Structured Review

Epicentre Biotechnologies dntps
Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM <t>dNTPs</t> (A, C, E and G) or <t>rNTPs</t> (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.
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1) Product Images from "Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity"

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

Journal: Nucleic Acids Research

doi: 10.1093/nar/gks233

Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM dNTPs (A, C, E and G) or rNTPs (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.
Figure Legend Snippet: Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM dNTPs (A, C, E and G) or rNTPs (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.

Techniques Used:

Discrimination against rNMP insertion by pol V mutants. Insertion of dA versus rA and T versus U by wild-type pol V ( A ), F10L ( B ), Y11A ( C ) or Y11F ( D ) were analyzed using a template containing five consecutive As. Insertion of dA/rA and dC/rC by wild-type pol V ( E ), F10L ( F ), Y11A ( G ) or Y11F ( H ) were analyzed using a template containing five consecutive Ts followed by two Gs. Reactions were performed in the presence of 100 µM dNTPs and/or 1 mM rNTPs for 10 min; half of each reaction mixture was subjected to alkaline hydrolysis (OH − ) (lanes 2, 4, 6, 8 and 10 in each panel) under conditions that completely hydrolyze DNA chains at the positions of rNTP insertion. The identity of the nucleotide present in the reaction is shown below each track (I NTP, II NTP). The extended sequence of the templates with five consecutive As (A–D) or Ts (E–H) is indicated to the right of the gel panels. Reactions in lanes 1 and 2 contained only ATP; lanes 3, 4 ATP and dATP; lanes 5, 6 ATP with UTP (A–D) or with CTP (E–H); lanes 7, 8 ATP and dATP with UTP and dTTP (A–D) or with CTP and dCTP (E–H); lanes 9, 10 ATP and dATP with dTTP (A–D) or with dCTP (E–H). Bands corresponding to incorporation of UTP (in A and D) or dTTP (in C) are indicated by stars.
Figure Legend Snippet: Discrimination against rNMP insertion by pol V mutants. Insertion of dA versus rA and T versus U by wild-type pol V ( A ), F10L ( B ), Y11A ( C ) or Y11F ( D ) were analyzed using a template containing five consecutive As. Insertion of dA/rA and dC/rC by wild-type pol V ( E ), F10L ( F ), Y11A ( G ) or Y11F ( H ) were analyzed using a template containing five consecutive Ts followed by two Gs. Reactions were performed in the presence of 100 µM dNTPs and/or 1 mM rNTPs for 10 min; half of each reaction mixture was subjected to alkaline hydrolysis (OH − ) (lanes 2, 4, 6, 8 and 10 in each panel) under conditions that completely hydrolyze DNA chains at the positions of rNTP insertion. The identity of the nucleotide present in the reaction is shown below each track (I NTP, II NTP). The extended sequence of the templates with five consecutive As (A–D) or Ts (E–H) is indicated to the right of the gel panels. Reactions in lanes 1 and 2 contained only ATP; lanes 3, 4 ATP and dATP; lanes 5, 6 ATP with UTP (A–D) or with CTP (E–H); lanes 7, 8 ATP and dATP with UTP and dTTP (A–D) or with CTP and dCTP (E–H); lanes 9, 10 ATP and dATP with dTTP (A–D) or with dCTP (E–H). Bands corresponding to incorporation of UTP (in A and D) or dTTP (in C) are indicated by stars.

Techniques Used: Sequencing

Gel images of primer extension reactions catalyzed by wild-type pol V in the presence of dNTPs ( A–D ) or rNTPs ( E–H ). Reactions in the presence of each individual nucleotide were carried out for 5 min. The identity of nucleotide incorporated is shown below the respective lanes. The extended sequence of templates with five consecutive Ts (A and E), As (B and F), Cs (C and G) or Gs (D and H), are indicated to the right of each gel panel. Determination of the specificity of nucleotide incorporation is complicated due to the efficient incorporation of ATP present in the reactions at 1 mM concentration. Lanes with reactions lacking polymerase are indicated as ‘pr’ (short for primer) and reactions with no additional nucleotide are indicated by dash (–).
Figure Legend Snippet: Gel images of primer extension reactions catalyzed by wild-type pol V in the presence of dNTPs ( A–D ) or rNTPs ( E–H ). Reactions in the presence of each individual nucleotide were carried out for 5 min. The identity of nucleotide incorporated is shown below the respective lanes. The extended sequence of templates with five consecutive Ts (A and E), As (B and F), Cs (C and G) or Gs (D and H), are indicated to the right of each gel panel. Determination of the specificity of nucleotide incorporation is complicated due to the efficient incorporation of ATP present in the reactions at 1 mM concentration. Lanes with reactions lacking polymerase are indicated as ‘pr’ (short for primer) and reactions with no additional nucleotide are indicated by dash (–).

Techniques Used: Sequencing, Concentration Assay

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DNA Extraction:

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Agarose Gel Electrophoresis:

Article Title: Distribution of selected gene polymorphisms of UGT1A1 in a Saudi population
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Article Title: Prevalence of UDP-glucuronosyltransferase polymorphisms (UGT1A6∗2, 1A7∗12, 1A8∗3, 1A9∗3, 2B7∗2, and 2B15∗2) in a Saudi population
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Amplification:

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Whole Genome Amplification:

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In Vitro:

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Fluorescence:

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Polymerase Chain Reaction:

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Software:

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Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer. .. The products were heat-denatured and immediately resolved by denaturing PAGE (8 M urea, 15% acrylamide), followed by visualization and quantification using a Fuji image analyzer FLA-5100 and MultiGauge software.

Neutralization:

Article Title: SIMPLE SEQUENCE REPEATS IN THE NATIONAL LONGITUDINAL STUDY OF ADOLESCENT HEALTH: AN ETHNICALLY DIVERSE RESOURCE FOR GENETIC ANALYSIS OF HEALTH AND BEHAVIOR
Article Snippet: Genomic DNA samples (50–100 ng) were dried in 96-well PCR plates, denatured for 5 minutes (2.5 μl of denaturation buffer), neutralized (2.5 μl of neutralization buffer) and placed on ice. .. The 20 μl of reaction mix consisted of 14.5 μl of Repli-g® reaction buffer, 1 μl of Repli-g® polymerase, 1.5 μl of dNTPs (Epicentre Biotechnologies, Madison, WI, 25 mM each, 1.5 mM final concentration), 0.5 μl of RepliPHI™ phi-29 polymerase (Epicentre, 1 unit/μl final concentration) and 3 μl of water.

Spectrophotometry:

Article Title: Distribution of selected gene polymorphisms of UGT1A1 in a Saudi population
Article Snippet: Genetic testing DNA extraction was carried out using Puregene Blood Core Kit C (Qiagen, Germantown, MD, USA) following the manufacturer's instructions and quantified using a Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). .. The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City CA, USA) in a total volume of 25 µl, containing 20 ng DNA, 0.25 µl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 µl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 µl (5 U/µl) of HotstarTaq DNA polymerase (Qiagen, Germantown, MD, USA).

Article Title: Prevalence of UDP-glucuronosyltransferase polymorphisms (UGT1A6∗2, 1A7∗12, 1A8∗3, 1A9∗3, 2B7∗2, and 2B15∗2) in a Saudi population
Article Snippet: DNA extraction was carried out using Puregene Blood Core Kit C (Qiagen, Germantown, MD, USA) following manufacturer’s instructions and quantified using Nanodrop 2000 Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). .. The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μl, containing 20 ng DNA, 0.25 μl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 μl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 μl (5 U/μl) of Hotstar Taq DNA polymerase (Qiagen, Germantown, MD, USA).

Purification:

Article Title: Distribution of selected gene polymorphisms of UGT1A1 in a Saudi population
Article Snippet: The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City CA, USA) in a total volume of 25 µl, containing 20 ng DNA, 0.25 µl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 µl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 µl (5 U/µl) of HotstarTaq DNA polymerase (Qiagen, Germantown, MD, USA). .. The PCR amplicons were evaluated by 2% agarose gel electrophoresis and then purified using an MCE-membrane MultiScreen plate (Millipore, Billerica, MA, USA) pre-packed with G-50 superfine Sephadex (GE Healthcare, Piscataway, NJ, USA).

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: RecA* (0.25 µM) was formed by incubating 4 µM RecA (New England Biolabs, Ipswich, MA, USA), 1 mM ATPγS and 0.25 µM 48-mer oligonucleotide in the 1× reaction buffer (20 mM Tris–HCl pH 7.5, 8 mM MgCl2 , 8 mM DTT, 80 µg/ml BSA, 4% glycerol) at 37°C for 5 min. Purified pol V variants (80 nM) were first combined with RecA* to form pol V Mut complexes ( ) and then added to the reaction mixture which had been pre-incubated for 3 min at 37°C. .. The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

Article Title: Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival
Article Snippet: Purified pol V variants (80 nM) were first combined with RecA*(0.25 µM) to form pol V Mut and then added to the main reaction mixture that had been preincubated for 3 min at 37°C. .. This mixture contained 1 mM ATP, 50 µM dNTPs (all four or each one individually) or rNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer.

Article Title: Prevalence of UDP-glucuronosyltransferase polymorphisms (UGT1A6∗2, 1A7∗12, 1A8∗3, 1A9∗3, 2B7∗2, and 2B15∗2) in a Saudi population
Article Snippet: The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μl, containing 20 ng DNA, 0.25 μl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 μl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 μl (5 U/μl) of Hotstar Taq DNA polymerase (Qiagen, Germantown, MD, USA). .. The PCR amplicons were evaluated by 2% agarose gel electrophoresis and then purified using MCE-membraned Multi-Screen plate (Millipore, Billerica, MA, USA) pre-packed with G-50 superfine cephadex (GE Healthcare, Piscataway, NJ, USA).

Concentration Assay:

Article Title: SIMPLE SEQUENCE REPEATS IN THE NATIONAL LONGITUDINAL STUDY OF ADOLESCENT HEALTH: AN ETHNICALLY DIVERSE RESOURCE FOR GENETIC ANALYSIS OF HEALTH AND BEHAVIOR
Article Snippet: .. The 20 μl of reaction mix consisted of 14.5 μl of Repli-g® reaction buffer, 1 μl of Repli-g® polymerase, 1.5 μl of dNTPs (Epicentre Biotechnologies, Madison, WI, 25 mM each, 1.5 mM final concentration), 0.5 μl of RepliPHI™ phi-29 polymerase (Epicentre, 1 unit/μl final concentration) and 3 μl of water. .. Reactions were incubated in a thermocycler (with a non-heated lid) at 30°C for 12–16 hours followed by 65°C for 5 minutes and a hold at 4°C.

Incubation:

Article Title: SIMPLE SEQUENCE REPEATS IN THE NATIONAL LONGITUDINAL STUDY OF ADOLESCENT HEALTH: AN ETHNICALLY DIVERSE RESOURCE FOR GENETIC ANALYSIS OF HEALTH AND BEHAVIOR
Article Snippet: The 20 μl of reaction mix consisted of 14.5 μl of Repli-g® reaction buffer, 1 μl of Repli-g® polymerase, 1.5 μl of dNTPs (Epicentre Biotechnologies, Madison, WI, 25 mM each, 1.5 mM final concentration), 0.5 μl of RepliPHI™ phi-29 polymerase (Epicentre, 1 unit/μl final concentration) and 3 μl of water. .. Reactions were incubated in a thermocycler (with a non-heated lid) at 30°C for 12–16 hours followed by 65°C for 5 minutes and a hold at 4°C.

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer. .. Reactions were incubated at 37°C for 0.5–20 min and terminated by adding 10 µl of 2× loading buffer (97% formamide, 10 mM EDTA, 0.1% xylene cyanol, 0.1% bromophenol blue).

Article Title: Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
Article Snippet: 4 mM RecA (New England Biolabs, Ipswich, MA) was incubated with 0.25 µM 48-mer single-stranded oligonucleotide in the presence of 1 mM adenosine 5′[γ-thio]triphosphate (ATPγS, Biolog Life Science Institute, Bremen, Germany) in the 1× reaction buffer [20 mM Tris-HCl pH 7.5, 8 mM MgCl2 , 8 mM DTT, 80 µg/ml BSA, 4% glycerol] at 37°C for 5 min to form RecA nucleoprotein filament on ssDNA (RecA*). .. Reaction mixture containing 1 mM ATP, 50 µM dNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer was preincubated for 3 min at 37°C.

Article Title: Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival
Article Snippet: 4 µM RecA (New England Biolabs, Ipswich, MA) was incubated with 1 mM ATPγS and 0.25 µM 48-mer single-stranded oligonucleotide in the 1× reaction buffer [20 mM Tris-HCl pH 7.5, 8 mM MgCl2 , 8 mM DTT, 80 µg/ml BSA, 4% glycerol] at 37 °C for 5 min to produce RecA*. .. This mixture contained 1 mM ATP, 50 µM dNTPs (all four or each one individually) or rNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer.

Labeling:

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: 5A17M, 5T17M, 5G17M and 5C17M primers were designed for hybridization to single stranded M13mp18 DNA to study the misincorporation specificity of pol V. 5′-32 P labeled primers were annealed to the single-stranded circular plasmids at a 1.5:1 molar ratio by heating the required mixture in an annealing buffer [50 mM Tris–HCl (pH 8), 5 mM MgCl2 , 50 µg/ml BSA, 1.42 mM 2-mercaptoethanol] for 10 min at 100°C followed by slow cooling to room temperature. .. The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

Article Title: Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
Article Snippet: 5′-32 P labeled M13-TT (5′ – GAT-CGA-TGG-TAC-GGA-CG) primer was annealed to pSOcpd ssDNA templates at a 1.5∶1 molar ratio by heating in an annealing buffer (50 mM Tris-HCl (pH 8), 5 mM MgCl2 , 50 µg/ml BSA, 1.42 mM 2-mercaptoethanol) for 10 min at 100°C followed by slow cooling to room temperature. .. Reaction mixture containing 1 mM ATP, 50 µM dNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer was preincubated for 3 min at 37°C.

Article Title: Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival
Article Snippet: 5'-32 P labeled M13-TT (5’ – GAT-CGA-TGG-TAC-GGA-CG) and SSP1 (5’-TGG-TAC-GGA-CGT-GCT-T) primers were annealed to pSO and pSOcpd ssDNA templates at a 1.5:1 molar ratio by heating in an annealing buffer (50 mM Tris-HCl (pH 8), 5 mM MgCl2 , 50 µg/ml BSA, 1.42 mM 2-mercaptoethanol) for 10 min at 100°C followed by slow cooling to room temperature. .. This mixture contained 1 mM ATP, 50 µM dNTPs (all four or each one individually) or rNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer.

Polyacrylamide Gel Electrophoresis:

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer. .. The products were heat-denatured and immediately resolved by denaturing PAGE (8 M urea, 15% acrylamide), followed by visualization and quantification using a Fuji image analyzer FLA-5100 and MultiGauge software.

Article Title: Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival
Article Snippet: This mixture contained 1 mM ATP, 50 µM dNTPs (all four or each one individually) or rNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer. .. The products were heat-denatured and resolved by denaturing PAGE (8 M urea, 15% acrylamide), followed by visualization on a Fuji image analyzer FLA-5100.

Construct:

Article Title: Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
Article Snippet: In vitro replication assays Wild-type pol V, the UmuC_Y11A variant and pol IV, β-clamp and γ-complex were purified as previously described . pSOcpd plasmid, containing a unique CPD adduct, was also constructed as previously described . .. Reaction mixture containing 1 mM ATP, 50 µM dNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer was preincubated for 3 min at 37°C.

Article Title: Escherichia coli UmuC active site mutants: effects on translesion DNA synthesis, mutagenesis and cell survival
Article Snippet: Undamaged pSO and pSOcpd, containing a unique CPD adduct, were constructed as previously described [ ]. .. This mixture contained 1 mM ATP, 50 µM dNTPs (all four or each one individually) or rNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer.

Sequencing:

Article Title: Distribution of selected gene polymorphisms of UGT1A1 in a Saudi population
Article Snippet: The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City CA, USA) in a total volume of 25 µl, containing 20 ng DNA, 0.25 µl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 µl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 µl (5 U/µl) of HotstarTaq DNA polymerase (Qiagen, Germantown, MD, USA). .. The purified PCR amplicons were then sequenced by dye termination sequencing using BigDye Terminator Cycle Sequencing V3.1 Kit and 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Article Title: Prevalence of UDP-glucuronosyltransferase polymorphisms (UGT1A6∗2, 1A7∗12, 1A8∗3, 1A9∗3, 2B7∗2, and 2B15∗2) in a Saudi population
Article Snippet: The indicated polymorphic variants were amplified in a Veriti® 96-Well Fast Thermal Cycler (Applied Biosystems, Foster City, CA, USA) in a total volume of 25 μl, containing 20 ng DNA, 0.25 μl (2.5 mM) of dNTPs (Epicentre Biotechnologies, Madison, WI, USA), 2 μl (10 pM) of primers (Metabion, Martinsried, Germany) and 0.3 μl (5 U/μl) of Hotstar Taq DNA polymerase (Qiagen, Germantown, MD, USA). .. The purified PCR amplicons were then sequenced by dye termination sequencing using BigDye Terminator Cycle Sequencing V3.1 Kit and 3730xl DNA Analyzer (Applied Biosystems, Foster City, CA, USA).

Gel Extraction:

Article Title: Construction and engineering of large biochemical pathways via DNA assembler
Article Snippet: QIAquick Gel Extraction Kit (QIAGEN, Valencia, CA, USA). .. Failsafe PCR 2× Premix G: Containing dNTPs and PCR reaction buffer (EPICENTRE Biotechnologies, Madison, WI, USA).

Variant Assay:

Article Title: Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
Article Snippet: In vitro replication assays Wild-type pol V, the UmuC_Y11A variant and pol IV, β-clamp and γ-complex were purified as previously described . pSOcpd plasmid, containing a unique CPD adduct, was also constructed as previously described . .. Reaction mixture containing 1 mM ATP, 50 µM dNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer was preincubated for 3 min at 37°C.

Plasmid Preparation:

Article Title: Mechanisms Employed by Escherichia coli to Prevent Ribonucleotide Incorporation into Genomic DNA by Pol V
Article Snippet: In vitro replication assays Wild-type pol V, the UmuC_Y11A variant and pol IV, β-clamp and γ-complex were purified as previously described . pSOcpd plasmid, containing a unique CPD adduct, was also constructed as previously described . .. Reaction mixture containing 1 mM ATP, 50 µM dNTPs, 2 nM DNA templates, 100 nM SSB (Epicentre Biotechnologies, Madison WI), 50 nM β clamp, and 5 nM γ complex in the 1× reaction buffer was preincubated for 3 min at 37°C.

Hybridization:

Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity
Article Snippet: 5A17M, 5T17M, 5G17M and 5C17M primers were designed for hybridization to single stranded M13mp18 DNA to study the misincorporation specificity of pol V. 5′-32 P labeled primers were annealed to the single-stranded circular plasmids at a 1.5:1 molar ratio by heating the required mixture in an annealing buffer [50 mM Tris–HCl (pH 8), 5 mM MgCl2 , 50 µg/ml BSA, 1.42 mM 2-mercaptoethanol] for 10 min at 100°C followed by slow cooling to room temperature. .. The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

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    Epicentre Biotechnologies dntps
    Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM <t>dNTPs</t> (A, C, E and G) or <t>rNTPs</t> (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.
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    Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM dNTPs (A, C, E and G) or rNTPs (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.

    Journal: Nucleic Acids Research

    Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

    doi: 10.1093/nar/gks233

    Figure Lengend Snippet: Role of UmuC F10 and Y11 residues on sugar discrimination by pol V. Primer extension reactions catalyzed by wild-type pol V ( A and B ), F10L ( C and D ), Y11A ( E and F ) and Y11F ( G and H ) were compared in the presence of 100 µM dNTPs (A, C, E and G) or rNTPs (B, D, F and H). Reactions were performed for 20 s, 1, 2, 4, 8 or 16 min (lanes 1–6, respectively) under optimal conditions for pol V Mut. The numbers shown next to the nucleotide identity represent primer elongation as a percent of total primer termini.

    Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

    Techniques:

    Discrimination against rNMP insertion by pol V mutants. Insertion of dA versus rA and T versus U by wild-type pol V ( A ), F10L ( B ), Y11A ( C ) or Y11F ( D ) were analyzed using a template containing five consecutive As. Insertion of dA/rA and dC/rC by wild-type pol V ( E ), F10L ( F ), Y11A ( G ) or Y11F ( H ) were analyzed using a template containing five consecutive Ts followed by two Gs. Reactions were performed in the presence of 100 µM dNTPs and/or 1 mM rNTPs for 10 min; half of each reaction mixture was subjected to alkaline hydrolysis (OH − ) (lanes 2, 4, 6, 8 and 10 in each panel) under conditions that completely hydrolyze DNA chains at the positions of rNTP insertion. The identity of the nucleotide present in the reaction is shown below each track (I NTP, II NTP). The extended sequence of the templates with five consecutive As (A–D) or Ts (E–H) is indicated to the right of the gel panels. Reactions in lanes 1 and 2 contained only ATP; lanes 3, 4 ATP and dATP; lanes 5, 6 ATP with UTP (A–D) or with CTP (E–H); lanes 7, 8 ATP and dATP with UTP and dTTP (A–D) or with CTP and dCTP (E–H); lanes 9, 10 ATP and dATP with dTTP (A–D) or with dCTP (E–H). Bands corresponding to incorporation of UTP (in A and D) or dTTP (in C) are indicated by stars.

    Journal: Nucleic Acids Research

    Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

    doi: 10.1093/nar/gks233

    Figure Lengend Snippet: Discrimination against rNMP insertion by pol V mutants. Insertion of dA versus rA and T versus U by wild-type pol V ( A ), F10L ( B ), Y11A ( C ) or Y11F ( D ) were analyzed using a template containing five consecutive As. Insertion of dA/rA and dC/rC by wild-type pol V ( E ), F10L ( F ), Y11A ( G ) or Y11F ( H ) were analyzed using a template containing five consecutive Ts followed by two Gs. Reactions were performed in the presence of 100 µM dNTPs and/or 1 mM rNTPs for 10 min; half of each reaction mixture was subjected to alkaline hydrolysis (OH − ) (lanes 2, 4, 6, 8 and 10 in each panel) under conditions that completely hydrolyze DNA chains at the positions of rNTP insertion. The identity of the nucleotide present in the reaction is shown below each track (I NTP, II NTP). The extended sequence of the templates with five consecutive As (A–D) or Ts (E–H) is indicated to the right of the gel panels. Reactions in lanes 1 and 2 contained only ATP; lanes 3, 4 ATP and dATP; lanes 5, 6 ATP with UTP (A–D) or with CTP (E–H); lanes 7, 8 ATP and dATP with UTP and dTTP (A–D) or with CTP and dCTP (E–H); lanes 9, 10 ATP and dATP with dTTP (A–D) or with dCTP (E–H). Bands corresponding to incorporation of UTP (in A and D) or dTTP (in C) are indicated by stars.

    Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

    Techniques: Sequencing

    Gel images of primer extension reactions catalyzed by wild-type pol V in the presence of dNTPs ( A–D ) or rNTPs ( E–H ). Reactions in the presence of each individual nucleotide were carried out for 5 min. The identity of nucleotide incorporated is shown below the respective lanes. The extended sequence of templates with five consecutive Ts (A and E), As (B and F), Cs (C and G) or Gs (D and H), are indicated to the right of each gel panel. Determination of the specificity of nucleotide incorporation is complicated due to the efficient incorporation of ATP present in the reactions at 1 mM concentration. Lanes with reactions lacking polymerase are indicated as ‘pr’ (short for primer) and reactions with no additional nucleotide are indicated by dash (–).

    Journal: Nucleic Acids Research

    Article Title: Critical amino acids in Escherichia coli UmuC responsible for sugar discrimination and base-substitution fidelity

    doi: 10.1093/nar/gks233

    Figure Lengend Snippet: Gel images of primer extension reactions catalyzed by wild-type pol V in the presence of dNTPs ( A–D ) or rNTPs ( E–H ). Reactions in the presence of each individual nucleotide were carried out for 5 min. The identity of nucleotide incorporated is shown below the respective lanes. The extended sequence of templates with five consecutive Ts (A and E), As (B and F), Cs (C and G) or Gs (D and H), are indicated to the right of each gel panel. Determination of the specificity of nucleotide incorporation is complicated due to the efficient incorporation of ATP present in the reactions at 1 mM concentration. Lanes with reactions lacking polymerase are indicated as ‘pr’ (short for primer) and reactions with no additional nucleotide are indicated by dash (–).

    Article Snippet: The reaction mixture contained 1 mM ATP, 50 µM dNTPs or rNTPs (unless otherwise indicated), 2 nM primed ssDNA templates (expressed as primer termini), 100 nM (as tetramer) SSB Epicentre Biotechnologies, (Madison, WI, USA), 50 nM (as a dimer) β clamp and 5 nM γ complex in the 1× reaction buffer.

    Techniques: Sequencing, Concentration Assay