dntps  (Thermo Fisher)


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    Name:
    MgCl2 magnesium chloride
    Description:
    Thermo Scientific PCR reagents meet high quality standards to generate accurate results from even the most challenging molecular biology applications Magnesium ions Mg2 are an important component of PCR reactions where they interact with the DNA template dNTPs and Taq DNA Polymerase
    Catalog Number:
    ab0359
    Price:
    None
    Applications:
    PCR|PCR & Real-Time PCR|Routine PCR
    Category:
    Lab Reagents and Chemicals
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    Structured Review

    Thermo Fisher dntps
    Schematic illustration of sequencing with MiSeq FGx and Ion Torrent, a MiSeq: Forenseq is a library prep used for STR and SNP sequencing (autosomal STRs, sex, geographical ancestry, and phenotypic SNPs); alternatively, Nextera is utilized for mtDNA sequencing. In the process of sample preparation, adaptors are added to DNA fragments in a two-step PCR reaction in order to enable DNA binding to a glass slide. In the next step, the fragments are clonally amplified on the slide and sequenced. The template strand is extended with one nucleotide at a time. The reaction of polymerization is halted due to the use of 3′- O <t>-azidomethyl-dNTPs</t> that are <t>fluorescently</t> labeled. The base incorporation is followed by removal of unincorporated bases and imaging using CCD camera. Subsequently, the 3′ block and the fluorescent tag on the incorporated nucleotide are removed and the reaction proceeds to the next cycle. b Ion Torrent: sample preparation of DNA fragments for sequencing on Ion Torrent is similar to the workflow utilized by Roche 454 sequencer, followed by amplification of adaptor-ligated DNA hybridized to beads using emulsion PCR (Margulies et al. 2005) [ 1 ]. The beads are distributed to microwells, where sequencing by synthesis occurs. The sensor located at the bottom of the well converts the changes in pH into a voltage signal proportional to the number of incorporated bases
    Thermo Scientific PCR reagents meet high quality standards to generate accurate results from even the most challenging molecular biology applications Magnesium ions Mg2 are an important component of PCR reactions where they interact with the DNA template dNTPs and Taq DNA Polymerase
    https://www.bioz.com/result/dntps/product/Thermo Fisher
    Average 94 stars, based on 5648 article reviews
    Price from $9.99 to $1999.99
    dntps - by Bioz Stars, 2020-07
    94/100 stars

    Images

    1) Product Images from "Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects"

    Article Title: Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects

    Journal: International Journal of Legal Medicine

    doi: 10.1007/s00414-020-02294-0

    Schematic illustration of sequencing with MiSeq FGx and Ion Torrent, a MiSeq: Forenseq is a library prep used for STR and SNP sequencing (autosomal STRs, sex, geographical ancestry, and phenotypic SNPs); alternatively, Nextera is utilized for mtDNA sequencing. In the process of sample preparation, adaptors are added to DNA fragments in a two-step PCR reaction in order to enable DNA binding to a glass slide. In the next step, the fragments are clonally amplified on the slide and sequenced. The template strand is extended with one nucleotide at a time. The reaction of polymerization is halted due to the use of 3′- O -azidomethyl-dNTPs that are fluorescently labeled. The base incorporation is followed by removal of unincorporated bases and imaging using CCD camera. Subsequently, the 3′ block and the fluorescent tag on the incorporated nucleotide are removed and the reaction proceeds to the next cycle. b Ion Torrent: sample preparation of DNA fragments for sequencing on Ion Torrent is similar to the workflow utilized by Roche 454 sequencer, followed by amplification of adaptor-ligated DNA hybridized to beads using emulsion PCR (Margulies et al. 2005) [ 1 ]. The beads are distributed to microwells, where sequencing by synthesis occurs. The sensor located at the bottom of the well converts the changes in pH into a voltage signal proportional to the number of incorporated bases
    Figure Legend Snippet: Schematic illustration of sequencing with MiSeq FGx and Ion Torrent, a MiSeq: Forenseq is a library prep used for STR and SNP sequencing (autosomal STRs, sex, geographical ancestry, and phenotypic SNPs); alternatively, Nextera is utilized for mtDNA sequencing. In the process of sample preparation, adaptors are added to DNA fragments in a two-step PCR reaction in order to enable DNA binding to a glass slide. In the next step, the fragments are clonally amplified on the slide and sequenced. The template strand is extended with one nucleotide at a time. The reaction of polymerization is halted due to the use of 3′- O -azidomethyl-dNTPs that are fluorescently labeled. The base incorporation is followed by removal of unincorporated bases and imaging using CCD camera. Subsequently, the 3′ block and the fluorescent tag on the incorporated nucleotide are removed and the reaction proceeds to the next cycle. b Ion Torrent: sample preparation of DNA fragments for sequencing on Ion Torrent is similar to the workflow utilized by Roche 454 sequencer, followed by amplification of adaptor-ligated DNA hybridized to beads using emulsion PCR (Margulies et al. 2005) [ 1 ]. The beads are distributed to microwells, where sequencing by synthesis occurs. The sensor located at the bottom of the well converts the changes in pH into a voltage signal proportional to the number of incorporated bases

    Techniques Used: Sequencing, Sample Prep, Polymerase Chain Reaction, Binding Assay, Amplification, Labeling, Imaging, Blocking Assay

    2) Product Images from "Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs"

    Article Title: Novel thermostable Y-family polymerases: applications for the PCR amplification of damaged or ancient DNAs

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkj512

    Primer extension on CPD and abasic site-containing templates. Primer SSHTP2 (5′-GCG GTG TAG AGA CGA GTG CGG AG-3′) was annealed to the undamaged template, HTU50 (Un) (5′-CTC TCA CAA GCA GCC AGG CAT TCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′); the CPD-containing template, HMTT50 (CPD) (5′-CTC TCA CAA GCA GCC AGG CA T T CT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′) (where the T-T CPD is underlined); or the abasic site-containing template, HTX50 (Abasic) (5′-CTC TCA CAA GCA GCC AGG CAT XCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′), (where X denotes the position of the abasic site). Primer extension assays utilized 10 nM of the three different primer/templates. The reactions containing the undamaged template (HTU50) was incubated at 60°C for 3 min, whereas the reactions containing the CPD (HMTT50) and abasic site (HTX50) templates were incubated at 60°C for 10 min. The concentrations of each enzyme utilized for the undamaged template (HTU50) reactions is as follows: Taq , 0.0025 U; Sso, 0.1 nM; Ste, 0.33 nM; Ssh, 0.4 nM; Ain, 0.2 nM; Ain/Sso, 0.5 nM; Ain/Ste, 0.5 nM. The concentrations of each enzyme utilized for the CPD-containing template (HMTT50) reactions was 50 times higher than that used for the undamaged template and is as follows: Taq , 0.125 U; Sso, 5 nM; Ste, 16.5 nM; Ssh, 20 nM; Ain, 10 nM; Ain/Sso, 25 nM; Ain/Ste, 25 nM. The concentrations of each enzyme utilized for the abasic site-containing template (HMTT50) reactions was five times higher than used for the undamaged template and is as follows: Taq , 0.0125 U; Sso, 0.5 nM; Ste, 1.65 nM; Ssh, 2 nM; Ain, 1 nM; Ain/Sso, 2.5 nM; Ain/Ste, 2.5 nM. Reactions were initiated by the addition of 100 µM of all four dNTPs ( 4 ) or 100 µM of individual dNTPs (G, A, T, C) (indicated below each lane). Replication products were separated on12%/8 M urea polyacrylamide gels and visualized by PhosphorImager analysis.
    Figure Legend Snippet: Primer extension on CPD and abasic site-containing templates. Primer SSHTP2 (5′-GCG GTG TAG AGA CGA GTG CGG AG-3′) was annealed to the undamaged template, HTU50 (Un) (5′-CTC TCA CAA GCA GCC AGG CAT TCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′); the CPD-containing template, HMTT50 (CPD) (5′-CTC TCA CAA GCA GCC AGG CA T T CT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′) (where the T-T CPD is underlined); or the abasic site-containing template, HTX50 (Abasic) (5′-CTC TCA CAA GCA GCC AGG CAT XCT CCG CAC TCG TCT CTA CAC CGC TCC GC-3′), (where X denotes the position of the abasic site). Primer extension assays utilized 10 nM of the three different primer/templates. The reactions containing the undamaged template (HTU50) was incubated at 60°C for 3 min, whereas the reactions containing the CPD (HMTT50) and abasic site (HTX50) templates were incubated at 60°C for 10 min. The concentrations of each enzyme utilized for the undamaged template (HTU50) reactions is as follows: Taq , 0.0025 U; Sso, 0.1 nM; Ste, 0.33 nM; Ssh, 0.4 nM; Ain, 0.2 nM; Ain/Sso, 0.5 nM; Ain/Ste, 0.5 nM. The concentrations of each enzyme utilized for the CPD-containing template (HMTT50) reactions was 50 times higher than that used for the undamaged template and is as follows: Taq , 0.125 U; Sso, 5 nM; Ste, 16.5 nM; Ssh, 20 nM; Ain, 10 nM; Ain/Sso, 25 nM; Ain/Ste, 25 nM. The concentrations of each enzyme utilized for the abasic site-containing template (HMTT50) reactions was five times higher than used for the undamaged template and is as follows: Taq , 0.0125 U; Sso, 0.5 nM; Ste, 1.65 nM; Ssh, 2 nM; Ain, 1 nM; Ain/Sso, 2.5 nM; Ain/Ste, 2.5 nM. Reactions were initiated by the addition of 100 µM of all four dNTPs ( 4 ) or 100 µM of individual dNTPs (G, A, T, C) (indicated below each lane). Replication products were separated on12%/8 M urea polyacrylamide gels and visualized by PhosphorImager analysis.

    Techniques Used: Cellular Antioxidant Activity Assay, Incubation

    Primer extension on the hydantoin-containing template. Primer SSHydP (5′-AGA TCA GTC ACG-3′) was annealed to the undamaged template, HydU22 (Un) (5′-CAC TTC GGA TCG TGA CTG ATC T-3′), and the 5-hydroxy-5-methylhydantoin-containing template, (5′-CAC TTC GGA HCG TGA CTG ATC T-3′), (where H indicates the position of the Hydantoin). Primer extension assays utilized 10 nM of these primer/templates and the reactions were incubated at 37°C for 5 min. The concentrations of each enzyme utilized for the undamaged template (HTU50) and hydantoin-containing template reactions is as follows: Taq , 0.0083 U; Sso, 0.33 nM; Ste, 0.75 nM; Ssh, 1 nM; Ain, 0.5 nM; Ain/Sso, 1.25 nM; Ain/Ste, 1.25 nM. Reactions were initiated by the addition of 100 µM of all four dNTPs ( 4 ) or 100 µM of individual dNTPs (G, A, T, C) (indicated below each lane). Replication products were separated on 18%/8 M urea polyacrylamide gels and visualized by PhosphorImager analysis.
    Figure Legend Snippet: Primer extension on the hydantoin-containing template. Primer SSHydP (5′-AGA TCA GTC ACG-3′) was annealed to the undamaged template, HydU22 (Un) (5′-CAC TTC GGA TCG TGA CTG ATC T-3′), and the 5-hydroxy-5-methylhydantoin-containing template, (5′-CAC TTC GGA HCG TGA CTG ATC T-3′), (where H indicates the position of the Hydantoin). Primer extension assays utilized 10 nM of these primer/templates and the reactions were incubated at 37°C for 5 min. The concentrations of each enzyme utilized for the undamaged template (HTU50) and hydantoin-containing template reactions is as follows: Taq , 0.0083 U; Sso, 0.33 nM; Ste, 0.75 nM; Ssh, 1 nM; Ain, 0.5 nM; Ain/Sso, 1.25 nM; Ain/Ste, 1.25 nM. Reactions were initiated by the addition of 100 µM of all four dNTPs ( 4 ) or 100 µM of individual dNTPs (G, A, T, C) (indicated below each lane). Replication products were separated on 18%/8 M urea polyacrylamide gels and visualized by PhosphorImager analysis.

    Techniques Used: CTG Assay, Incubation

    3) Product Images from "DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells"

    Article Title: DNA polymerase mu (Pol ?), homologous to TdT, could act as a DNA mutator in eukaryotic cells

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.7.1731

    Fig. 6. Pol μ-catalysed misinsertion at the four template bases. The four template–primer structures used, which differ only in the first template base (outlined), are indicated on the left. The single-stranded oligonucleotide corresponding to the primer strand was assayed in parallel as a control of DNA-independent nucleotide insertion. Mg 2+ -activated nucleotide insertion on each 5′–labelled DNA substrate (3.2 nM) was analysed in the presence of either the complementary nucleotide (10 μM) or each of the three incorrect dNTPs (100 μM), as described in Materials and methods. Mn 2+ -activated nucleotide insertion was assayed with each of the four dNTPs (0.1 μM). After incubation for 15 min at 30°C in the presence of 20 ng of human Pol μ, extension of the 5′–labelled (*) strand was analysed by electrophoresis in an 8 M urea–20% polyacrylamide gel and autoradiography.
    Figure Legend Snippet: Fig. 6. Pol μ-catalysed misinsertion at the four template bases. The four template–primer structures used, which differ only in the first template base (outlined), are indicated on the left. The single-stranded oligonucleotide corresponding to the primer strand was assayed in parallel as a control of DNA-independent nucleotide insertion. Mg 2+ -activated nucleotide insertion on each 5′–labelled DNA substrate (3.2 nM) was analysed in the presence of either the complementary nucleotide (10 μM) or each of the three incorrect dNTPs (100 μM), as described in Materials and methods. Mn 2+ -activated nucleotide insertion was assayed with each of the four dNTPs (0.1 μM). After incubation for 15 min at 30°C in the presence of 20 ng of human Pol μ, extension of the 5′–labelled (*) strand was analysed by electrophoresis in an 8 M urea–20% polyacrylamide gel and autoradiography.

    Techniques Used: Incubation, Electrophoresis, Autoradiography

    Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.
    Figure Legend Snippet: Fig. 5. Inhibition of DNA-directed synthesis by non-complementary dNTPs. ( A ) Inhibition of [α– 32 P]dATP labelling of activated (gapped) DNA by addition of different concentrations of a mixture of dC, dG and dTTP, in the presence of 1 mM MnCl 2 (a scheme is depicted). Under the standard conditions described in Materials and methods, only dATP (13 nM) is used as substrate for this assay. After incubation for 15 min at 37°C in the presence of either TdT (2.5 U/41 ng), Klenow (1 U) or Pol μ (20 ng), and the concentration indicated of dNTPs, dAMP incorporation on activated DNA was expressed as a percentage of that obtained under standard assay conditions: 100% represents either 73 (TdT), 13 (Klenow) or 8 (Pol μ) fmol of incorporated dAMP. ( B ) A similar analysis was carried out, but using a poly(dT)/oligo(dA) hybrid to provide a homopolymeric template (dT)n. The assay was carried out in the presence of 1 mM MnCl 2 , 13 nM [α– 32 P]dATP as the correct nucleotide, either 20 ng of Pol μ (circles) or 1 U of Klenow (squares), and the concentration indicated (on the abscissa) of individual non-complementary dNTPs. After 5 min at 37°C, dAMP incorporation on poly(dT)/oligo(dA) was expressed as a percentage of that obtained when non-complementary nucleotides were added: 100% represents either 23 (Pol μ) or 127 (Klenow) fmol of incorporated dAMP.

    Techniques Used: Inhibition, Incubation, Concentration Assay

    4) Product Images from "Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response"

    Article Title: Gene Silencing Using 4′-thioDNA as an Artificial Template to Synthesize Short Hairpin RNA Without Inducing a Detectable Innate Immune Response

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1038/mtna.2015.48

    Schematic of gene silencing using an intelligent shRNA expression device (iRed) . Step1: A region of plasmid DNA (pDNA) encoding the U6 promoter and a short hairpin RNA (shRNA) was amplified using PCR in the presence of one type of 2'-deoxy-4'-thionucleoside triphosphate (dSNTP) and three other dNTPs to synthesize an iRed. Step 2: The iRed was delivered into the nucleus. Step 3: Numerous shRNAs were transcribed from the iRed to possess a potent RNA interference (RNAi) activity.
    Figure Legend Snippet: Schematic of gene silencing using an intelligent shRNA expression device (iRed) . Step1: A region of plasmid DNA (pDNA) encoding the U6 promoter and a short hairpin RNA (shRNA) was amplified using PCR in the presence of one type of 2'-deoxy-4'-thionucleoside triphosphate (dSNTP) and three other dNTPs to synthesize an iRed. Step 2: The iRed was delivered into the nucleus. Step 3: Numerous shRNAs were transcribed from the iRed to possess a potent RNA interference (RNAi) activity.

    Techniques Used: shRNA, Expressing, Plasmid Preparation, Amplification, Polymerase Chain Reaction, Activity Assay

    5) Product Images from "Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †"

    Article Title: Functional Domains of Tat Required for Efficient Human Immunodeficiency Virus Type 1 Reverse Transcription †

    Journal: Journal of Virology

    doi:

    NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat virus trans -complemented with wild-type tat (lanes 2), Δ tat virus (lanes 3), or Δ tat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).
    Figure Legend Snippet: NERT assay for HIV-1 wild-type and tat mutant viruses. Virus stocks for wild-type virus (lanes 1), Δ tat virus trans -complemented with wild-type tat (lanes 2), Δ tat virus (lanes 3), or Δ tat virus produced in the presence of tat mutants [E2G, D5G, E9G], P3L, P[6, 10]L, P[10, 14]L, C27S, K41A, and K/R[50-57]G (lanes 4 to 10, respectively) were analyzed for endogenous reverse transcription. Culture supernatant (200 μl) containing approximately 0.75 mU of RT activity was treated with 100 U of DNase I. Half of each reaction mixture was added to 150 μl of stop solution, incubated at 37°C for 10 min, and then boiled for 10 min (B). The remaining half of each reaction mixture was supplemented with 50 μM dNTPs and incubated at 37°C for 90 minutes before the reaction was terminated as described above. (A) PCR to detect HIV-1 negative-strand strong-stop DNA was performed on NERT reaction mixtures as described in Materials and Methods. All PCRs were performed within the linear range of the assay as determined by assays of HIV-1 DNA copy number (10, 10 2 , 10 3 , and 10 4 ).

    Techniques Used: Mutagenesis, Produced, Activity Assay, Incubation, Polymerase Chain Reaction

    6) Product Images from "Reconstituting ParA/ParB-mediated transport of DNA cargo"

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

    Journal: Methods in cell biology

    doi: 10.1016/bs.mcb.2015.01.021

    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.
    Figure Legend 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.

    Techniques Used: Labeling, Plasmid Preparation, Flow Cytometry, Ligation

    7) Product Images from "Subtype-associated differences in HIV-1 reverse transcription affect the viral replication"

    Article Title: Subtype-associated differences in HIV-1 reverse transcription affect the viral replication

    Journal: Retrovirology

    doi: 10.1186/1742-4690-7-85

    The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation . A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24 CA -normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of dNTPs and permeabilizing agent melittin. Samples without dNTPs were used as a control. DNA was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.
    Figure Legend Snippet: The presence of RT functional domains from HIV-1 subtype C leads to decreased cDNA accumulation . A - Endogenous reverse transcription (ERT) in permeabilized virions. Purified and p24 CA -normalized virus particles of either the backbone NL4-3 or NL-based chimeric viruses were subjected to ERT with addition of dNTPs and permeabilizing agent melittin. Samples without dNTPs were used as a control. DNA was harvested after the indicated time of incubation. The relative amounts of negative-strand strong-stop DNA were measured using quantitative real-time PCR. Data from the control samples were subtracted. Levels of cDNA are shown as percentages of the peak accumulation detected in virions of NL4-3 at 5 h after initiation of incubation. Error bars show the standard deviation from three independent viral preparations. B - Accumulation of early or strong-stop viral DNA in Sup-T1 cells at 24 h p.i. Untreated or treated with 10 μM nevirapine cells were infected with backbone NL4-3 or the chimeric viruses, containing pol fragments from subtype C 1084i isolate using spinoculation. Relative amounts of reverse transcription products were measured using quantitative real-time PCR analysis of DNA from infected cells after incubation with or without 10 μM nevirapine. Data from nevirapine-treated samples were subtracted. Levels of cDNA are shown as percentages of the maximal accumulation detected for cDNA in cells infected with NL4-3 virus strain. Error bars show the standard deviation from three independent viral preparations. C - Accumulation of early and late reverse transcription products in Sup-T1 cells infected with recombinant viruses carrying protease and RT polymerase domain from 1084i, 2669i, and 1984i isolates of subtype C at 24 h p.i. The cells were infected with the indicated viruses as described in B. Harvested DNA was measured using quantitative real-time PCR analysis. Levels of cDNA are shown as percentages of the maximal accumulation detected for negative strand strong-stop cDNA in cells infected with NL4-3. Error bars indicate the standard deviation from three independent viral preparations.

    Techniques Used: Functional Assay, Purification, Incubation, Real-time Polymerase Chain Reaction, Standard Deviation, Infection, Recombinant

    8) Product Images from "High-Throughput Plasmid Content Analysis of Borrelia burgdorferi B31 by Using Luminex Multiplex Technology ▿ B31 by Using Luminex Multiplex Technology ▿ †"

    Article Title: High-Throughput Plasmid Content Analysis of Borrelia burgdorferi B31 by Using Luminex Multiplex Technology ▿ B31 by Using Luminex Multiplex Technology ▿ †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.01877-10

    Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and dNTPs in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates biotin-dCTP. (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.
    Figure Legend Snippet: Overview of Luminex procedure for determining plasmid content. (1) Plasmid-specific regions are amplified by multiplex PCR, using genomic DNA or B. burgdorferi culture as the template. Unincorporated primers and dNTPs in amplified PCR products are removed by treatment with exonuclease I and alkaline phosphatase. (2) Primers that contain an xTAG sequence are utilized in an asymmetric PCR that incorporates biotin-dCTP. (3) Biotinylated products are hybridized with xTAG microspheres that are coupled to antitag sequences and detected by binding of streptavidin-R-phycoerythrin. MFI, mean fluorescence intensity.

    Techniques Used: Luminex, Plasmid Preparation, Amplification, Multiplex Assay, Polymerase Chain Reaction, Sequencing, Binding Assay, Fluorescence

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    Amplification:

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    Spectrophotometry:

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    SYBR Green Assay:

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    Concentration Assay:

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    Incubation:

    Article Title: A unique insertion in STARD9's motor domain regulates its stability
    Article Snippet: .. After 3 h, a master mix containing all the ubiquitination components (ubiquitin [Enzo Life Sciences], ROC1, E1, E2, Skp1, with or without β-TrCP, with or without CUL1, in a buffer containing 20 mM HEPES, 5 mM NaCl, 5 mM MgCl2 , DTT, MG132, and protease and phosphatase inhibitor cocktail (Thermo Scientific) and ATP regeneration system was added to the tubes and further incubated for 90 min at 30°C. ..

    Polymerase Chain Reaction:

    Article Title: Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods ▿ Gene Expression in Foods ▿ †
    Article Snippet: .. The 50-μl PCR mixture (10 ng DNA, 0.5 μM each primer, 1.5 mM MgCl2 , 0.4 mM each deoxynucleoside triphosphate [dNTP], 1.25 U ThermoStart Taq polymerase [all from ABgene]) was activated (95°C for 15 min), followed by 30 amplification cycles (95°C for 30 s, 60°C for 45 s, and 72°C for 1 min) and an elongation step (72°C for 5 min). .. Total DNA was isolated as described previously , and plasmid DNA was prepared using standard procedures.

    Article Title: Association of COX-2 Promoter Polymorphisms -765G/C and -1195A/G with Migraine
    Article Snippet: .. Therefore, the final volume of 25 μl PCR reactions in 0.2 ml tubes containing 500 ng/μl of DNA template, 2 mM of MgCl2 concentration, 2 mM dNTPs, 10 pmol/μl of each primer, 5 μl of 10X PCR buffer and 1 U of Taq DNA polymerase (Fermentas, Germany) was performed. .. Thermal PCR conditions consisted of denaturation phase for 5 min at 95 °C, followed by 30 cycles of 94 °C for 1 min, temperature of 59 °C for COX-2-1195A>G (rs689466) primer and 56 °C for COX-2-765G>C (rs20417) primer for 1 min, and 72 °C for 1 min, with a final extension for 5 min at 72 °C.

    Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
    Article Snippet: .. Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl. ..

    Article Title: Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish
    Article Snippet: .. Reaction volumes (50 μl) for the PCR consisted of 5 μl of template DNA; 5.0 mM MgCl2 ; 5 μl of 10×TaqMan buffer A; 200 nM each primer; 200 μM each dATP, dCTP, and dGTP; 400 μM dUTP; 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems); 0.5 U of uracil- N -glycosidase (AmpErase UNG; Applied Biosystems); and 100 nM TaqMan probe. .. Amplification and detection were performed with the ABI PRISM 7700 sequence detector (Applied Biosystems).

    Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
    Article Snippet: .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

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  • 92
    Thermo Fisher dntp
    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ <t>cDNA)</t> or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated <t>dNTP</t> concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 5312 article reviews
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    dntp - by Bioz Stars, 2020-07
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    90
    Thermo Fisher fluorescein labelled dntp s
    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of <t>dNTP’s</t> on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .
    Fluorescein Labelled Dntp S, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labelled dntp s/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
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    fluorescein labelled dntp s - by Bioz Stars, 2020-07
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    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Journal: Nucleic Acids Research

    Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

    doi: 10.1093/nar/gkx1206

    Figure Lengend Snippet: Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Article Snippet: In vitro gap-filling assay In vitro gap-filling assays were performed in 1X Ampligase buffer (Epicentre) with 10 nM padlock probes (XC1149 and XC1151) and 10 nM cDNA template (XC1498), 20 μM dNTP, 0.012 U/μl Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) or Stoffel fragment (DNA Gdansk), additional 50 mM KCl, 20% formamide, and glycerol to a final concentration of 10%.

    Techniques: In Situ, Sequencing, Amplification, Infection, In Vitro

    Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Journal: Scientific Reports

    Article Title: Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

    doi: 10.1038/srep39985

    Figure Lengend Snippet: Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Article Snippet: dNTP Quantification by UPLC-MS/MS Chromatography and Mass Spectrometry Method Analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic AX 5 μm, 100 × 2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a mixture of 10 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 6.0 (buffer A), and 1 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 10.5 (buffer B).

    Techniques: Activity Assay, In Vitro, Incubation, Colorimetric Assay, Expressing, Mass Spectrometry

    Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: Flow Cytometry, Cytometry

    ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: High Performance Liquid Chromatography, Labeling

    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Infection, Binding Assay, Fluorescence

    The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Fluorescence, Binding Assay

    ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Recombinant