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Promega taq dna polymerase
Superimposition of the thumb domains of <t>Taq</t> <t>DNA</t> polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Taq Dna Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 97/100, based on 40 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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1) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

2) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

3) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

4) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

5) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

6) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Journal: Nucleic Acids Research

doi:

Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

7) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

8) Product Images from "Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis"

Article Title: Rapid Typing of Human Adenoviruses by a General PCR Combined with Restriction Endonuclease Analysis

Journal: Journal of Clinical Microbiology

doi: 10.1128/JCM.39.2.498-505.2001

Comparison of Taq I restriction profiles of amplified DNA of prototype strains of Ad types 21, 51, 40, and 41.
Figure Legend Snippet: Comparison of Taq I restriction profiles of amplified DNA of prototype strains of Ad types 21, 51, 40, and 41.

Techniques Used: Amplification

9) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

10) Product Images from "Visual and modular detection of pathogen nucleic acids with enzyme–DNA molecular complexes"

Article Title: Visual and modular detection of pathogen nucleic acids with enzyme–DNA molecular complexes

Journal: Nature Communications

doi: 10.1038/s41467-018-05733-0

Visual and modular detection of pathogen nucleic acids. a The enVision system consists of a series of enzyme–DNA nanostructures to enable target recognition, target-independent signaling, and visual detection. The nanostructures are designed to decouple recognition from signaling. The recognition nanostructure is a hybrid complex, composed of an inactivating aptamer and a Taq DNA polymerase. In the presence of complementary target DNA, the complex dissociates to activate the polymerase activity. The active polymerase proceeds to elongate a universal, self-priming signaling nanostructure, in a target-independent manner. Modified deoxynucleotides (dNTPs) are incorporated to immobilize horseradish peroxidase (HRP) onto the signaling nanostructures. Upon the addition of optical substrate, visual signals can be enzymatically enhanced, detected by the naked eye and quantified with a smartphone camera. Photograph (inset) shows an example of the actual visual readouts in the presence of none (−) and varying (+) amounts of target DNA on a smartphone application. b Schematic of the enVision microfluidic system. The platform is designed to complement the modular enVision workflow. Independent assay cassettes, preloaded with specific recognition nanostructures at the inlets, can be mounted on-demand onto a common signaling cartridge. The common cartridge houses the universal signaling nanostructures, which are immobilized on embedded membranes, for target-independent signaling and visual detection. Direction of cassette sliding is indicated by a red arrow. c Photograph of the microfluidic enVision prototype, developed for versatile assay integration and parallel processing. Scale bar indicates 1 cm
Figure Legend Snippet: Visual and modular detection of pathogen nucleic acids. a The enVision system consists of a series of enzyme–DNA nanostructures to enable target recognition, target-independent signaling, and visual detection. The nanostructures are designed to decouple recognition from signaling. The recognition nanostructure is a hybrid complex, composed of an inactivating aptamer and a Taq DNA polymerase. In the presence of complementary target DNA, the complex dissociates to activate the polymerase activity. The active polymerase proceeds to elongate a universal, self-priming signaling nanostructure, in a target-independent manner. Modified deoxynucleotides (dNTPs) are incorporated to immobilize horseradish peroxidase (HRP) onto the signaling nanostructures. Upon the addition of optical substrate, visual signals can be enzymatically enhanced, detected by the naked eye and quantified with a smartphone camera. Photograph (inset) shows an example of the actual visual readouts in the presence of none (−) and varying (+) amounts of target DNA on a smartphone application. b Schematic of the enVision microfluidic system. The platform is designed to complement the modular enVision workflow. Independent assay cassettes, preloaded with specific recognition nanostructures at the inlets, can be mounted on-demand onto a common signaling cartridge. The common cartridge houses the universal signaling nanostructures, which are immobilized on embedded membranes, for target-independent signaling and visual detection. Direction of cassette sliding is indicated by a red arrow. c Photograph of the microfluidic enVision prototype, developed for versatile assay integration and parallel processing. Scale bar indicates 1 cm

Techniques Used: Activity Assay, Modification

11) Product Images from "An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11"

Article Title: An ABC Transporter System of Yersinia pestis Allows Utilization of Chelated Iron by Escherichia coli SAB11

Journal: Journal of Bacteriology

doi:

PCR of DNAs derived from whole cells of Yersinia spp. or E. coli DH5α. (A) Oligonucleotide primers derived from a region of Y. enterocolitica yfuA were used to PCR amplify genomic DNAs from the indicated strains. The predicted product is indicated by the arrow. (B) Oligonucleotide primers were derived from a region of Y. pestis yfeA . The predicted amplicon is designated with an arrow. Reactions were performed with Taq DNA polymerase for 25 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s.
Figure Legend Snippet: PCR of DNAs derived from whole cells of Yersinia spp. or E. coli DH5α. (A) Oligonucleotide primers derived from a region of Y. enterocolitica yfuA were used to PCR amplify genomic DNAs from the indicated strains. The predicted product is indicated by the arrow. (B) Oligonucleotide primers were derived from a region of Y. pestis yfeA . The predicted amplicon is designated with an arrow. Reactions were performed with Taq DNA polymerase for 25 cycles of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s.

Techniques Used: Polymerase Chain Reaction, Derivative Assay, Amplification

12) Product Images from "Nitric oxide-induced Cl− secretion in isolated rat colon is mediated by the release of thromboxane A2"

Article Title: Nitric oxide-induced Cl− secretion in isolated rat colon is mediated by the release of thromboxane A2

Journal: The Journal of Physiology

doi: 10.1113/jphysiol.2002.021287

Expression of TXA 2 receptor mRNA in colonic crypt cells Gel analysis of the RT-PCR product from isolated colonic crypts. A single band of 478 bp was detected by ethidium bromide staining (colonic crypts). No band was detected in negative control experiments of RT-PCR without reverse transcriptase (RT(-)) or Taq DNA polymerase (PCR(-)). Inset, a scheme of this PCR experiment is shown. TXA 2 R, TXA 2 receptor; sense, a sense primer; antisense, an antisense primer.
Figure Legend Snippet: Expression of TXA 2 receptor mRNA in colonic crypt cells Gel analysis of the RT-PCR product from isolated colonic crypts. A single band of 478 bp was detected by ethidium bromide staining (colonic crypts). No band was detected in negative control experiments of RT-PCR without reverse transcriptase (RT(-)) or Taq DNA polymerase (PCR(-)). Inset, a scheme of this PCR experiment is shown. TXA 2 R, TXA 2 receptor; sense, a sense primer; antisense, an antisense primer.

Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Staining, Negative Control, Polymerase Chain Reaction

13) Product Images from "Rapid Detection of Penicillin-Resistant Streptococcus pneumoniae in Cerebrospinal Fluid by a Seminested-PCR Strategy"

Article Title: Rapid Detection of Penicillin-Resistant Streptococcus pneumoniae in Cerebrospinal Fluid by a Seminested-PCR Strategy

Journal: Journal of Clinical Microbiology

doi:

Agarose gel electrophoresis of PCR-amplified DNA fragments of the pbp2B gene from S. pneumoniae . Lane M, molecular size marker (in base pairs). Lane 1, negative control; lane 2, penicillin-susceptible S. pneumoniae . Primer combinations are as follows: R1 + P5 + P6 (lane 3), R3 + P5 + P6 (lane 4), R1 + R3 + P5 + P6 (lane 5), R2 + P5 + P6 (lane 6), R4 + P5 + P6 (lane 7), and R2 + R4 + P5 + P6 (band C is poorly visible) (lane 8). (A) A 682-bp species-specific product arising from amplification with primers P5 and P6. (B) A 328- to 334-bp products arising from amplification with primers R1 to R3 and P6. (C) A 214-bp product arising from amplification with primers R4 and P6. (D) Amplification products produced as a result of annealing between a resistance product(s) and the 682-bp product and which are subsequently extended by Taq DNA polymerase to produce a larger product (±900 to 1,000 bp).
Figure Legend Snippet: Agarose gel electrophoresis of PCR-amplified DNA fragments of the pbp2B gene from S. pneumoniae . Lane M, molecular size marker (in base pairs). Lane 1, negative control; lane 2, penicillin-susceptible S. pneumoniae . Primer combinations are as follows: R1 + P5 + P6 (lane 3), R3 + P5 + P6 (lane 4), R1 + R3 + P5 + P6 (lane 5), R2 + P5 + P6 (lane 6), R4 + P5 + P6 (lane 7), and R2 + R4 + P5 + P6 (band C is poorly visible) (lane 8). (A) A 682-bp species-specific product arising from amplification with primers P5 and P6. (B) A 328- to 334-bp products arising from amplification with primers R1 to R3 and P6. (C) A 214-bp product arising from amplification with primers R4 and P6. (D) Amplification products produced as a result of annealing between a resistance product(s) and the 682-bp product and which are subsequently extended by Taq DNA polymerase to produce a larger product (±900 to 1,000 bp).

Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Negative Control, Produced

14) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

15) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Journal: Nucleic Acids Research

doi:

Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

16) Product Images from "Relating genes in the biosynthesis of the polyphenol composition of Andean colored potato collection"

Article Title: Relating genes in the biosynthesis of the polyphenol composition of Andean colored potato collection

Journal: Food Science & Nutrition

doi: 10.1002/fsn3.69

Genomic DNA of Durazno Imilla 30CN-010 sample was amplified by Bch6 , 21BA , UGPasa , CT-203 , Chi , GP-24 , Stan1 , Chs , and Zep primers, restricted with Taq DNA polymerase, and electrophoresed through a 2% agarose gel. The primers are listed in ascending order in a range of 400–1600 bp.
Figure Legend Snippet: Genomic DNA of Durazno Imilla 30CN-010 sample was amplified by Bch6 , 21BA , UGPasa , CT-203 , Chi , GP-24 , Stan1 , Chs , and Zep primers, restricted with Taq DNA polymerase, and electrophoresed through a 2% agarose gel. The primers are listed in ascending order in a range of 400–1600 bp.

Techniques Used: Amplification, Agarose Gel Electrophoresis

17) Product Images from "Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR"

Article Title: Typing of Dengue Viruses in Clinical Specimens and Mosquitoes by Single-Tube Multiplex Reverse Transcriptase PCR

Journal: Journal of Clinical Microbiology

doi:

Detection and typing of dengue virus by using two versions of the RT-PCR assay. (A) Reverse transcription with RAV-2 RT and amplification with Taq DNA polymerase; (B) reverse transcription and amplification with the bifunctional enzyme r Tth . (A and B) Lanes 1, dengue-2 (den-2); lanes 2, dengue-3 (den-3); lanes 3, dengue-4 (den-4); lanes 4, dengue-1 (den-1); lanes M, 100-bp ladder (lowest band shown, 100 bp); lanes 5 to 8, dengue-3 at 1,000, 100, 10, and 1 PFU, respectively. (A) Lane 9, 0 pfu; lane 10, water (negative control). (B) Lane 9, water. Expected product sizes are as follows: dengue-2, 119 bp; dengue-3, 290 bp; dengue-4, 389 bp; dengue-1, 482 bp.
Figure Legend Snippet: Detection and typing of dengue virus by using two versions of the RT-PCR assay. (A) Reverse transcription with RAV-2 RT and amplification with Taq DNA polymerase; (B) reverse transcription and amplification with the bifunctional enzyme r Tth . (A and B) Lanes 1, dengue-2 (den-2); lanes 2, dengue-3 (den-3); lanes 3, dengue-4 (den-4); lanes 4, dengue-1 (den-1); lanes M, 100-bp ladder (lowest band shown, 100 bp); lanes 5 to 8, dengue-3 at 1,000, 100, 10, and 1 PFU, respectively. (A) Lane 9, 0 pfu; lane 10, water (negative control). (B) Lane 9, water. Expected product sizes are as follows: dengue-2, 119 bp; dengue-3, 290 bp; dengue-4, 389 bp; dengue-1, 482 bp.

Techniques Used: Reverse Transcription Polymerase Chain Reaction, Amplification, Negative Control

18) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

19) Product Images from "Relating genes in the biosynthesis of the polyphenol composition of Andean colored potato collection"

Article Title: Relating genes in the biosynthesis of the polyphenol composition of Andean colored potato collection

Journal: Food Science & Nutrition

doi: 10.1002/fsn3.69

Genomic DNA of Durazno Imilla 30CN-010 sample was amplified by Bch6 , 21BA , UGPasa , CT-203 , Chi , GP-24 , Stan1 , Chs , and Zep primers, restricted with Taq DNA polymerase, and electrophoresed through a 2% agarose gel. The primers are listed in ascending order in a range of 400–1600 bp.
Figure Legend Snippet: Genomic DNA of Durazno Imilla 30CN-010 sample was amplified by Bch6 , 21BA , UGPasa , CT-203 , Chi , GP-24 , Stan1 , Chs , and Zep primers, restricted with Taq DNA polymerase, and electrophoresed through a 2% agarose gel. The primers are listed in ascending order in a range of 400–1600 bp.

Techniques Used: Amplification, Agarose Gel Electrophoresis

20) Product Images from "Proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure which can be targeted by G-quadruplex-interactive agents"

Article Title: Proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure which can be targeted by G-quadruplex-interactive agents

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-07-2119

Taq DNA polymerase stop assay showing the stabilization of G-quadruplex structures by K + and TMPyP2, TMPyP4, and Se2SAP. (A) Sequence of the single-stranded DNA template annealed with primer used in DNA polymerase stop assay. (B) KCl-dependent pausing
Figure Legend Snippet: Taq DNA polymerase stop assay showing the stabilization of G-quadruplex structures by K + and TMPyP2, TMPyP4, and Se2SAP. (A) Sequence of the single-stranded DNA template annealed with primer used in DNA polymerase stop assay. (B) KCl-dependent pausing

Techniques Used: Sequencing

21) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Journal: Nucleic Acids Research

doi:

Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

22) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: bioRxiv

doi: 10.1101/2020.05.27.120238

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.
Figure Legend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

Techniques Used: Quantitative RT-PCR, Amplification

TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

Techniques Used: Quantitative RT-PCR, Amplification

23) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

24) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

25) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

26) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

27) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: Biochemistry

doi: 10.1021/acs.biochem.0c00778

Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

Techniques Used: Quantitative RT-PCR, Incubation, Amplification

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

28) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

29) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: Biochemistry

doi: 10.1021/acs.biochem.0c00778

Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

Techniques Used: Quantitative RT-PCR, Incubation, Amplification

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

30) Product Images from "Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'"

Article Title: Quantitative target display: a method to screen yeast mutants conferring quantitative phenotypes by 'mutant DNA fingerprints'

Journal: Nucleic Acids Research

doi:

Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.
Figure Legend Snippet: Selective and quantitative amplification of targets. ( A ) Selective amplification of DNA flanking Tn insertions. Genomic DNA of 16 individual mutants were used to amplify DNA fragments until nearby Taq I restriction sites, and resolved on a sequencing gel. The mutants analyzed were: lane 1, SSA1 (V45B4); lane 2, YDJ1 (V6A2); lane 3, DDR48 (V6G5); lane 4, SSA2 (V18E7); lane 5, SSA3 (V41F1); lane 6, SSA4 (V5E8); lane 7, SSB1 (V23F11); lane 8, SSB2 (V32E7); lane 9, HSP35 (V18D3); lane 10, SSA4 (V3B8); lane 11, SOD2 (V4D11); lane 12, SSB2 (V47A3); lane 13, UBI4 (V36G6); lane 14, TPS2 (V2C2); lane 15, HSP104 (V8D8); lane 16, HSP104 (V22A9). Two bands each are seen for most of the mutants, consistent with specific amplification of DNA from both sides of each Tn insertion. ( B ) Quantitative amplification of targets, demonstrated by a reconstruction experiment. Ten different Tn insertion mutants were grown individually and then mixed together in equal proportions to obtain a pool of eight mutants (pool A, lane 1) and two mutants (pool B, lane 2). These two pools were then mixed at different ratios such that the abundance of the two mutants from pool B, with respect to the other mutants, was the same (lane 3) or was decreased 2-fold (lane 4), 4-fold (lane 5), 8-fold (lane 6) or 16-fold (lane 7). Genomic DNA was isolated from all the pools immediately and processed to amplify the targets. Equal volumes of PCR products were loaded, except for lane 2 where it was one-fifth of other lanes. While the intensity of the bands from eight mutants remained constant in lanes 3–7, that of two mutants (arrows) decreased in proportion to the abundance of the mutants in the pools, confirming quantitative amplification of the targets.

Techniques Used: Amplification, Sequencing, Isolation, Polymerase Chain Reaction

31) Product Images from "Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2"

Article Title: Catalytic amplification by transition-state molecular switches for direct and sensitive detection of SARS-CoV-2

Journal: Science Advances

doi: 10.1126/sciadv.abe5940

Catalytic amplification by transition-state molecular switch. ( A ) Schematic representation of the CATCH assay. The CATCH assay leverages the specific binding of nucleic acid targets (SARS-CoV-2 viral RNA) to activate molecular switches. Each molecular switch consists of a Taq DNA polymerase and an inhibitory DNA complex, comprising an inhibitor strand and an enhancer strand, that binds and inactivates the polymerase. As the viral RNA target hybridizes with the enhancer strand, it destabilizes the inhibitory complex and releases the active polymerase (left). By adjusting the ratio of molecular constituents in individual switches, we prepare molecular switches in different states of target responsiveness: closed, transition, and open (right). In the closed state, switches are fully inactivated, due to excess inhibitory complexes, and cannot be readily activated by sparse RNA targets. In the open state, switches are fully activated and largely unresponsive to targets due to a high initial background. In the transition state, different forms of switches exist in a delicate equilibrium—that a small amount of RNA targets can readily shift this equilibrium to favor the formation of more activated switches. The transition-state switches thus demonstrate maximal responsiveness (i.e., the largest change in polymerase activity within the shortest time span). ( B ) Signal generation. To enhance the detection signal, the CATCH assay recruits additional enzymatic cascades to transduce and amplify the target-induced polymerase activity as a fluorescence readout (see Fig. 3A for more details). As compared to that using the closed- or open-state molecular switches, the CATCH assay (transition state) generates strong signals from clinical samples with a low viral load. ( C ) Different assay formats. The CATCH assay can be performed in a 96-well format for high-throughput applications (top) or a miniaturized microfluidic device for portable, smartphone-based detection (bottom).
Figure Legend Snippet: Catalytic amplification by transition-state molecular switch. ( A ) Schematic representation of the CATCH assay. The CATCH assay leverages the specific binding of nucleic acid targets (SARS-CoV-2 viral RNA) to activate molecular switches. Each molecular switch consists of a Taq DNA polymerase and an inhibitory DNA complex, comprising an inhibitor strand and an enhancer strand, that binds and inactivates the polymerase. As the viral RNA target hybridizes with the enhancer strand, it destabilizes the inhibitory complex and releases the active polymerase (left). By adjusting the ratio of molecular constituents in individual switches, we prepare molecular switches in different states of target responsiveness: closed, transition, and open (right). In the closed state, switches are fully inactivated, due to excess inhibitory complexes, and cannot be readily activated by sparse RNA targets. In the open state, switches are fully activated and largely unresponsive to targets due to a high initial background. In the transition state, different forms of switches exist in a delicate equilibrium—that a small amount of RNA targets can readily shift this equilibrium to favor the formation of more activated switches. The transition-state switches thus demonstrate maximal responsiveness (i.e., the largest change in polymerase activity within the shortest time span). ( B ) Signal generation. To enhance the detection signal, the CATCH assay recruits additional enzymatic cascades to transduce and amplify the target-induced polymerase activity as a fluorescence readout (see Fig. 3A for more details). As compared to that using the closed- or open-state molecular switches, the CATCH assay (transition state) generates strong signals from clinical samples with a low viral load. ( C ) Different assay formats. The CATCH assay can be performed in a 96-well format for high-throughput applications (top) or a miniaturized microfluidic device for portable, smartphone-based detection (bottom).

Techniques Used: Amplification, Binding Assay, Activity Assay, Transduction, Fluorescence, High Throughput Screening Assay

32) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: bioRxiv

doi: 10.1101/2020.05.27.120238

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.
Figure Legend Snippet: SARS-CoV-2 N1 TaqMan RT-qPCR assays performed using NEB Taq DNA polymerase and N gene armored RNA in indicated buffers. Buffer compositions are detailed in Table 2 . Amplification curves resulting from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces), 30 (green traces), and 0 (gray) copies of SARS-CoV-2 N gene armored RNA are depicted.

Techniques Used: Quantitative RT-PCR, Amplification

TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.
Figure Legend Snippet: TaqMan RT-qPCR analysis of SARS-CoV-2 viral genomic RNA and RNaseP armored RNA using Taq DNA polymerase-based one-enzyme assays. CDC SARS-CoV-2 N gene assays, N1, N2, and N3, and RNaseP assay were performed using Taq DNA polymerase from either NEB (panels A-H) or Thermo Fisher (panels I-P). Assays were performed either using the companion commercial buffer (panels A-D and panels I-L) or using Gen 6 A buffer (panels E-H and panels M-P). Amplification curves from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of viral genomic RNA are depicted in panels A-C, E-G, I-K, and M-O. Amplification curves from 3 × 10 5 (black traces), 3 × 10 4 (red traces), 3 × 10 3 (blue traces), 3 × 10 2 (pink traces) and 0 (gray traces) copies of armored RNaseP RNA are depicted in panes D, H, L, and P.

Techniques Used: Quantitative RT-PCR, Amplification

33) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

34) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

35) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

36) Product Images from "Proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure which can be targeted by G-quadruplex-interactive agents"

Article Title: Proximal promoter region of the human vascular endothelial growth factor gene has a G-quadruplex structure which can be targeted by G-quadruplex-interactive agents

Journal: Molecular cancer therapeutics

doi: 10.1158/1535-7163.MCT-07-2119

Taq DNA polymerase stop assay showing the stabilization of G-quadruplex structures by K + and TMPyP2, TMPyP4, and Se2SAP. (A) Sequence of the single-stranded DNA template annealed with primer used in DNA polymerase stop assay. (B) KCl-dependent pausing
Figure Legend Snippet: Taq DNA polymerase stop assay showing the stabilization of G-quadruplex structures by K + and TMPyP2, TMPyP4, and Se2SAP. (A) Sequence of the single-stranded DNA template annealed with primer used in DNA polymerase stop assay. (B) KCl-dependent pausing

Techniques Used: Sequencing

37) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: Biochemistry

doi: 10.1021/acs.biochem.0c00778

Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

Techniques Used: Quantitative RT-PCR, Incubation, Amplification

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

38) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

39) Product Images from "Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase"

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

Journal: Nucleic Acids Research

doi:

Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.
Figure Legend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

Techniques Used: Sequencing

Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.
Figure Legend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

Techniques Used: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.
Figure Legend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

Techniques Used: Incubation

The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.
Figure Legend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

Techniques Used: Activity Assay, Labeling, Concentration Assay

40) Product Images from "One enzyme reverse transcription qPCR using Taq DNA polymerase"

Article Title: One enzyme reverse transcription qPCR using Taq DNA polymerase

Journal: Biochemistry

doi: 10.1021/acs.biochem.0c00778

Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.
Figure Legend Snippet: Effect of RNase treatment on Taq DNA polymerase-mediated RT-qPCR assays. Taq DNA polymerase (NEB) was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA that had been pre-incubated either with zero RNase units (panels A-C) or with a combination of 1 unit of RNase A and 40 units of RNase T1 (panels D-F). Representative amplification curves from duplicate experiments using 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA are depicted.

Techniques Used: Quantitative RT-PCR, Incubation, Amplification

Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.
Figure Legend Snippet: Effect of DNase I treatment on Taq DNA polymerase-mediated RT-qPCR assay. Taq DNA polymerase purchased from NEB was used to operate CDC SARS-CoV-2 N1, N2, and N3 TaqMan RT-qPCR assays using SARS-CoV-2 viral genomic RNA (panels A-C) or N gene armored RNA (panels D-F) treated with DNase I in duplicate experiments. Amplification curves shown in panels A-C resulted from 6000 (black traces), 600 (red traces), 60 (blue traces), 6 (pink traces), and 0 (gray traces) copies of SARS-CoV-2 genomic RNA. Amplification curves in panels D-F resulted from 30,000 (black traces), 3,000 (red traces), 300 (blue traces), 30 (pink traces) and 0 (gray traces) copies of N gene armored RNA. Representative Ct values for RT-qPCR amplification of indicated copies of untreated and DNase I treated SARS-CoV-2 genomic RNA and N gene armored RNA are tabulated.

Techniques Used: Quantitative RT-PCR, Amplification

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  • 99
    Promega taq polymerase
    PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the <t>C7-amino-modified</t> 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using <t>Taq</t> polymerase.
    Taq Polymerase, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 846 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/taq polymerase/product/Promega
    Average 99 stars, based on 846 article reviews
    Price from $9.99 to $1999.99
    taq polymerase - by Bioz Stars, 2022-09
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    99
    Promega promega taq buffers
    T1942C PCR-SSCP. Three DNA samples, 1942T/T (homozygous normal), 1942C/C (homozygous polymorphic), 1942T/C (heterozygous), were amplified with AmpliTaq Gold polymerase and AmpliTaq Gold buffer ( A ), <t>Promega</t> <t>Taq</t> and Promega buffer ( B ), Promega Taq and AmpliTaq Gold buffer ( C ) and with AmpliTaq Gold and Promega buffer (no PCR products obtained). All samples were amplified and SSCP-electrophoresed in the same experiment. Arrows point to the polymorphic SSCP fragment. Note that the polymorphic fragments are clearly visible only after amplification in AmpliTaq Gold buffer ( A , C ).
    Promega Taq Buffers, supplied by Promega, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.

    Journal: Nucleic Acids Research

    Article Title: Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

    doi:

    Figure Lengend Snippet: PAGE gel image of the ethidium bromide-stained and UV-visualised 62 nt PCR fragment derived from pUC19 and the C7-amino-modified 7-deaza-dATP analogues 9 – 11 and dUTP imidazole conjugates 12 and 13 . Lane 1, molecular weight markers; lane 2, a PCR containing all four natural triphosphates, dATP, dCTP, dGTP and dTTP; lane 3, a PCR containing dCTP and dGTP does not result in the formation of any product; lane 4, a PCR containing dTTP, dCTP, dGTP and 9 ; lane 5, a PCR containing dATP, dCTP, dGTP and 12 ; lane 6, a PCR dCTP, dGTP, 9 and 12 ; lane 7, a PCR dCTP, dGTP, 9 and 13 ; lane 8, molecular weight markers. Full-length 62 nt PCR products were formed using 9 and 12 , and 9 and 13 in place of dATP and dUTP, respectively, using Taq polymerase.

    Article Snippet: We have studied the incorporation of the C7-modified triphosphates, which replace dATP during PCR mediated by Taq polymerase, using pUC19 template DNA with the appropriate primers to yield 62 or 98 nt products.

    Techniques: Polyacrylamide Gel Electrophoresis, Staining, Polymerase Chain Reaction, Derivative Assay, Modification, Molecular Weight

    Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Superimposition of the thumb domains of Taq DNA polymerase (blue) with T7 DNA polymerase (pink). The arrows indicate the site of insertion of the T3 TBD (yellow). The primary amino acid sequence of Taq DNA polymerase from residue 470–507 is indicated below (blue) with the sequence of T3 TBD in yellow and the deleted region in red.

    Article Snippet: As Taq DNA polymerase has only 25% amino acid identity with T3 DNA polymerase, this lack of similarity between the proteins at the primary sequence supports the notion that the TBD of T3 polymerase is by itself sufficient to confer processivity.

    Techniques: Sequencing

    Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Slippage chromatograms were obtained from PCR products amplified with either Taq DNA polymerase or Taq DNA pol/TBD. One primer was labeled with 6-FAM fluorophore and the PCR product was digested with EcoRI. The DNA was gel purified and slippage polymorphisms detected using an automated DNA sequencer (model 377; Applied Biosystems) and GENESCAN 672 software. The result is one representative of three experiments.

    Article Snippet: As Taq DNA polymerase has only 25% amino acid identity with T3 DNA polymerase, this lack of similarity between the proteins at the primary sequence supports the notion that the TBD of T3 polymerase is by itself sufficient to confer processivity.

    Techniques: Polymerase Chain Reaction, Amplification, Labeling, Purification, Software

    Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: Streptavidin processivity assay. An immobilized single-stranded DNA molecule of 2000 nt in length was incubated in a reaction containing a primer hybridized to the 5′ end, and polymerase. Extension was initiated by the addition of dNTPs including [α- 32 P]dGTP, Mg 2+ and 0.8 mg/ml activated calf thymus DNA as described in Materials and Methods. Cleavage with restriction enzymes located 18, 96, 492, 1122 and 1898 nt, respectively, from the primer terminus only occurs if primer extension results in a double-stranded DNA substrate. Full extension with 5 U Promega Taq DNA polymerase in the absence of trap DNA allowed the percentage of primers extended to be determined.

    Article Snippet: As Taq DNA polymerase has only 25% amino acid identity with T3 DNA polymerase, this lack of similarity between the proteins at the primary sequence supports the notion that the TBD of T3 polymerase is by itself sufficient to confer processivity.

    Techniques: Incubation

    The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Journal: Nucleic Acids Research

    Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase

    doi:

    Figure Lengend Snippet: The effect of thioredoxin on processivity of the hybrid Taq DNA pol/TBD. ( A ) Extension assays were performed with a molar excess of template corresponding to a primer/template ratio of 470 for Taq DNA polymerase and Taq DNA polymerase (exo–) and 67 for Taq DNA pol/TBD and Taq DNA pol/TBD(exo–). Different ratios for the enzymes were used to ensure equal activity was loaded on the gel. (+) 100 µM thioredoxin, (–) no thioredoxin. No enzyme control shows the labeled primer alone. ( B ) Extension assay showing the effect of increasing concentrations of thioredoxin and enzyme dilution for Taq DNA pol/TBD(exo–). For each thioredoxin concentration (0.2, 2 and 20 µM), three enzyme concentrations were used (56, 28 and 5.6 pM) corresponding to a primer/template ratio of 67, 134 and 670.

    Article Snippet: As Taq DNA polymerase has only 25% amino acid identity with T3 DNA polymerase, this lack of similarity between the proteins at the primary sequence supports the notion that the TBD of T3 polymerase is by itself sufficient to confer processivity.

    Techniques: Activity Assay, Labeling, Concentration Assay

    T1942C PCR-SSCP. Three DNA samples, 1942T/T (homozygous normal), 1942C/C (homozygous polymorphic), 1942T/C (heterozygous), were amplified with AmpliTaq Gold polymerase and AmpliTaq Gold buffer ( A ), Promega Taq and Promega buffer ( B ), Promega Taq and AmpliTaq Gold buffer ( C ) and with AmpliTaq Gold and Promega buffer (no PCR products obtained). All samples were amplified and SSCP-electrophoresed in the same experiment. Arrows point to the polymorphic SSCP fragment. Note that the polymorphic fragments are clearly visible only after amplification in AmpliTaq Gold buffer ( A , C ).

    Journal: British Journal of Cancer

    Article Title: Frequent loss of the AXIN1 locus but absence of AXIN1 gene mutations in adenocarcinomas of the gastro-oesophageal junction with nuclear β-catenin expression

    doi: 10.1038/sj.bjc.6601589

    Figure Lengend Snippet: T1942C PCR-SSCP. Three DNA samples, 1942T/T (homozygous normal), 1942C/C (homozygous polymorphic), 1942T/C (heterozygous), were amplified with AmpliTaq Gold polymerase and AmpliTaq Gold buffer ( A ), Promega Taq and Promega buffer ( B ), Promega Taq and AmpliTaq Gold buffer ( C ) and with AmpliTaq Gold and Promega buffer (no PCR products obtained). All samples were amplified and SSCP-electrophoresed in the same experiment. Arrows point to the polymorphic SSCP fragment. Note that the polymorphic fragments are clearly visible only after amplification in AmpliTaq Gold buffer ( A , C ).

    Article Snippet: To determine whether the SSCP conditions have influence on the detection of the T1942C SNP, we amplified DNA samples with AmpliTaq Gold and with Promega Taq DNA polymerases (Promega, Madison, WI, USA) in both AmpliTaq Gold and Promega Taq buffers.

    Techniques: Polymerase Chain Reaction, Amplification