antibody specific to p-Stat3, C/EBPβ or C/EBPα, as described in Fig. 2 . ( B and D ) the recovered, ChIP DNA was amplified by semi-quantitative PCR using primers that flank the Stat3 and C/EBP binding sites in the miR-21 and miR-181b promoters. An IgG-immunoprecipitated samples is shown as a negative control. The results are representative of three experiments. ( C and E ) the recovered DNA was amplified by real-time PCR. Samples values were normalized to the “input” DNA values and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. of three experiments. (F) Levels of c-Myc protein after the Rb knockdown in sepsis Gr1 + CD11b + cells isolated from the bone marrow. The results are representative of two experiments.

    Journal: Immunology and cell biology

    Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

    doi: 10.1038/icb.2016.63

    Figure Lengend Snippet: Knockdown of cyclin D1, CDK4, or Rb disrupts Stat3 and C/EBPβ binding and simultaneously induces C/EBPα binding to the miR-21 and miR-181b promoters in sepsis Gr1 + CD11b + MDSCs Gr1 + CD11b + cells were isolated from the bone marrow of late septic mice and transfected with cyclin D1-, CDK4-, Rb-specific or control siRNA for 36 hr. ( A ) Knockdown of cyclin D1 or CDK4 inhibits Rb phosphorylation. Levels of Rb and p-Rb in cell lysate were determined by immunoblot. Cells were fixed, lyzed, and chromatin immunoprecipitation (ChIP) was performed with antibody specific to p-Stat3, C/EBPβ or C/EBPα, as described in Fig. 2 . ( B and D ) the recovered, ChIP DNA was amplified by semi-quantitative PCR using primers that flank the Stat3 and C/EBP binding sites in the miR-21 and miR-181b promoters. An IgG-immunoprecipitated samples is shown as a negative control. The results are representative of three experiments. ( C and E ) the recovered DNA was amplified by real-time PCR. Samples values were normalized to the “input” DNA values and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. of three experiments. (F) Levels of c-Myc protein after the Rb knockdown in sepsis Gr1 + CD11b + cells isolated from the bone marrow. The results are representative of two experiments.

    Article Snippet: The PCR reaction (25 μl) contained 5 μl ChIP DNA, 12.5 μl of 2x TaqMan real-time PCR Master Mix containing DNA polymerase and dNTPs (Applied Biosystem, Foster City, CA) and 100 nM of primer/probe mix (Integrated DNA Technologies).

    Techniques: Binding Assay, Isolation, Mouse Assay, Transfection, Chromatin Immunoprecipitation, Amplification, Real-time Polymerase Chain Reaction, Immunoprecipitation, Negative Control

    Stat3 and C/EBPβ, but not C/EBPα, bind to miR-21 and miR-181 promoter in sepsis Gr1 + CD11b + MDSCs ( A ) Protein binding in total Gr1 + CD11b + cell population. Gr1 + CD11b + cells were isolated and pooled (n = 6 mice per group) from the bone marrow of sham and septic mice. Cells were fixed in 1% formaldehyde to cross-link protein-DNA complexes. Cells were lyzed and the pelleted nuclei were digested with chromatin shearing enzymatic cocktail. The chromatin solution was then immunoprecipitated with antibodies specific to p-Stat3 (Tyr 705 ), C/EBPβ, C/EBPα, p-Rb (Ser 780 ), or IgG isotype control antibody. Next, chromatin cross-links were reversed to recover the protein-bound DNA. The purified DNA was amplified by qPCR to measure the level of enrichment of miR-21 and miR-181b sequences in the immunoprecipitated complexes using promoter-specific primer/probe sets. PCR reactions were performed in triplicate. Samples were normalized to the “input” DNA (i.e., DNA isolated before immunoprecipitation) and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. (* p ≤ 0.05) of three experiments. *, compare with early sepsis. ( B ) Stat3 and C/EBPβ binding to the miR-21 and miR-181 promoters in Gr1 + CD11b + MDSCs is restriced to the CD31 + subset. The CD31 + cell were then purified from the total Gr1 + CD11b + cell population by positive selection. Cells were treated, and chromatin was immunoprecipitated as described above. PCR was performed to measure the levels of the miR-21 and miR-181b promoter DNA sequence enrichment in chromatin immunoprecipitated from the CD31 + -enriched Gr1 + CD11b + cells from the bone marrow and spleens. Data are expressed as mean ± s.d. of three experiment s .

    Journal: Immunology and cell biology

    Article Title: Stat3 and C/EBPβ synergize to induce miR-21 and miR-181b expression during sepsis

    doi: 10.1038/icb.2016.63

    Figure Lengend Snippet: Stat3 and C/EBPβ, but not C/EBPα, bind to miR-21 and miR-181 promoter in sepsis Gr1 + CD11b + MDSCs ( A ) Protein binding in total Gr1 + CD11b + cell population. Gr1 + CD11b + cells were isolated and pooled (n = 6 mice per group) from the bone marrow of sham and septic mice. Cells were fixed in 1% formaldehyde to cross-link protein-DNA complexes. Cells were lyzed and the pelleted nuclei were digested with chromatin shearing enzymatic cocktail. The chromatin solution was then immunoprecipitated with antibodies specific to p-Stat3 (Tyr 705 ), C/EBPβ, C/EBPα, p-Rb (Ser 780 ), or IgG isotype control antibody. Next, chromatin cross-links were reversed to recover the protein-bound DNA. The purified DNA was amplified by qPCR to measure the level of enrichment of miR-21 and miR-181b sequences in the immunoprecipitated complexes using promoter-specific primer/probe sets. PCR reactions were performed in triplicate. Samples were normalized to the “input” DNA (i.e., DNA isolated before immunoprecipitation) and are presented as fold enrichment relative to the IgG-immunoprecipitated samples (set at 1-fold). Data are expressed as mean ± s.d. (* p ≤ 0.05) of three experiments. *, compare with early sepsis. ( B ) Stat3 and C/EBPβ binding to the miR-21 and miR-181 promoters in Gr1 + CD11b + MDSCs is restriced to the CD31 + subset. The CD31 + cell were then purified from the total Gr1 + CD11b + cell population by positive selection. Cells were treated, and chromatin was immunoprecipitated as described above. PCR was performed to measure the levels of the miR-21 and miR-181b promoter DNA sequence enrichment in chromatin immunoprecipitated from the CD31 + -enriched Gr1 + CD11b + cells from the bone marrow and spleens. Data are expressed as mean ± s.d. of three experiment s .

    Article Snippet: The PCR reaction (25 μl) contained 5 μl ChIP DNA, 12.5 μl of 2x TaqMan real-time PCR Master Mix containing DNA polymerase and dNTPs (Applied Biosystem, Foster City, CA) and 100 nM of primer/probe mix (Integrated DNA Technologies).

    Techniques: Protein Binding, Isolation, Mouse Assay, Immunoprecipitation, Purification, Amplification, Real-time Polymerase Chain Reaction, Polymerase Chain Reaction, Binding Assay, Selection, Sequencing

    Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Journal: Matrix biology : journal of the International Society for Matrix Biology

    Article Title: CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

    doi: 10.1016/j.matbio.2016.04.002

    Figure Lengend Snippet: Detection and determination of Has2 gene mutation A 454 bp region within exon2 of rat Has2 that contains the Cas9 mutation site was PCR amplified from genomic DNA of each cell clone as a template. The amplified product was gel-purified and then digested with Pas1. The final products were analyzed by agarose gel electrophoresis. DNA standards (std) are labeled. Panel A. Products amplified from RCS-o WT cells and clones 1 thru 7. Panel B. Pas1 digestion products of the amplified 454 bp products shown in panel A. Panel C. Products amplified from RCS-Cas9 WT cells and clones 3, 7, 39, 43, 53, 80 and 88. Panel D. Pas1 digestion products of the amplified 454 bp products shown in panel C. Panel E. Alignment of the targeted rat Has2 genomic sequences from RCS-Cas9 clone #7, allele 1 and allele 2 with the WT RCS-Cas9 cells. Deleted bases are indicated.

    Article Snippet: To identify the mutation in the RCS-Cas9 clone #7, the same PCR product was also amplified but with a different DNA polymerase, TaKaRa LA Taq® DNA polymerase (Clontech), and this PCR product cloned into the pCR4-TOPO vector (Invitrogen) according to the manufacturer's instructions.

    Techniques: Mutagenesis, Polymerase Chain Reaction, Amplification, Purification, Agarose Gel Electrophoresis, Labeling, Clone Assay, Genomic Sequencing

    The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Journal: Nucleic Acids Research

    Article Title: Nuclear MxA proteins form a complex with influenza virus NP and inhibit the transcription of the engineered influenza virus genome

    doi: 10.1093/nar/gkh192

    Figure Lengend Snippet: The effect of transiently expressed nuclear MxA proteins. ( A ) The effect of nuclear MxA proteins in Swiss3T3 cells. The cells were transfected with pHMP1-vNS-Luc (0.2 µg), pSEAP2-control (0.1 µg), plasmids encoding three RNA polymerase subunits and NP (0.05 µg of each plasmid) and either parental vector, pVP16-MxA or pHMG-TMxA (0.3 µg each). Luciferase activity was determined and shown as described in Materials and Methods. Error bars represent standard deviation (n = 3). ( B ) Dose-dependent effect of wild-type MxA and VP16-MxA. The same procedure for (A) was carried out in the presence of increasing amounts of wild-type MxA or VP16-MxA. Error bars represent standard deviation (n = 3). ( C ) The effect of C-terminal (MxAΔC) and internal (MxAΔM) deletion VP16-MxA mutant proteins on reporter gene expression. The same procedure for (A) was carried out with mutant MxA proteins. To generate a VP16-MxA deletion mutant (MxAΔC) for expression of MxA lacking its C-terminal region (362–662), we amplified a fragment by PCR with specific primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATCGCA-3′ and 5′-CGCGGATCCTTAACCATACTTTTGTAGCTCCTCTGT-3′ and pVP16-MxA as template. To generate a VP16-MxA deletion mutant (MxAΔM) lacking its internal region (362–573), a fragment was amplified by PCR with primers, 5′-GGCATCCATATGGTTGTTTCCGAAGTGGACATC GCA-3′ and 5′-CGCGGATCCTTAACCGGGGAACTGGGCAAGCCGGCG-3′ and pCHA-MxAΔM plasmid as a template. pCHA-MxAΔM was derived from previously constructed plasmid, pCHA-MxA by removal of an internal part of MxA by digestion with SalI (TOYOBO) and NcoI (TOYOBO) restriction enzymes. The main part of the plasmid was blunted with Klenow fragment and self-ligated. MxA fragments thus prepared were digested with NdeI, blunted with Klenow fragment and then digested with BamHI. These fragments were cloned into pVP16 plasmid digested with EcoRI followed by Klenow treatment and subsequent digestion with BamHI. Error bars represent standard deviation (n = 3).

    Article Snippet: A fragment containing MxA gene was prepared from pET14b-MxA by digestion with NdeI followed by treatment with Klenow fragment and digestion with BamHI.

    Techniques: Transfection, Plasmid Preparation, Luciferase, Activity Assay, Standard Deviation, Mutagenesis, Expressing, Amplification, Polymerase Chain Reaction, Derivative Assay, Construct, Clone Assay

    Assembled HXB2 plasmid, varying base caller and polymerase. Each pane (n=6) summarizes the results of contig curation. Tan boxes contain alignments of reference/blastn hits to contig (Black line). Divergence from reference decreases with newer base callers, and with long amplicon DNA polymerase (Sigma-Aldrich Taq vs. LA Taq by Takara). Annotations below the black lines were automatically imported with SnapGene and represent sequences with minimal/no divergence from common cloning features. The regions corresponding to HXB2 are between red boxes (LTRs). Viewed in SnapGene.

    Journal: bioRxiv

    Article Title: Full-coverage sequencing of HIV-1 provirus from a reference plasmid

    doi: 10.1101/611848

    Figure Lengend Snippet: Assembled HXB2 plasmid, varying base caller and polymerase. Each pane (n=6) summarizes the results of contig curation. Tan boxes contain alignments of reference/blastn hits to contig (Black line). Divergence from reference decreases with newer base callers, and with long amplicon DNA polymerase (Sigma-Aldrich Taq vs. LA Taq by Takara). Annotations below the black lines were automatically imported with SnapGene and represent sequences with minimal/no divergence from common cloning features. The regions corresponding to HXB2 are between red boxes (LTRs). Viewed in SnapGene.

    Article Snippet: Two DNA polymerases were evaluated (barcode 10 used LA Taq (Takara); barcode 11 Taq (Sigma-Aldrich).

    Techniques: Plasmid Preparation, Amplification, Clone Assay

    The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Journal: PLoS ONE

    Article Title: An Efficient Strategy for Broad-Range Detection of Low Abundance Bacteria without DNA Decontamination of PCR Reagents

    doi: 10.1371/journal.pone.0020303

    Figure Lengend Snippet: The principle of PE-PCR for bacterial DNA amplification and detection. A fusion probe is designed with the sequences at the 3′-end corresponding to the bacterial genomic sequences and a non-bacterial tag sequence at the 5′-end. The reaction is initiated by annealing the fusion probe to the template bacterial DNA after heat-denaturing at 95°C for 5 min (Step 1 and 2). An enzyme mix (EK mix) of exo I and Klenow DNA polymerase is then added into the reaction mixture and incubated at 37°C for 2 h (Step 3a and 3b). Following heat-inactivation of EK mix at 80°C for 20 min (Step 3c), a forward primer (non-bac-F) corresponding to the non-bacterial sequence of the fusion probe and a reverse primer (bac-R) targeting bacterial genomic sequence downstream of the fusion probe are used for PCR amplification of the primer extension product (Step 4). In this setting, only template bacterial DNA but not the endogenous contaminated bacterial DNA is amplified (Step 5).

    Article Snippet: Materials The exo I and Klenow DNA polymerase were purchased from New England Biolab (Ipswich, MA).

    Techniques: Polymerase Chain Reaction, Amplification, Genomic Sequencing, Sequencing, Incubation, BAC Assay

    Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Journal: Nature

    Article Title: Tissue–selective effects of nucleolar stress and rDNA damage in developmental disorders

    doi: 10.1038/nature25449

    Figure Lengend Snippet: Inhibition of Pol I results in DNA damage in a subset of cells a , Representative immunofluorescence images of wild-type and TCOF1 +/− cNCCs stained with an antibody against γH2A.X; quantification is shown in b . c , Representative immunofluorescence images of DNA-damaged wild-type cNCCs stained with an antibody against γH2A.X after 1 h treatment with iPol I or actinomycin D (ActD); quantification is shown in d . e , Representative immunofluorescence images of DNA-damaged HeLa cells stained with an antibody against γH2A.X after 1 h treatment with iPol I; quantification is shown in f . For a – f , cells were collected from n = 3 biologically independent experiments. Boxes represent median value and 25th and 75th percentiles, whiskers are minimum to maximum, crosses are outliers. ***P

    Article Snippet: After the NT2 wash, DDX21-bound RNA–protein complexes were dephosphorylated with T4 PNK (NEB, catalogue number M0210) for 30 min in an Eppendorf Thermomixer at 37 °C, 15 s at 1,400 r.p.m., 90 s rest in a 30 μl reaction, pH 6.5, containing 10 units of T4 PNK, 0.1 μl SUPERase-IN, and 6 μl of PEG-400 (16.7% final).

    Techniques: Inhibition, Immunofluorescence, Staining

    Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Analysis of structurally heterogeneous BRCA1 Ex1a and transcript truncation to obtain a homogeneous structure. ( A ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript treated as follows: lane 1, dissolved in water and incubated at 20°C for 30 min; lane 2, heated to 75°C for 1 min (denaturation) and cooled slowly to 20°C (renaturation); lane 3, dissolved in the structure-probing buffer (10 mM Tris–HCl pH 7.2, 10 mM magnesium ions, 40 mM NaCl) and incubated at 20°C for 30 min; lane 4, dissolved as described for lane 3 and subjected to the denaturation/renaturation procedure; lane 5, dissolved as described for lane 3, carrier RNA added to a final concentration of 8 µM, and incubated at 20°C for 30 min; lane 6, carrier RNA added and subjected to denaturation/renaturation. ( B ) CE in non-denaturing conditions of Ex1a transcript fluorescently labeled at its 3′ end with TdT and: [R110]dUTP, [RG6]dUTP, [TAMRA]dUTP (shadowed peaks); TAMRA-500 internal standard (gray line). ( C ) CE in non-denaturing polymer at temperatures: 30, 45 and 60°C of Ex1a transcript end labeled with [R110]dUTP and Klenow fragment. ( D ) Non-denaturing 10% polyacrylamide gel electrophoresis of 5′-end radiolabeled Ex1a transcript (0.5 µM) (lane 1), and the same transcript hybridized with 18 nt of Rex1a oligodeoxynucleotide complementary to its 3′ end (lane 2). Hybridization of transcript (1 µM) with oligodeoxynucleotide (1 µM) was performed in 15 mM Tris–HCl (pH 7.2), 10 mM MgCl 2 , 1.5 mM DTT by heating the sample at 90°C for 1 min and fast cooling. Arrowhead indicates the position of hybrid migration. ( E ) CE in non-denaturing conditions of Ex1a102nt transcript labeled with TdT and [RG6]dUTP (gray line indicates ROX-500 internal standard).

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: Polyacrylamide Gel Electrophoresis, Incubation, Concentration Assay, Labeling, Hybridization, Migration

    Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Journal: Nucleic Acids Research

    Article Title: RNA structure analysis assisted by capillary electrophoresis

    doi:

    Figure Lengend Snippet: Efficiency of fluorescent 3′-end labeling of the BRCA1 transcripts. ( A ) Three rhodamine derivatives of dUTP used for 3′-end labeling of RNAs: [R110]dUTP (left), [RG6]dUTP (middle), [TAMRA]dUTP (right). The maximum emission wavelengths of fluorochromes used are 525, 549 and 572 nm for R110, RG6 and TAMRA, respectively. ( B ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a47nt, Ex1b64nt and Ex1a-2, labeled with Klenow fragment and [R110]dUTP (top), [RG6]dUTP (middle) and [TAMRA]dUTP (bottom); TAMRA-500 or ROX-1000 internal standard (gray lines). ( C ) CE in denaturing conditions of three BRCA1 transcripts: Ex1a102nt, Ex1a and Ex1a-2, labeled with TdT and [R110]dUTP (top), [RG6]dUTP (middle), [TAMRA]dUTP (bottom). ( D ) Relative labeling efficiency of three different RNA molecules with three fluorescent dUTP derivatives using Klenow fragment. The shown labeling efficiency with [TAMRA]dUTP was multiplied by a factor of 4, as the emission intensity of this fluorochrome is four times lower than that for the other two rhodamine derivatives used. The data represent average values obtained in three independent experiments, and [R110]dUTP incorporation is taken as 100%. ( E ) As in (D), but using TdT to label five different RNAs.

    Article Snippet: In the reaction with Klenow fragment any RNA of known sequence may be extended by a single labeled deoxynucleotide in a DNA template-dependent manner ( ).

    Techniques: End Labeling, Labeling

    Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli DNA polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.

    Journal: Journal of Virology

    Article Title: Relationship between 3?-Azido-3?-Deoxythymidine Resistance and Primer Unblocking Activity in Foscarnet-Resistant Mutants of Human Immunodeficiency Virus Type 1 Reverse Transcriptase

    doi: 10.1128/JVI.77.11.6127-6137.2003

    Figure Lengend Snippet: Effect of the W88G mutation on removal of AZTMP from blocked primer-template. (A) AZTMP-terminated [5′- 32 P]L33 primer-WL50 template was incubated with the indicated WT or mutant RT in the absence (−) or presence (+) of 3.2 mM ATP for the indicated times at 37°C. The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with an exonuclease-free Klenow fragment of E. coli DNA polymerase I. The products were separated on a 20% denaturing polyacrylamide gel. The positions of unextended primer (primer) and of products formed after elongation to the end of the template (ext. primer) are shown to the left of the figure. (B) Radioactivity in products longer than 34 nucleotides (rescued primers) from experiments whose results are shown in panel A were quantitated by PhosphorImager analysis, expressed as a percentage of total radioactivity for each lane, and plotted against time. (C) Experiments were performed as described for panel A, except that the ATP concentration was varied from 0.2 to 6.4 mM and the time of incubation (2 to 90 min) was chosen for each RT to allow a maximum of 40% of the primer to be rescued. (D) Rescue experiments were performed as described for panel A, except that 50 μM PP i was used instead of ATP. For panels B, C, and D, the symbols represent data points obtained in a typical experiment with the RTs indicated at the bottom of the figure, and the lines represent the best fit of the data to a hyperbola.

    Article Snippet: The RT was inactivated by heat treatment, and the unblocked primer was extended by incubation with the exonuclease-free Klenow fragment of Escherichia coli DNA polymerase I (0.3 U; USB Corp.) and all four dNTPs (100 μM each).

    Techniques: Mutagenesis, Incubation, Radioactivity, Concentration Assay

    The full length bicistronic RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23] . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).

    Journal: PLoS ONE

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    doi: 10.1371/journal.pone.0035031

    Figure Lengend Snippet: The full length bicistronic RNA is expressed from the dl VAR vectors. (A) Schematic representation of the experimental procedure to detect the full length bicistronic mRNA showing the primers and the size of the expected amplicons [23] . (B and D) HeLa cells were transfected with 200 ng of the dl HIV-1, dl ΔEMCV, or the different dl VAR plasmids. Total RNA was extracted from transfected cells and quantified. (B) Extracted RNA (3 µg) was used as template in a one-step RT-PCR designed to specifically detect the bicistronic RNAs (lanes 4, 6, 8, 10, 14, and 16). To assay for DNA contamination the same reaction was conducted in the absence of reverse transcriptase (lanes 5, 7, 9, 11, 13, 15, 17). In vitro transcribed dl HIV-1 IRES RNAs (lane 2) and water (lane 3) were included as RT-PCR controls. (C) Schematic representation of the experimental procedure to detect the RLuc-RNA and FLuc-RNA showing the primers and the size of the expected amplicons (D) Total RNA (200 ng) extracted from transfected HeLa cells was used as template in parallel RT-qPCR reactions designed to specifically detect the RLuc or FLuc containing RNAs. The RNA-FLuc concentration (pmol)/RNA-RLuc (pmol) ratio was calculated. Values are the means +/- SEM from three independent experiments (each RNA sample was amplified in three independent reactions).

    Article Snippet: To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Techniques: Transfection, Reverse Transcription Polymerase Chain Reaction, In Vitro, Quantitative RT-PCR, Concentration Assay, Amplification

    Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples. ( A ) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. ( B ) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described [23] . Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in Fig. 2B ) [23] . In vitro transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. ( C ) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described [23] . Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.

    Journal: PLoS ONE

    Article Title: Functional and Structural Analysis of the Internal Ribosome Entry Site Present in the mRNA of Natural Variants of the HIV-1

    doi: 10.1371/journal.pone.0035031

    Figure Lengend Snippet: Analysis of a promoterless bicistronic construct containing the HIV-1 5′UTR sequences recovered from clinical samples. ( A ) Schematic representation of the bicistronic constructs. The SV40 promoter from dl ΔEMCV (lane 4), dl HIV-1 IRES, or dl VAR was removed to generate the equivalent promoterless (ΔSV40) vectors. ( B ) HeLa cells were transfected with DNA (200 ng) corresponding to the vectors depicted in (A) as previously described [23] . Total DNA was extracted from transfected cells and the presence of the transfected plasmids was confirmed by PCR (upper panel). The dl HIV-1 IRES plasmid (100 ng) was used as an amplification control. Total RNA was extracted from transfected cells and the presence of transcripts for the ΔSV40-dl ΔEMCV, ΔSV40-dl HIV-1 IRES, or the ΔSV40-dl VAR plasmids was evaluated by a one step RT-PCR designed to detect the bicistronic RNA (depicted in Fig. 2B ) [23] . In vitro transcribed RNA (100 ng) generated from plasmids dl HIV-1 IRES (lane 2) were used as amplification controls. ( C ) HeLa cells were transfected with either the 200 ng of SV40 or ΔSV40 version of dl ΔEMCV, dl HIV-1 IRES, or the different dl VAR plasmids as previously described [23] . Cells were processed and RLuc and FLuc activities were measured. For each data point the [RLuc/(total protein)] (left panel) and the [FLuc/(total protein)] (right panel) for the SV40 positive plasmids was arbitrary set to 100%. Values are the means +/- SD from three independent experiments.

    Article Snippet: To generate plasmids without the SV40 mammalian promoter, the bicistronic vectors were digested with StuI and MluI (Fermentas), treated with the E. coli DNA Polymerase I Klenow fragment (Fermentas) to generate blunt ends, and ligated using T4 DNA ligase (Fermentas).

    Techniques: Construct, Transfection, Polymerase Chain Reaction, Plasmid Preparation, Amplification, Reverse Transcription Polymerase Chain Reaction, In Vitro, Generated

    The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Journal: Nucleic Acids Research

    Article Title: A functional interaction of Ku with Werner exonuclease facilitates digestion of damaged DNA

    doi:

    Figure Lengend Snippet: The effect of Ku on the 3′→5′ exonuclease activity of WRN, Klenow and exo III on a substrate containing 8-oxoA. DNA substrate containing 8-oxoA was incubated without enzyme (–enzyme) or with WRN (180 fmol), Klenow (2 U) or exo III (1 U) in the absence or presence of Ku (64 fmol) at 37°C for 1 h. The labeled reaction products were analyzed and visualized as described earlier.

    Article Snippet: Exonuclease III (exo III) and the Klenow fragment of Escherichia coli were purchased from Boehringer Mannheim and T4 polynucleotide kinase was from New England Biolabs (Beverly, MA).

    Techniques: Activity Assay, Incubation, Labeling

    PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

    Journal: Applied and Environmental Microbiology

    Article Title: Molecular Characterization of Natural Erwinia pyrifoliae Strains Deficient in Hypersensitive Response

    doi: 10.1128/AEM.69.1.679-682.2003

    Figure Lengend Snippet: PFGE analysis to show the relatedness of E. pyrifoliae strains Ep2/97 and Ep4/97. Genomic DNA of agar-embedded cells was digested with restriction enzyme Xba I, and the PFGE was performed in a 1% agarose gel (Ultra Pure DNA grade agarose; Bio-Rad) with running buffer (3.5 mM HEPES, 3.5 mM sodium acetate, 0.35 mM EDTA [pH 8.3]) for 22 h at 14°C and a ramping time from 1 to 25 s. M, positions of marker DNA in kilobases. Multimeric λ genomes were run in a separate lane. Lanes: 1, E. pyrifoliae Ep8/95 (pattern identical with those of strains Ep1/96 and Ep16/96 in Xba I digests); 2, SLR21 (from Asian pear; not E. pyrifoliae ); 3, Ep2/97; 4, Ep4/97; 5, E. amylovora Ea1/79.

    Article Snippet: To reconfirm the hrpL sequences, DNA from strains Ep2/97, Ep4/97, and Ep16/96 was also amplified with a proofreading DNA polymerase (Accu Taq ; Sigma).

    Techniques: Agarose Gel Electrophoresis, Marker