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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: 98/100, based on 34 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 "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

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 "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

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 "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

11) 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

12) 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

13) 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

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 "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

16) 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

17) Product Images from "Molecular cloning of the human PDGFR-? promoter and drug targeting of the G-quadruplex-forming region to repress PDGFR-? expression"

Article Title: Molecular cloning of the human PDGFR-? promoter and drug targeting of the G-quadruplex-forming region to repress PDGFR-? expression

Journal: Biochemistry

doi: 10.1021/bi100330w

(A) Effect of NaCl and KCl on the PDGFR-β NHE Pu38-mer CD spectra. The CD signals of the NHE Pu38-mer in 100 mM NaCl, 100 mM KCl, and water are shown in black, red, and blue respectively. All CD data were obtained with a 5 μM DNA concentration at 25 °C. The sequence of the NHE Pu38-mer is shown under the CD spectra. (B) Effect of increasing concentrations of NaCl and KCl on the stability of G-quadruplex structures within the NHE Pu38-mer using the Taq polymerase stop assay. Lanes 1 and 2 represent the guanine and cytosine sequencing reactions respectively. Lane 3 is the 32 P-labeled primer without Taq polymerase. The primer extension reaction in lane 4 was performed in the buffer without KCl or NaCl. The samples in lanes 5–9 were pre-incubated with different concentrations of KCl or NaCl before the primer extension reaction. The three primer extension stop products are designated 5′-end, mid-3′, and 3′-end. The corresponding sites for the stop products are indicated on the core G-tract sequence of NHE Pu38-mer at the bottom of gel. (C) DMS footprinting of intramolecular G-quadruplex structures in the NHE. The NHE Pu38-mer (full length) was incubated in a Tris buffer without KCl (lanes 1 and 3) or in the presence of 140 mM of KCl (lanes 2 and 4) before treatment with DMS. The seven runs of two or more guanines are indicated by brackets. DMS methylation patterns are indicated by open circles (protected), half-shaded circles (partially protected), and full-shaded circles (unprotected).
Figure Legend Snippet: (A) Effect of NaCl and KCl on the PDGFR-β NHE Pu38-mer CD spectra. The CD signals of the NHE Pu38-mer in 100 mM NaCl, 100 mM KCl, and water are shown in black, red, and blue respectively. All CD data were obtained with a 5 μM DNA concentration at 25 °C. The sequence of the NHE Pu38-mer is shown under the CD spectra. (B) Effect of increasing concentrations of NaCl and KCl on the stability of G-quadruplex structures within the NHE Pu38-mer using the Taq polymerase stop assay. Lanes 1 and 2 represent the guanine and cytosine sequencing reactions respectively. Lane 3 is the 32 P-labeled primer without Taq polymerase. The primer extension reaction in lane 4 was performed in the buffer without KCl or NaCl. The samples in lanes 5–9 were pre-incubated with different concentrations of KCl or NaCl before the primer extension reaction. The three primer extension stop products are designated 5′-end, mid-3′, and 3′-end. The corresponding sites for the stop products are indicated on the core G-tract sequence of NHE Pu38-mer at the bottom of gel. (C) DMS footprinting of intramolecular G-quadruplex structures in the NHE. The NHE Pu38-mer (full length) was incubated in a Tris buffer without KCl (lanes 1 and 3) or in the presence of 140 mM of KCl (lanes 2 and 4) before treatment with DMS. The seven runs of two or more guanines are indicated by brackets. DMS methylation patterns are indicated by open circles (protected), half-shaded circles (partially protected), and full-shaded circles (unprotected).

Techniques Used: Concentration Assay, Sequencing, Labeling, Incubation, Footprinting, Methylation

18) 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

19) 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

20) 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

21) 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

22) 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

23) 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

Related Articles

Clone Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Single read sequences were obtained from 88 individual clones totaling 24 531 and 19 631 bases sequenced for Taq DNA polymerase and Taq DNA pol/TBD, respectively. .. In order to determine the effect of processivity on slippage during PCR, we utilized a frame shift reporter assay described previously ( ).

Amplification:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. A decrease of 4-fold in slippage peak was observed in DNA obtained from processive amplification using Taq DNA pol/TBD compared with Taq DNA polymerase. ..

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR products were obtained by nested amplification of 10 ng of human genomic DNA at the D2S123 loci using reaction mixtures as above with either Taq DNA polymerase or Taq DNA pol/TBD. .. Two rounds of PCR was performed, each with 35 cycles using primers D2S123F (5′-CAAAAATTGACTCAAGAAGAA-3′), D2S123R (5′-TTAGGAGCTCTTTTGAATTG-3′) and nested primers, D2S123nestF (3′-AAATCTGAACAAACCTATGC-5′) and D2S123nestR (5′-GGGACTGTGTCATCTACAAT-3′).

DNA Synthesis:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: DNA synthesis was initiated by the addition of dNTPs, including [α-32 P]dGTP and a large excess of activated calf thymus trap DNA (final concentration of 0.8 mg/ml). .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. We have shown that insertion of the T3 bacteriophage TBD into the Taq DNA polymerase at the analogous position in the thumb domain, resulted in the formation of a hybrid protein that becomes stimulated by thioredoxin to produce highly processive DNA synthesis at elevated temperature. .. Insertion of the T3 thioredoxin loop resulted in both full-length and N-terminally truncated hybrid Taq DNA pol/TBD having ∼15% of the activity of their wild-type counterparts at 60°C in the absence of thioredoxin.

Polymerase Chain Reaction:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR was performed using λ phage as the template DNA and the primers listed in Materials and Methods with equivalent units of both Taq DNA polymerase and Taq DNA pol/TBD + thioredoxin activity (2.5 U per reaction). .. The Taq DNA pol/TBD polymerase was as able as Taq DNA polymerase to perform PCR when thioredoxin was included in the reaction mix (Fig. ) producing up to a 5 kb fragment.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Two methods were employed to determine the slippage frequency obtained during PCR with the highly processive Taq DNA pol/TBD versus Taq DNA polymerase. .. PCR fragments containing A10, G10 or CA11 repeats generated with Taq DNA polymerase or Taq DNA pol/TBD in the presence of thioredoxin were made.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR fragments containing A10, G10 or CA11 repeats generated with Taq DNA polymerase or Taq DNA pol/TBD in the presence of thioredoxin were made. .. Cloning into the frame shift reporter vector enabled determination of frame shifting frequencies in the PCR samples.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. The Taq DNA pol/TBD polymerase was as able as Taq DNA polymerase to perform PCR when thioredoxin was included in the reaction mix (Fig. ) producing up to a 5 kb fragment. .. In the absence of the thioredoxin cofactor, the Taq DNA pol/TBD enzyme is unable to amplify even the 1 kb fragment and is incapable of amplification of any fragments larger than 350 bp (data not shown).

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Huang M.M., Arnheim,N. and Goodman,M.F. (1992) Extension of base mispairs by Taq DNA polymerase: implications for single nucleotide discrimination in PCR. ..

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR products were obtained by nested amplification of 10 ng of human genomic DNA at the D2S123 loci using reaction mixtures as above with either Taq DNA polymerase or Taq DNA pol/TBD. .. Two rounds of PCR was performed, each with 35 cycles using primers D2S123F (5′-CAAAAATTGACTCAAGAAGAA-3′), D2S123R (5′-TTAGGAGCTCTTTTGAATTG-3′) and nested primers, D2S123nestF (3′-AAATCTGAACAAACCTATGC-5′) and D2S123nestR (5′-GGGACTGTGTCATCTACAAT-3′).

Mutagenesis:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Insertion of 76 residues encompassing the T3 bacteriophage gene5 TBD into the Taq DNA polymerase at the analogous position in the thumb domain, resulted in a hybrid enzyme that when expressed from a single copy plasmid, fully complemented the PolA temperature-sensitive mutation of E.coli strain JS200 at the restrictive temperature (data not shown). .. Purification of both Taq /TDB hybrid and Taq DNA polymerase full-length and N-terminal truncated 5′–3′ exonuclease-deficient enzymes were achieved using heparin and immobilized metal affinity chromatography.

Incubation:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage. ..

Concentration Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: DNA synthesis was initiated by the addition of dNTPs, including [α-32 P]dGTP and a large excess of activated calf thymus trap DNA (final concentration of 0.8 mg/ml). .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage.

Generated:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: Only double-stranded radiolabeled fragments generated during single binding and extension events, were released into solution for quantitation. .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR fragments containing A10, G10 or CA11 repeats generated with Taq DNA polymerase or Taq DNA pol/TBD in the presence of thioredoxin were made. .. Cloning into the frame shift reporter vector enabled determination of frame shifting frequencies in the PCR samples.

Activity Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Polymerase activity was compared between Taq DNA polymerase, Taq DNA polymerase (exo–) enzyme and the cognate Taq DNA pol/TBD hybrid proteins using primed M13mp18 DNA in the presence and absence of thioredoxin (Table ). .. In the absence of thioredoxin, both Taq DNA pol/TBD and Taq DNA pol/TBD(exo–) display ∼15% activity compared with the unmodified wild-type Taq DNA polymerase.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. PCR was performed using λ phage as the template DNA and the primers listed in Materials and Methods with equivalent units of both Taq DNA polymerase and Taq DNA pol/TBD + thioredoxin activity (2.5 U per reaction). .. The Taq DNA pol/TBD polymerase was as able as Taq DNA polymerase to perform PCR when thioredoxin was included in the reaction mix (Fig. ) producing up to a 5 kb fragment.

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Thioredoxin stimulates the activity of Taq DNA pol/TBD by ∼4-fold and Taq DNA pol/TBD(exo–) enzymatic activities by > 30-fold but had no effect on Taq DNA polymerase. .. Both Taq DNA pol/TBD and Taq DNA pol/TBD(exo–) are as thermostable as the Taq DNA polymerase and Taq DNA polymerase (exo–) enzymes, respectively, following exposure to 94°C for 30 min (Fig. ).

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Unlike these polymerases, Taq DNA polymerase does not have a proof-reading 3′–5′ exonuclease activity, relying instead on an extremely low mismatch extension efficiency ( ). .. That fewer C or G nucleotides were misincorporated across from template A or T positions may be due to a higher stringency against mismatched T-G or C-A extensions or alternatively an increased discrimination at the active site for misinsertion of C or G opposite A or T. In addition to the more accurate replication of non-iterated DNA, processive synthesis of microsatellite DNA is also reflected in the reduced frequency of slippage during PCR by 6–7-fold for the poly(A/T) and poly(CA/TG).

Sequencing:

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

Quantitation Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: Only double-stranded radiolabeled fragments generated during single binding and extension events, were released into solution for quantitation. .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage.

Binding Assay:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: Only double-stranded radiolabeled fragments generated during single binding and extension events, were released into solution for quantitation. .. Untrapped fully extended control reactions were incubated with 5 U Taq DNA polymerase (Promega) and used to quantitate the maximum signal obtained for each restriction cleavage.

Plasmid Preparation:

Article Title: Insertion of the T3 DNA polymerase thioredoxin binding domain enhances the processivity and fidelity of Taq DNA polymerase
Article Snippet: .. Insertion of 76 residues encompassing the T3 bacteriophage gene5 TBD into the Taq DNA polymerase at the analogous position in the thumb domain, resulted in a hybrid enzyme that when expressed from a single copy plasmid, fully complemented the PolA temperature-sensitive mutation of E.coli strain JS200 at the restrictive temperature (data not shown). .. Purification of both Taq /TDB hybrid and Taq DNA polymerase full-length and N-terminal truncated 5′–3′ exonuclease-deficient enzymes were achieved using heparin and immobilized metal affinity chromatography.

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