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PerkinElmer mulv
Mulv, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 91/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 91 stars, based on 10 article reviews
Price from $9.99 to $1999.99
mulv - by Bioz Stars, 2020-09
91/100 stars

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Polymerase Chain Reaction:

Article Title: Elevated Atherosclerosis-Related Gene Expression, Monocyte Activation and Microparticle-Release Are Related to Increased Lipoprotein-Associated Oxidative Stress in Familial Hypercholesterolemia
Article Snippet: .. Subsequently, 4 μL of undiluted RNA solution was mixed with 10 μL MgCl2 25 mM, (Roche A/S, Hvidovre, Denmark), 4 μL 10 × PCR buffer II (Roche A/S), 2 μL Oligo d(T)16 50 μM (DNA Technology A/S, Aarhus, Denmark), 16 μL dNTPmix (dATP/dTTP/dCTP/dGTP) (Pharmacia Biotech, Hilleroed, Denmark), 2 μL MuLV (Moloney murine Leukaemia Virus) reverse transcriptase, (50 U/ml) (PerkinElmer Denmark A/S, Hvidovre, Denmark) and 2 μL RNase inhibitor, (20 U/ml) (PerkinElmer Denmark A/S) for a final volume of 40 μL. cDNA was synthesized by incubation at 42°C for 30 min and the process was stopped by increasing incubation temperature to 99°C for 5 min. cDNA was either used immediately or stored at -20°C. .. Quantitative Real-time RT-PCR was performed using the Lightcycler 480 instrument (Roche Diagnostics, Penzberg, Germany).

Real-time Polymerase Chain Reaction:

Article Title: Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Dependent Viral Penetration into the Cytosol and Not for Viral Uncoating
Article Snippet: .. However, after infection, the early HIV DNA products were quantitated in quantitative PCR experiments as described previously for ASLV and MLV , with the primers ert2f, ert2r , and a FAM-labeled probe ERT2 (FAM TaqMan probe; Perkin-Elmer). .. A dilution series of proviral DNA contained in a plasmid vector (pR9ΔEnv) was used to construct a standard curve to quantify viral DNA.

Incubation:

Article Title: Elevated Atherosclerosis-Related Gene Expression, Monocyte Activation and Microparticle-Release Are Related to Increased Lipoprotein-Associated Oxidative Stress in Familial Hypercholesterolemia
Article Snippet: .. Subsequently, 4 μL of undiluted RNA solution was mixed with 10 μL MgCl2 25 mM, (Roche A/S, Hvidovre, Denmark), 4 μL 10 × PCR buffer II (Roche A/S), 2 μL Oligo d(T)16 50 μM (DNA Technology A/S, Aarhus, Denmark), 16 μL dNTPmix (dATP/dTTP/dCTP/dGTP) (Pharmacia Biotech, Hilleroed, Denmark), 2 μL MuLV (Moloney murine Leukaemia Virus) reverse transcriptase, (50 U/ml) (PerkinElmer Denmark A/S, Hvidovre, Denmark) and 2 μL RNase inhibitor, (20 U/ml) (PerkinElmer Denmark A/S) for a final volume of 40 μL. cDNA was synthesized by incubation at 42°C for 30 min and the process was stopped by increasing incubation temperature to 99°C for 5 min. cDNA was either used immediately or stored at -20°C. .. Quantitative Real-time RT-PCR was performed using the Lightcycler 480 instrument (Roche Diagnostics, Penzberg, Germany).

Infection:

Article Title: Low pH Is Required for Avian Sarcoma and Leukosis Virus Env-Dependent Viral Penetration into the Cytosol and Not for Viral Uncoating
Article Snippet: .. However, after infection, the early HIV DNA products were quantitated in quantitative PCR experiments as described previously for ASLV and MLV , with the primers ert2f, ert2r , and a FAM-labeled probe ERT2 (FAM TaqMan probe; Perkin-Elmer). .. A dilution series of proviral DNA contained in a plasmid vector (pR9ΔEnv) was used to construct a standard curve to quantify viral DNA.

Synthesized:

Article Title: Elevated Atherosclerosis-Related Gene Expression, Monocyte Activation and Microparticle-Release Are Related to Increased Lipoprotein-Associated Oxidative Stress in Familial Hypercholesterolemia
Article Snippet: .. Subsequently, 4 μL of undiluted RNA solution was mixed with 10 μL MgCl2 25 mM, (Roche A/S, Hvidovre, Denmark), 4 μL 10 × PCR buffer II (Roche A/S), 2 μL Oligo d(T)16 50 μM (DNA Technology A/S, Aarhus, Denmark), 16 μL dNTPmix (dATP/dTTP/dCTP/dGTP) (Pharmacia Biotech, Hilleroed, Denmark), 2 μL MuLV (Moloney murine Leukaemia Virus) reverse transcriptase, (50 U/ml) (PerkinElmer Denmark A/S, Hvidovre, Denmark) and 2 μL RNase inhibitor, (20 U/ml) (PerkinElmer Denmark A/S) for a final volume of 40 μL. cDNA was synthesized by incubation at 42°C for 30 min and the process was stopped by increasing incubation temperature to 99°C for 5 min. cDNA was either used immediately or stored at -20°C. .. Quantitative Real-time RT-PCR was performed using the Lightcycler 480 instrument (Roche Diagnostics, Penzberg, Germany).

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  • 88
    PerkinElmer multilamellar vesicles mlv
    FC mole percentage in rHDL of various sizes as a function of initial FC mole percentage in <t>MLV.</t> rHDL were formed from stoichiometric ratios of <t>DMPC</t> and apo A-II (A) and excess DMPC (B). Compositions were calculated from the data depicted in Figure 4 . The dashed gray line is a plot of the expected rHDL FC mole percentage vs the initial FC mole percentage if FC and DMPC were incorporated into rHDL at the ratio in the starting MLV.
    Multilamellar Vesicles Mlv, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 88 stars, based on 1 article reviews
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    85
    PerkinElmer popc c16 c1p mlvs
    ( a ) Intermolecular I e /I m for PPDPC ( X =0.01) measured for <t>MLVs</t> composed of <t>POPC</t> and the indicated contents of C 16 -ceramide (■) or C 16 <t>-C1P</t> (○). ( b ) Fluorescence anisotropy (r) for DPH ( X = 0.002) residing in binary MLVs
    Popc C16 C1p Mlvs, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/popc c16 c1p mlvs/product/PerkinElmer
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    85
    PerkinElmer mlv virions
    ATPγS inhibits retroviral natural endogenous reverse transcription. Purified <t>HIV-1</t> NL4-3 , SIV MAC239 , and <t>MLV</t> NCA virions were incubated in natural endogenous reverse transcription buffer with MgCl 2 , [α- 32 P]dCTP, and unlabeled deoxynucleoside
    Mlv Virions, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mlv virions/product/PerkinElmer
    Average 85 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    89
    PerkinElmer mulv rt
    Misincorporation assay with IAV Pol, bacterial phage T7 RNA polymerase, <t>HIV-1</t> RT and <t>MuLV</t> 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.
    Mulv Rt, supplied by PerkinElmer, used in various techniques. Bioz Stars score: 89/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/mulv rt/product/PerkinElmer
    Average 89 stars, based on 3 article reviews
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    FC mole percentage in rHDL of various sizes as a function of initial FC mole percentage in MLV. rHDL were formed from stoichiometric ratios of DMPC and apo A-II (A) and excess DMPC (B). Compositions were calculated from the data depicted in Figure 4 . The dashed gray line is a plot of the expected rHDL FC mole percentage vs the initial FC mole percentage if FC and DMPC were incorporated into rHDL at the ratio in the starting MLV.

    Journal: Biochemistry

    Article Title: Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A-II and Phospholipid Membranes

    doi: 10.1021/bi3011994

    Figure Lengend Snippet: FC mole percentage in rHDL of various sizes as a function of initial FC mole percentage in MLV. rHDL were formed from stoichiometric ratios of DMPC and apo A-II (A) and excess DMPC (B). Compositions were calculated from the data depicted in Figure 4 . The dashed gray line is a plot of the expected rHDL FC mole percentage vs the initial FC mole percentage if FC and DMPC were incorporated into rHDL at the ratio in the starting MLV.

    Article Snippet: Multilamellar Vesicle Preparation and Labeling Multilamellar vesicles (MLV) were prepared from [3 H]DMPC synthesized as described previously and [14 C]FC (Perkin-Elmer, Inc.).

    Techniques:

    Kinetics of solubilization of DMPC MLV by apolipoproteins according to the reduction in MLV turbidity at 24 °C as a function of FC mole percentage. Experiments were conducted with stoichiometric amounts of DMPC and apo A-II, i.e., 65/1 molar ratio: (A) apo A-II and (B) rcm apo A-II. For both apolipoproteins, decay curves for 0–15 mol % FC are closely spaced and are distinct from curves at 20 and 30 mol % FC. (C) Plots of the rate constants for the solubilization by apo A-II (●) and rcm apo A-II (○) vs FC mole percentage.

    Journal: Biochemistry

    Article Title: Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A-II and Phospholipid Membranes

    doi: 10.1021/bi3011994

    Figure Lengend Snippet: Kinetics of solubilization of DMPC MLV by apolipoproteins according to the reduction in MLV turbidity at 24 °C as a function of FC mole percentage. Experiments were conducted with stoichiometric amounts of DMPC and apo A-II, i.e., 65/1 molar ratio: (A) apo A-II and (B) rcm apo A-II. For both apolipoproteins, decay curves for 0–15 mol % FC are closely spaced and are distinct from curves at 20 and 30 mol % FC. (C) Plots of the rate constants for the solubilization by apo A-II (●) and rcm apo A-II (○) vs FC mole percentage.

    Article Snippet: Multilamellar Vesicle Preparation and Labeling Multilamellar vesicles (MLV) were prepared from [3 H]DMPC synthesized as described previously and [14 C]FC (Perkin-Elmer, Inc.).

    Techniques:

    Distribution of FC between rHDL (supernatant) and MLV (pellet). (A–C) Fraction of DMPC incorporated into rHDL, calculated as the total counts in the sample minus the counts in the MLV pellet obtained by centrifugation. (A) rHDL formation with stoichiometric amounts of DMPC and apolipoproteins. (B) Cholate-catalyzed rHDL formation of stoichiometric amounts of DMPC and apolipoproteins. (C) rHDL formation in the presence of excess DMPC: (●) apo A-II and (○) rcm apo A-II. (D) Stoichiometric DMPC/apolipoprotein ratio and (E) excess DMPC. Both panels show the FC mole percentage in the supernatant (rHDL) and pellet (residual MLV), calculated from the ratio of 14 C to 3 H radioactivity in the supernatant and pellet, as a function of FC mole percentage in the starting MLV, as labeled in panel D. The dashed gray lines in D and E represent the expected FC mole percentage in rHDL if FC were incorporated in proportion to the initial FC mole percentage.

    Journal: Biochemistry

    Article Title: Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A-II and Phospholipid Membranes

    doi: 10.1021/bi3011994

    Figure Lengend Snippet: Distribution of FC between rHDL (supernatant) and MLV (pellet). (A–C) Fraction of DMPC incorporated into rHDL, calculated as the total counts in the sample minus the counts in the MLV pellet obtained by centrifugation. (A) rHDL formation with stoichiometric amounts of DMPC and apolipoproteins. (B) Cholate-catalyzed rHDL formation of stoichiometric amounts of DMPC and apolipoproteins. (C) rHDL formation in the presence of excess DMPC: (●) apo A-II and (○) rcm apo A-II. (D) Stoichiometric DMPC/apolipoprotein ratio and (E) excess DMPC. Both panels show the FC mole percentage in the supernatant (rHDL) and pellet (residual MLV), calculated from the ratio of 14 C to 3 H radioactivity in the supernatant and pellet, as a function of FC mole percentage in the starting MLV, as labeled in panel D. The dashed gray lines in D and E represent the expected FC mole percentage in rHDL if FC were incorporated in proportion to the initial FC mole percentage.

    Article Snippet: Multilamellar Vesicle Preparation and Labeling Multilamellar vesicles (MLV) were prepared from [3 H]DMPC synthesized as described previously and [14 C]FC (Perkin-Elmer, Inc.).

    Techniques: Centrifugation, Radioactivity, Labeling

    Apo A-II concentration dependence of rHDL formation. (A) Determination of the DMPC/apo A-II stoichiometry according to the increase in turbidity produced by excess MLV. The inset shows the maximal stoichiometry of 65/1 for solubilization of DMPC by apo A-II was determined by extrapolation. (B) Kinetics of formation of rHDL from DMPC as a function of added apo A-II according to the disappearance of MLV turbidity. The DMPC/apo A-II molar ratios for the top (70/1) to bottom (20/1) curves are given. The black and gray curves are the data and the fit of the data, respectively. The inset shows the first-order rate constant calculated from curve fits of Figure 1 B as a function of DMPC/apo A-II molar ratio.

    Journal: Biochemistry

    Article Title: Cholesterol Determines and Limits rHDL Formation from Human Plasma Apolipoprotein A-II and Phospholipid Membranes

    doi: 10.1021/bi3011994

    Figure Lengend Snippet: Apo A-II concentration dependence of rHDL formation. (A) Determination of the DMPC/apo A-II stoichiometry according to the increase in turbidity produced by excess MLV. The inset shows the maximal stoichiometry of 65/1 for solubilization of DMPC by apo A-II was determined by extrapolation. (B) Kinetics of formation of rHDL from DMPC as a function of added apo A-II according to the disappearance of MLV turbidity. The DMPC/apo A-II molar ratios for the top (70/1) to bottom (20/1) curves are given. The black and gray curves are the data and the fit of the data, respectively. The inset shows the first-order rate constant calculated from curve fits of Figure 1 B as a function of DMPC/apo A-II molar ratio.

    Article Snippet: Multilamellar Vesicle Preparation and Labeling Multilamellar vesicles (MLV) were prepared from [3 H]DMPC synthesized as described previously and [14 C]FC (Perkin-Elmer, Inc.).

    Techniques: Concentration Assay, Produced

    ( a ) Intermolecular I e /I m for PPDPC ( X =0.01) measured for MLVs composed of POPC and the indicated contents of C 16 -ceramide (■) or C 16 -C1P (○). ( b ) Fluorescence anisotropy (r) for DPH ( X = 0.002) residing in binary MLVs

    Journal: Biophysical Journal

    Article Title: Ceramide-1-Phosphate, in Contrast to Ceramide, Is Not Segregated into Lateral Lipid Domains in Phosphatidylcholine Bilayers

    doi: 10.1016/j.bpj.2008.11.060

    Figure Lengend Snippet: ( a ) Intermolecular I e /I m for PPDPC ( X =0.01) measured for MLVs composed of POPC and the indicated contents of C 16 -ceramide (■) or C 16 -C1P (○). ( b ) Fluorescence anisotropy (r) for DPH ( X = 0.002) residing in binary MLVs

    Article Snippet: Fluorescence emission spectra for POPC/C16 -ceramide and POPC/C16 -C1P MLVs labeled with PPDPC ( X = 0.01) were recorded with a Perkin-Elmer (Waltham, MA) LS55 spectrofluorometer equipped with a magnetically stirred, thermostated cuvette compartment.

    Techniques: Fluorescence

    ATPγS inhibits retroviral natural endogenous reverse transcription. Purified HIV-1 NL4-3 , SIV MAC239 , and MLV NCA virions were incubated in natural endogenous reverse transcription buffer with MgCl 2 , [α- 32 P]dCTP, and unlabeled deoxynucleoside

    Journal:

    Article Title: ATP?S Disrupts Human Immunodeficiency Virus Type 1 Virion Core Integrity

    doi: 10.1128/JVI.79.9.5557-5567.2005

    Figure Lengend Snippet: ATPγS inhibits retroviral natural endogenous reverse transcription. Purified HIV-1 NL4-3 , SIV MAC239 , and MLV NCA virions were incubated in natural endogenous reverse transcription buffer with MgCl 2 , [α- 32 P]dCTP, and unlabeled deoxynucleoside

    Article Snippet: HIV-1, SIV, and MLV virions were pelleted through a 25% sucrose cushion and incubated in natural endogenous reverse transcription buffer composed of 10 mM Tris-HCl (pH 7.4), 100 mM NaCl, 1 mM MgCl2 , 50 μCi of [α-32 P]dCTP (3,000 Ci/mmol, Perkin Elmer) and 0.1 mM deoxynucleoside triphosphates (1 mM for MLV).

    Techniques: Purification, Incubation

    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: For HIV and MuLV RT, a mixture of 200 nM 48-mer RNA template annealed to 100 nM 18-mer DNA primer , 500 µM dNTPs, 0.16 µM α-32 P-dNTPs (PerkinElmer, 3000 Ci/mmol) were incubated with RT for 1 hour at 37°C under conditions described previously (16).

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