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  • 99
    Thermo Fisher dntp set
    Dntp Set, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 296 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 99 stars, based on 296 article reviews
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    99
    Jena Bioscience dntp
    PrimPol catalyzes repriming downstream of 3′ incorporated CTNAs and templating abasic or thymine glycol lesions. PrimPol (1μM) was incubated for 15 min at 37° C with <t>dNTPs</t> (250 μM), <t>FAM-dNTPs</t> (dATP, dCTP, dUTP) (2.5 μM), and mixed sequence primer-templates (1 μM) (as shown in the schematic). Primers containing a 3′ dideoxynucleotide were annealed upstream of the lesion on templates containing a single Ap site (Ap) or thymine glycol (Tg) to allow us to assay for repriming, rather than TLS, activity. In the case of CTNAs, a single CTNA (acyclovir (ACV) or carbovir (CBV)) was located at the 3′ end of the primer in place of the dideoxnucleotide. The length of primase reaction products extended to the end of the template allows analysis of the priming location by PrimPol; the near identical extension products produced in each case show close-coupled repriming by PrimPol downstream of the lesion or CTNA. Oligonucleotide nucleotide (Nt) length markers are shown in the left panel. Priming and extension are represented in the schematic as green and blue, respectively. “C” indicates the no enzyme control. “ND” indicates the non-damaged template without an annealed primer.
    Dntp, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 99/100, based on 50 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp  (TaKaRa)
    99
    TaKaRa dntp
    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), <t>10×</t> buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L <t>dNTP</t> (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.
    Dntp, supplied by TaKaRa, used in various techniques. Bioz Stars score: 99/100, based on 11475 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher dntp
    Creating a supercoiled and fluorescent-labeled <t>sopC-plasmid.</t> The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with <t>dNTPs</t> and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 31074 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    Bio-Rad dntps
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntps, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 95/100, based on 706 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Bio-Rad dntp mix
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp Mix, supplied by Bio-Rad, used in various techniques. Bioz Stars score: 99/100, based on 219 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dntp mix
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp Mix, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 3181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Thermo Fisher geneamp dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Geneamp Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 87 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    geneamp dntp - by Bioz Stars, 2020-04
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    95
    Applichem dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Applichem, used in various techniques. Bioz Stars score: 95/100, based on 26 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Bioteke Corporation dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Bioteke Corporation, used in various techniques. Bioz Stars score: 93/100, based on 36 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Boehringer Mannheim dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Boehringer Mannheim, used in various techniques. Bioz Stars score: 92/100, based on 756 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Eppendorf AG dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 97/100, based on 502 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp - by Bioz Stars, 2020-04
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    90
    Eurobio dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Eurobio, used in various techniques. Bioz Stars score: 90/100, based on 168 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Fisher Scientific dntp
    Incorporation of <t>Fc1-dUTP</t> into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of <t>dNTPs</t> as indicated. DNA fragment lengths in nucleotides are shown on the left.
    Dntp, supplied by Fisher Scientific, used in various techniques. Bioz Stars score: 94/100, based on 163 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    GE Healthcare dntp
    Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of <t>dNTPs</t> (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding <t>50μM</t> of dNTPs for 15 min.
    Dntp, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 93/100, based on 1823 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    95
    GeneDireX dntp
    Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of <t>dNTPs</t> (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding <t>50μM</t> of dNTPs for 15 min.
    Dntp, supplied by GeneDireX, used in various techniques. Bioz Stars score: 95/100, based on 13 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp - by Bioz Stars, 2020-04
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    93
    Kaneka Corp dntp
    Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of <t>dNTPs</t> (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding <t>50μM</t> of dNTPs for 15 min.
    Dntp, supplied by Kaneka Corp, used in various techniques. Bioz Stars score: 93/100, based on 387 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp - by Bioz Stars, 2020-04
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    99
    Meridian Life Science dntp
    Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of <t>dNTPs</t> (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding <t>50μM</t> of dNTPs for 15 min.
    Dntp, supplied by Meridian Life Science, used in various techniques. Bioz Stars score: 99/100, based on 2045 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore dntp
    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM <t>oligo-dT,</t> and 1 mM <t>dNTP.</t>
    Dntp, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 2634 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    PerkinElmer dntp
    Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM <t>α-</t> 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM <t>dNTPs</t> mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.
    Dntp, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 94/100, based on 2063 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Promega dntp
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntp, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 11528 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Protech Technology Enterprise dntp
    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. <t>bRT</t> is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and <t>dNTPs,</t> including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
    Dntp, supplied by Protech Technology Enterprise, used in various techniques. Bioz Stars score: 93/100, based on 42 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp  (Roche)
    99
    Roche dntp
    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
    Dntp, supplied by Roche, used in various techniques. Bioz Stars score: 99/100, based on 4651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    dntp - by Bioz Stars, 2020-04
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    Shanghai Generay Biotech dntp
    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Transgenomic dntp
    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Viogene dntp
    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using <t>rNTPs</t> and <t>dNTPs</t> opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.
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    Image Search Results


    PrimPol catalyzes repriming downstream of 3′ incorporated CTNAs and templating abasic or thymine glycol lesions. PrimPol (1μM) was incubated for 15 min at 37° C with dNTPs (250 μM), FAM-dNTPs (dATP, dCTP, dUTP) (2.5 μM), and mixed sequence primer-templates (1 μM) (as shown in the schematic). Primers containing a 3′ dideoxynucleotide were annealed upstream of the lesion on templates containing a single Ap site (Ap) or thymine glycol (Tg) to allow us to assay for repriming, rather than TLS, activity. In the case of CTNAs, a single CTNA (acyclovir (ACV) or carbovir (CBV)) was located at the 3′ end of the primer in place of the dideoxnucleotide. The length of primase reaction products extended to the end of the template allows analysis of the priming location by PrimPol; the near identical extension products produced in each case show close-coupled repriming by PrimPol downstream of the lesion or CTNA. Oligonucleotide nucleotide (Nt) length markers are shown in the left panel. Priming and extension are represented in the schematic as green and blue, respectively. “C” indicates the no enzyme control. “ND” indicates the non-damaged template without an annealed primer.

    Journal: Cell Cycle

    Article Title: Repriming by PrimPol is critical for DNA replication restart downstream of lesions and chain-terminating nucleosides

    doi: 10.1080/15384101.2016.1191711

    Figure Lengend Snippet: PrimPol catalyzes repriming downstream of 3′ incorporated CTNAs and templating abasic or thymine glycol lesions. PrimPol (1μM) was incubated for 15 min at 37° C with dNTPs (250 μM), FAM-dNTPs (dATP, dCTP, dUTP) (2.5 μM), and mixed sequence primer-templates (1 μM) (as shown in the schematic). Primers containing a 3′ dideoxynucleotide were annealed upstream of the lesion on templates containing a single Ap site (Ap) or thymine glycol (Tg) to allow us to assay for repriming, rather than TLS, activity. In the case of CTNAs, a single CTNA (acyclovir (ACV) or carbovir (CBV)) was located at the 3′ end of the primer in place of the dideoxnucleotide. The length of primase reaction products extended to the end of the template allows analysis of the priming location by PrimPol; the near identical extension products produced in each case show close-coupled repriming by PrimPol downstream of the lesion or CTNA. Oligonucleotide nucleotide (Nt) length markers are shown in the left panel. Priming and extension are represented in the schematic as green and blue, respectively. “C” indicates the no enzyme control. “ND” indicates the non-damaged template without an annealed primer.

    Article Snippet: Briefly, PrimPol (1 μM) was incubated at 37°C for 15 mins in 20 μl reactions volumes containing 10 mM Bis-Tris-Propane-HCl pH 7.0, 10 mM MgCl2 , 1 mM DTT, 250 μM dNTPs or rNTPs, and 2.5 μM FAM dNTPs (dATP, dCTP, dUTP) (Jena-Biosciences).

    Techniques: Incubation, Sequencing, Activity Assay, Produced

    Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Journal: International Journal of Clinical and Experimental Pathology

    Article Title: Knockdown of Gli1 by small-interfering RNA enhances the effects of BCNU on the proliferation and apoptosis of glioma U251 cells

    doi:

    Figure Lengend Snippet: Gli1, cyclin D1, Bcl-2 and Bax mRNA levels in blank, Lipofectamine, siRNA-Gli1, BCNU and combination groups. Semi-quantitative real-time polymerase chain reaction was performed using SuperScript III One-Step RT-PCR kit (12574-018, Invitrogen, Thermo Scientific, Waltham, MA, USA) on PCR thermo cycler (C1000, Bio-Rad, Hercules, CA, USA). The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl 2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each). M, markers; 1, blank group; 2, Lipofectamine group; 3, BCNU group; 4, siRNA-Gli1 group; 5, combination group.

    Article Snippet: The 20 μl reaction system was formed by cDNA (5 μl), 10× buffer (2 μl), 25 mmol/L MgCl2 (0.8 μl), 2.5 mmol/L dNTP (2 μl), DNA polymerase (0.2 μl; Takara, Tokyo, Japan), and upstream and downstream primers (1 μl each).

    Techniques: Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction

    Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Primer extension from the CTNA-blocked termini by the Klenow fragment of Escherichia coli DNA polymerase I, with or without its proofreading 3′–5′ exonuclease activity (KF + or KF − , respectively, from Takara Bio, Inc., Shiga, Japan), in the ( A ) presence or ( B ) absence of dNTPs. ( A ) The 32 P-labeled oligonucleotides, 32 P-d(TCCGTTGAAGCCTGCTTT)X, where X represents no added nucleoside (OH, lanes 1–3), 2’-deoxyadenosine (lanes 4–6), acyclovir (ACV, lanes 7–9), abacavir (ABC, lanes 10–12), carbovir (CBV, lanes 13–15) or lamivudine ((−)3TC, lanes 16–18), were hybridized with their complementary strands, d(CTCGTCAGCTANAAAGCAGGCTTCAACGGA), where N represents A (for ABC and an oligonucleotide without CTNAs), G (for A and (−)3TC) or C (for ACV and CBV). Each substrate was incubated at 37 °C for 10 min, in the absence (lanes 1, 4, 7, 10, 13 and 16) or presence of KF − (0.1 unit, lanes 2, 5, 8, 11, 14 and 17) or KF + (0.1 unit, lanes 3, 6, 9, 12, 15 and 18), in 10 mM Tris-HCl buffer (pH 7.9) containing 50 mM NaCl, 10 mM MgCl 2 , 10 mM DTT and 100 µM dNTPs; ( B ) The 32 P-labeled substrates were incubated with KF + at 37 °C for the indicated incubation time, in the same reaction buffer without dNTPs.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Activity Assay, Labeling, Incubation

    Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Journal: Molecules

    Article Title: Chemical Incorporation of Chain-Terminating Nucleoside Analogs as 3′-Blocking DNA Damage and Their Removal by Human ERCC1-XPF Endonuclease

    doi: 10.3390/molecules21060766

    Figure Lengend Snippet: Repair of the CTNA-containing oligonucleotides by human ERCC1-XPF endonuclease and DNA polymerase. Panels A and B represent the reaction scheme and the results, respectively. First, the 32 P-labeled substrates (400 fmol) were incubated at 25 °C for 16 h, in the absence (lanes 1, 2, 5, 6, 9, 10, 13, 14, 17, 18, 21 and 22) or presence (the other lanes) of ERCC1-XPF (230 fmol), in 10 µL of 50 mM Tris-HCl buffer (pH 8.0) containing 2 mM MgCl 2 , 0.5 mM DTT and 0.1 mg·mL −1 BSA. Then, a polymerase reaction mixture (5 µL, containing 30 mM Tris-HCl, (pH 7.9), 150 mM NaCl, 30 mM MgCl 2 , 30 mM DTT, 300 µM dNTPs and 0.1 unit of KF − for even lanes) or 5 mM EDTA (5 µL for odd lanes) was added to the reaction mixture, and the total reaction mixtures (15 µL) were incubated at 37 °C for 10 min. The cleavage sites observed with ERCC1-XPF are indicated by black triangles. The fully extended products were quantified, and the values are shown.

    Article Snippet: Resumption of DNA Synthesis by DNA Polymerase after the Removal of CTNAs The hybridized oligonucleotides (400 fmol) were first treated with ERCC1-XPF (230 fmol) in 10 µL of 50 mM Tris-HCl buffer (pH 8.0), containing 2 mM MgCl2 , 0.5 mM DTT and 0.1 mg·mL−1 BSA, at 25 °C for 16 h. To the reaction mixture was added 5 µL of 30 mM Tris-HCl buffer (pH 7.9), containing 150 mM NaCl, 30 mM MgCl2 , 30 mM DTT, 300 µM dNTPs and the Klenow fragment of Escherichia coli DNA polymerase I, lacking the 3′–5′ exonuclease activity (KF− , 0.1 unit, Takara Bio).

    Techniques: Labeling, Incubation

    Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Journal: Methods in cell biology

    Article Title: Reconstituting ParA/ParB-mediated transport of DNA cargo

    doi: 10.1016/bs.mcb.2015.01.021

    Figure Lengend Snippet: Creating a supercoiled and fluorescent-labeled sopC-plasmid. The plasmid pBR322:: sopC is fluorescently labeled to visualize its movement over the DNA-carpeted flow cell. We have developed an efficient labeling protocol that does not require intercalating dyes and produces a negatively supercoiled plasmid. The restriction enzyme Nt.BspQ1 nicks the pBR322 backbone at a site located approximately 180° from sopC . DNA polymerase I is used with dNTPs and Alexa647-labeled dCTP to label the DNA. Ethidium Bromide promotes negative supercoiling before a final ligation reaction that covalently closes the nick. The final product is a negatively supercoiled and fluorescently labeled plasmid bearing the sopC centromere site. This protocol can be used to incorporate a variety of dyes without significant perturbation to plasmid topology.

    Article Snippet: pBR322:: sopC plasmid (PCR template) PCR Primers for sopC DNA amplification: (Primers synthesized by IDT) 10 mM dNTPs (Life Technologies; Cat. # 18427–013) 100 mM MgSO4 (New England Biolabs, Cat. # M0254S) 2000 U/mL VENT Polymerase (New England Biolabs, Cat. # M0254S) SYBR Gold DNA Stain (Life Technologies, Cat. # S-11494) Illustra MicroSpin S-400HR columns (GE Healthcare, Cat # 27–5140-01) MyOne Streptavidin C1 Dynabeads (Life Technologies, Cat. # 65001) 20 U/μL PstI Restriction Enzyme (New England Biolabs, Cat. # R0140S) Phenol:CHCl3:Isoamyl Alcohol (UltraPure 25:24:1 v/v) Ethanol.

    Techniques: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

    Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.

    Journal: Stem cell research

    Article Title: Vpx mediated degradation of SAMHD1 has only a very limited effect on lentiviral transduction rate in ex vivo cultured HSPCs

    doi: 10.1016/j.scr.2015.06.012

    Figure Lengend Snippet: Vpx+ virus, dN and dNTP treatments result in minor increases in the dNTP pool of HSPCs. MDMs (A, C and E) and freshly isolated HSPCs (B, D and F) were pretreated for 2 h with Vpx+/− viruses, dNs or dNTPs, and cultured for 3 days before cell lysis. Quantification of dNTPs was done by a single nucleotide incorporation assay (see “Material and methods” section). Fold induction was calculated based on the absolute values of vero- vs mock-treated samples (C–F). White dots represent Vpx− virus-pretreated samples, black dots represent Vpx+ virus-pretreated samples (A–B). White bars represent dN-pretreated samples, black bars represent dNTP-pretreated samples (C–F). Data are from three donors and bars represent mean ± SEM. Statistical analysis was done using two-tailed paired t-test.

    Article Snippet: THP-1 cells transduced with lentiviral vectors encoding SAMHD1 shRNA were selected by culturing in a medium containing 0.8 μg/ml puromycin. dNs consisted of a mixture of dA (D8668), dC (D0776), dG (D0901) and dT (T1895; all from Sigma-Aldrich). dNTPs (R0192) were purchased from Fermentas (Glen Burnie, MD).

    Techniques: Isolation, Cell Culture, Lysis, Two Tailed Test

    Incorporation of Fc1-dUTP into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of dNTPs as indicated. DNA fragment lengths in nucleotides are shown on the left.

    Journal: Nucleic Acids Research

    Article Title: Ferrocene conjugates of dUTP for enzymatic redox labelling of DNA

    doi:

    Figure Lengend Snippet: Incorporation of Fc1-dUTP into DNA by Klenow fragment and T4 DNA polymerase. The P18.1/T40.1 primer–template pair of Figure 3 was incubated with DNA polymerase and different sets of dNTPs as indicated. DNA fragment lengths in nucleotides are shown on the left.

    Article Snippet: To generate the duplex for HPLC–ECD, the P18.1 and T40.1 oligonucleotides (4 µM each) were annealed before incubation in 240 µl of reaction mixture containing 6.7 mM Tris–HCl pH 8.8, 6.6 mM MgCl2 , 1 mM DTT, 16.8 mM (NH4 )2 SO4 , 200 µM dNTPs (except dTTP), 200 µM Fc1-dUTP and 0.25 U/µl Klenow fragment at room temperature for 20 min. Low molecular weight components were removed with a Bio-Spin 30 chromatography column (Bio-Rad).

    Techniques: Incubation

    Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of dNTPs (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding 50μM of dNTPs for 15 min.

    Journal: DNA repair

    Article Title: Replication of 2-hydroxyadenine-containing DNA and recognition by human MutS?

    doi: 10.1016/j.dnarep.2006.11.002

    Figure Lengend Snippet: Elongation of terminal 2-OH-A:T, 2-OH-A: A, 2-OH-A:G and 2-OH-A:C base pairs. (A) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Kf exo− . Reactions contained 50nM primer/template and 2.5nM Kf exo− supplemented with an equimolar mixture of dNTPs (30μM). Controls were incubated without enzyme or dNTP. (B) Elongation of 2-OH-A containing terminal mismatches in the 6A * sequence by Dpo4. Primer/template (50 nM) were pre-incubated with 150nM Dpo4 enzyme at 55 °C for 3min. The elongation was performed by adding 50μM of dNTPs for 15 min.

    Article Snippet: The subsequent elongation was performed by adding 50μM of dNTPs for further 15 min. Fluorescent bands were visualized by Typhoon 9200 Gel Imager (Amersham Bio-sciences Europe GmbH) and quantitated by ImageQuant TL software.

    Techniques: Sequencing, Incubation

    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: Directly lysed cells were incubated in 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo-dT (Metabion), and 2.5 μM random hexamers (Metabion) at 65°C for 5 min and then chilled on ice.

    Techniques: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: Directly lysed cells were incubated in 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo-dT (Metabion), and 2.5 μM random hexamers (Metabion) at 65°C for 5 min and then chilled on ice.

    Techniques: Lysis, Polymerase Chain Reaction

    Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.

    Journal: Nucleic Acids Research

    Article Title: Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    doi: 10.1093/nar/gkw170

    Figure Lengend Snippet: Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.

    Article Snippet: Reagents 8-oxodGTP was obtained from TriLink (TriLink BioTechnologies, San Diego, CA 92121, USA), dNTPs were from Sigma (Sigma-Aldrich, Corporate Offices St. Louis, MO 63103, USA) and 2-OH-dATP was purchased from Jena (Jena Bioscience GmbH 07749 Jena, DE).

    Techniques: Sequencing, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Software, CTG Assay

    Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.

    Journal: Nucleic Acids Research

    Article Title: Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    doi: 10.1093/nar/gkw170

    Figure Lengend Snippet: Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.

    Article Snippet: Reagents 8-oxodGTP was obtained from TriLink (TriLink BioTechnologies, San Diego, CA 92121, USA), dNTPs were from Sigma (Sigma-Aldrich, Corporate Offices St. Louis, MO 63103, USA) and 2-OH-dATP was purchased from Jena (Jena Bioscience GmbH 07749 Jena, DE).

    Techniques: Incubation, Concentration Assay, CTG Assay

    Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).

    Journal: Nucleic Acids Research

    Article Title: Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

    doi: 10.1093/nar/gkw170

    Figure Lengend Snippet: Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).

    Article Snippet: Reagents 8-oxodGTP was obtained from TriLink (TriLink BioTechnologies, San Diego, CA 92121, USA), dNTPs were from Sigma (Sigma-Aldrich, Corporate Offices St. Louis, MO 63103, USA) and 2-OH-dATP was purchased from Jena (Jena Bioscience GmbH 07749 Jena, DE).

    Techniques: Ligation, Activity Assay, Synthesized, Modification

    Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Journal: PLoS ONE

    Article Title: Biochemical Characterization of Enzyme Fidelity of Influenza A Virus RNA Polymerase Complex

    doi: 10.1371/journal.pone.0010372

    Figure Lengend Snippet: Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, HIV-1 RT and MuLV RT with biased nucleotide substrate pools. ( A ) Template sequences used for the misincorporation assay with IAV Pol, T7 RNA polymerase and RTs of HIV-1 and MuLV. The IAV Pol template used is a 30-nt sequence from the 3′ end of the viral PA sequence with a ApG primer binding site (P:primer, T:template,). The first UTP or TTP incorporation site of each template was marked with “X”, and the first stop site in the (−) UTP or TTP reaction was marked with “1*” under each template sequence (the second and third stop sites were marked “2*” and “3*” for the IAV template). The RNA synthesis initiation sites were marked in blue. ( B ) ApG-initiated RNA-dependent RNA polymerization by IAV Pol: ApG primer was extended with a 30-nt RNA template and three different amounts of H3N2 IAV Pol protein (1x, 2x and 3x) with 500 µM four NTPs (“4”) and 0.16 µM α- 32 P-GTP, and the same reactions were repeated except using two biased NTP pools, minus UTP (“- U”) or minus ATP (“- A”). The sequence of the incorporated nucleotides near the three UTP stop sites (red) is shown at the side. The extended products in the (−) UTP reaction, which was used for calculating the misincorporation efficiency, was marked as “]”.Dotted lines refer to RNAs. ( C ) DNA dependent RNA polymerization of bacterial phage T7 RNA polymerase: A 47 bp ds DNA (box) encoding T7 promoter (grey box) and 29 bp sequence (white box) was used for RNA synthesis with T7 RNA polymerase at 37°C for 60 mins. “]”: Fully extended misincorporated product. ( D ) and ( E ) RNA dependent DNA polymerization reaction by HIV-1 RT (D) and MuLV RT (E). A 48 mer RNA template annealed to a 18-mer single stranded DNA primer was used for the DNA polymerization by HIV-1 and MuLV RTs at 37°C for 60 mins. 500 µM dNTPs mixed with α- 32 P-dNTPs (0.16 µM), which is the same nucleotide concentration and ratio as used in the reactions with IAV Pol and T7 RNA polymerase, was used for DNA synthesis. “]”: Fully extended misincorporated product. Dotted lines refer to RNA and solid lines refer to DNA. ( F ) and ( G ) Comparison of the misincorporation efficiency of the four polymerases. For calculation of the misincorporation efficiency, the fully extended misincorporated product in (−) U/(−) T reactions in all four polymerases was normalized to the total extended product in the lanes with all four NTPs (dNTPs). The fold differences of the calculated misincorporation percentages between three activities of IAV Pol ( F ) and between IAV Pol and three other polymerases ( G ) were determined. At least five repeats of the assay were conducted in this analysis.

    Article Snippet: Misincorporation assay In the misincorporation assays with all four polymerases, an equal concentration of all the nucleotides (NTPs or dNTPs) was provided using a mix of four α-32 P-NTPs or dNTPs (0.16 µM, PerkinElmer, specific activity, 3000 Ci/mmol) and nonradioactive NTPs or dNTPs (500 µM).

    Techniques: Sequencing, Binding Assay, Concentration Assay, DNA Synthesis

    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

    Journal: Nucleic Acids Research

    Article Title: Molecular dissection of the domain architecture and catalytic activities of human PrimPol

    doi: 10.1093/nar/gku214

    Figure Lengend Snippet: Primase activity of human PrimPol. ( A ) Human PrimPol has primase activity and can produce de novo primers using rNTPs and dNTPs opposite a poly(dT) template. ( B ) PrimPol ZF-KO lacks de novo primer synthesis activity, suggesting that an intact zinc finger is required for primase activity. ( C ) PrimPol 1–487 also has primase activity similar to the wild-type PrimPol. The unstructured region that is downstream of the zinc finger is therefore not required for primase activity. ( D ) PrimPol 1–354 has no primase activity, which indicates that PrimPol requires a functional zinc finger for primer synthesis.

    Article Snippet: Typically detection of primase activity was started from incubation of 1μM of the enzyme to be tested in 20 μl reaction volume containing 500 nM homopolymeric ss DNA templates with a biotin modification at the 5′ end (see sequences 1–4 in Supplementary Table S2), 500 μM rNTPs (Invitrogen) or 500 μM dNTPs (Roche), 10 mM Bis-Tris-Propane-HCl (pH 7.0), 10 mM MgCl2 , 50 mM NaCl.

    Techniques: Activity Assay, Functional Assay