Structured Review

Promega dntps
Controlling the extent of pausing by <t>AMV-RT</t> at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM <t>dNTPs</t> at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
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Images

1) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

2) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

3) Product Images from "Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex"

Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky620

In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
Figure Legend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

Techniques Used: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.
Figure Legend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.
Figure Legend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

4) Product Images from "Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex"

Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky620

In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.
Figure Legend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

Techniques Used: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.
Figure Legend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

Techniques Used: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.
Figure Legend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

Techniques Used: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

5) Product Images from "Profiling proliferative cells and their progeny in damaged murine hearts"

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1805829115

Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Figure Legend Snippet: Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P

Techniques Used: Mouse Assay, Isolation, Amplification, Sequencing, Expressing, Staining, Flow Cytometry, Cytometry, Construct

6) Product Images from "Template-dependent multiple displacement amplification for profiling human circulating RNA"

Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA

Journal: BioTechniques

doi: 10.2144/000114566

Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U phi29 DNA polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.
Figure Legend Snippet: Real-time reverse transcription–template dependent multiple displacement amplification (RT-tdMDA) using three hepatitis C virus (HCV) patient serum samples and a negative control (H 2 O) These samples covered the range of the RNA yield extracted from 200 μL serum (7.5–19.2 ng), as quantitated in the final 14 μL of elution by the QIAGEN miRNA kit prior to HL-DNase digestion. An aliquot of 10.6 μL RNA was used for RT in a reaction containing 200 U SuperScript III, 80 μM 5′-end-blocked random pentamer primer (5′-/iSpC3/NNN*N*N-3′; asterisks denote phosphorothioate bonds), and 2 mM dNTPs in a 20-μL volume. An aliquot of 4 μL of the RT reaction was used in a 40-μL tdMDA reaction containing 300 U phi29 DNA polymerase (Epicentre), 80 μM primer, and 0.1× SYBR Green I (Thermo Fisher Scientific). The reaction was incubated at 28°C for 24 h on the ABI TaqMan 7500, in which fluorescent intensities were monitored through the SYBR Green channel. Estimated amount of cDNA input in tdMDA = [10.6 / (14 + 0.5 (HL-DNase) + 1.4 (buffer)] × [total RNA amount] × 0.2. Template-independent amplification was completely inhibited, as indicated by the negative control.

Techniques Used: Multiple Displacement Amplification, Negative Control, SYBR Green Assay, Incubation, Amplification

7) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

8) Product Images from "Profiling proliferative cells and their progeny in damaged murine hearts"

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1805829115

Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P
Figure Legend Snippet: Single-cell transcriptome analysis uncovers distinct proliferative populations within the murine heart. ( A ) Experimental timeline for tissue collection of hearts from wild-type and Mki67 RFP mice, either neonatal or adults, 14 d after sham, ischemia/reperfusion (I/R), or MI surgery ( n = 2–4 mice per condition). ( B ) Schematic representation of SORT-seq workflow. Hearts were isolated (1) and digested into single-cell suspension (2), and Ki67-RFP + and Ki67-RFP − cells were sorted into 384-well plates containing primers, dNTPs, and spike-ins (3). Retrotranscription mix was distributed using Nanodrop II, and material was pooled and amplified (4) before pair-end sequencing (5). Cells were clustered using RaceID2 (6). ( C ) Clustering of cardiac cells and cell-to-cell distances visualized by t -distributed stochastic neighbor-embedding ( t -SNE) map, highlighting identified major cardiac cell types. ( D ) Numbers of cells assigned to each cardiac cell lineage. ( E ) t -SNE map highlighting identified cell types based on previously described cellular markers (logarithmic scale of transcript expression). Markers expression is shown in Lower panel by immunofluorescent staining. (Scale bars: 50 μm.) ( F ) t -SNE map displaying cell cycle stage of each cell [S (red), G 2 /M (green), G 0 /G 1 (blue)] assigned by the cyclone algorithm. ( G ) t -SNE map showing the Ki67-RFP status from the flow cytometry data; Ki67-RFP + (red), Ki67-RFP − (black), or Mki67 wt/wt cells without TagRFP construct (gray) and radar plot showing Ki67-RFP + cells enriched for the cycling G 2 /M stage according to the cyclone algorithm. Asterisks indicate significance (χ 2 test: *** P

Techniques Used: Mouse Assay, Isolation, Amplification, Sequencing, Expressing, Staining, Flow Cytometry, Cytometry, Construct

9) Product Images from "Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA"

Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

Journal: ACS Chemical Biology

doi: 10.1021/cb500270x

Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
Figure Legend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

Techniques Used: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

Related Articles

Sequencing:

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: .. DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD). ..

Flow Cytometry:

Article Title: Profiling proliferative cells and their progeny in damaged murine hearts
Article Snippet: .. DAPI-negative and MitoTracker-positive living cells were either sorted into TRIzol reagent (Thermo Scientific) for bulk mRNA sequencing or into 384-well plates containing 96 or 384 unique molecular identifier barcode primer sets, ERCC92 spike-ins (Agilent) and dNTPs (Promega) for single-cell mRNA-sequencing (SORT-seq) ( ) using a flow sorter (FACSAriaII, FACSFusion, or FACSJazz; all BD). ..

other:

Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex
Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex
Article Snippet: Reverse transcription Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

Purification:

Article Title: Template-dependent multiple displacement amplification for profiling human circulating RNA
Article Snippet: .. Purified RNA (9.4 μL) was reverse transcribed in a 20 μL reaction consisting of 1× SuperScript III buffer (Life Technologies), 10 mM DTT, 2 mM dNTPs, 20 U RNasein RNase Inhibitor (Promega, Madison, WI), 200 U SuperScript III reverse transcriptase and 80 μM primers. ..

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  • 96
    Promega dntps
    Controlling the extent of pausing by <t>AMV-RT</t> at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM <t>dNTPs</t> at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.
    Dntps, supplied by Promega, used in various techniques. Bioz Stars score: 96/100, based on 2827 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    98
    Promega deoxynucleotide triphosphates
    Primer extension with <t>deoxynucleotide</t> triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.
    Deoxynucleotide Triphosphates, supplied by Promega, used in various techniques. Bioz Stars score: 98/100, based on 170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

    Journal: ACS Chemical Biology

    Article Title: Click Modification of RNA at Adenosine: Structure and Reactivity of 7-Ethynyl- and 7-Triazolyl-8-aza-7-deazaadenosine in RNA

    doi: 10.1021/cb500270x

    Figure Lengend Snippet: Controlling the extent of pausing by AMV-RT at a 7-EAA site. (A) Sequences of strands employed in primer extension assay, with N indicating the site of the variable nucleotide. (B) Primer extension results for reactions containing 10 μM dNTPs at 42 °C for 45 min. Lanes are labeled as follows for N: −, labeled primer only, no extension; A, adenosine; 7, 7-EAA; B, biotin triazole; and B + S, biotin triazole + monomeric streptavidin. (C) Primer extension results for reactions containing 1 μM dNTPs at 37 °C for 5 min; lanes are labeled the same as in panel B, and the arrow indicates a pause site. (D) Quantification of inhibition of primer extension under the conditions used in panel C; the average for at least three independent primer extension reactions ± standard deviation is plotted.

    Article Snippet: Samples were then mixed with AMV-RT and dNTP mix so that the final concentrations were as follows: 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 10 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the standard extension conditions protocol and 20 nM RNA, ∼80 nM 32 P-labeled GluR B pre-mRNA 18 nt primer, 1 μM dNTPs, 1× Promega AMV-RT buffer, and 5 units of AMV-RT for the low [dNTP] conditions protocol.

    Techniques: Primer Extension Assay, Labeling, Inhibition, Standard Deviation

    In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: In vitro template-primed cDNA synthesis. ( A ) Bordetella bacteriophage DGR diversification of Mtd. mtd contains a variable region ( VR ), which encodes the receptor-binding site of the Mtd protein. Downstream of VR is the template region ( TR ). Adenines in TR (‘A’) are frequently replaced by another base in VR (‘N’). TR is transcribed to produce TR- RNA, which is then reverse transcribed to TR- cDNA. During this process, adenines in TR are mutagenized, as depicted by ‘X’ in TR -cDNA. Adenine-mutagenized TR- cDNA homes to and replaces VR , resulting in diversification of Mtd. bRT is the DGR reverse transcriptase, and avd the DGR accessory variability determinant. ( B ) Sequence elements of the 580 nt DGR RNA template used for reverse transcription reactions. ( C ) bRT-Avd, bRT, or Avd was incubated with the 580 nt DGR RNA and dNTPs, including [α- 32 P]dCTP, for 2h. Products resulting from the incubation were untreated (U), or treated with RNase (+R), DNase (+D), or both RNase and DNase (+R+D), and resolved by 8% denaturing polyacrylamide gel electrophoresis (PAGE). Lane T corresponds to internally-labeled 580 nt DGR RNA as a marker for the size of the template. The positions of the 580 nt band, and 120 and 90 nt cDNA bands are indicated. Nuclease-treated samples were loaded at twice the amount as untreated samples, here and throughout unless otherwise indicated. Lane M here and throughout corresponds to radiolabeled, single-stranded DNA molecular mass markers (nt units). ( D ) DGR RNA templates containing internal truncations in TR . ( E ) Radiolabeled cDNA products resulting from bRT-Avd activity for 2 h with intact (WT) or internally truncated 580 nt DGR RNA as template. Samples were treated with RNase and resolved by denaturing PAGE. The positions of the 120 and 90 nt cDNAs produced from intact template are indicated by red and yellow circles, respectively, as are positions of the correspondingly shorter cDNAs produced from truncated RNA templates. ( F ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template. Prior to reverse transcription, the RNA template was mock-treated (–Per) or treated with periodate (+Per). Products of the reaction were untreated (U) or treated with RNase (+R), and resolved by 4% (top) or 8% (bottom) denaturing PAGE. In the top gel, the red arrowhead indicates the ∼580 nt species, and the green arrowheads the several ∼540 nt species. In the bottom gel, the black arrowheads indicate the 120 and 90 nt cDNA products. The black vertical line within the gel indicates irrelevant lanes that were removed for display purposes. A 2-fold higher quantity was loaded for +Per samples than –Per samples.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: In Vitro, Binding Assay, Sequencing, Incubation, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Activity Assay, Produced

    Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Core DGR RNA. ( A ) Schematic of core DGR RNA. ( B ) Radiolabeled products resulting from bRT-Avd activity for 2 h with the core DGR RNA as template. Prior to the reverse transcription reaction, the RNA template was untreated (-Per) or treated with periodate (+Per). Products from the reaction were untreated (U) or treated with RNase (+R), and resolved by 6% denaturing PAGE. Lane T corresponds to internally-labeled core DGR RNA as a marker for the size of the template. Red arrowheads indicate radiolabeled product bands that migrate at the same position or slower than the core DGR RNA, and green arrowheads ones that migrate faster. The positions of the 120 and 90 nt cDNA bands are indicated. The two panels are from the same gel, with the black line indicating that intermediate lanes were removed. ( C ) Internally-labeled core DGR RNA was not incubated (–), or incubated with bRT-Avd alone or bRT-Avd with 100 μM standard dNTPs (+dNTP), 100 μM dCTP (+CTP), 100 μM dNTPs excluding dCTP (+d(A,T,G)TP), or 100 μM nonhydrolyzeable analog of dCTP (+N-dCTP) for 2 h. Incubation products were resolved by denaturing PAGE. The band corresponding to the 5′ fragment of the cleaved core RNA containing either a deoxycytidine alone (5′+dC) or cDNA (5′+cDNA), and the band corresponding to the 3′ fragment of the RNA are indicated. ( D ) The core DGR RNA was biotinylated at its 3′ end (RNA-Bio), and either reacted with no protein or used as a template for reverse transcription with bRT-Avd. The core DGR RNA in its unbiotinylated form (RNA) was also used as a template for reverse transcription with bRT-Avd. Samples were then purified using streptavidin beads, and the presence of TR -cDNA in the purified samples was assessed by PCR. Products from the PCR reaction were resolved on an agarose gel. ( E ) Radiolabeled products resulting from bRT-Avd activity for 12 h with core, hybrid core dA56, or hybrid core A56 DGR RNA as template. Products were untreated (U) or treated with RNase (+R), and resolved by denaturing PAGE. Separate samples of core dA56 and A56 were 5′ 32 P-labeled for visualization of inputs (I). The positions of the 120 and 90 nt cDNAs are indicated.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Activity Assay, Polyacrylamide Gel Electrophoresis, Labeling, Marker, Incubation, Purification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Journal: Nucleic Acids Research

    Article Title: Template-assisted synthesis of adenine-mutagenized cDNA by a retroelement protein complex

    doi: 10.1093/nar/gky620

    Figure Lengend Snippet: Adenine mutagenesis and template-priming. ( A ) Covalently-linked RNA–cDNA molecule. The linkage is to Sp A56 of the RNA, and the first nucleotide reverse transcribed is TR G117. The RT-PCR product resulting from primers 1 and 2 (blue arrows) is indicated by the dashed red line. ( B ) RT-PCR amplicons from 580 nt DGR RNA reacted with no protein (–), bRT, Avd, or bRT-Avd, separated on a 2% agarose gel and ethidium bromide-stained. The specific amplicon produced from reaction with bRT-Avd shown by the red arrowhead. ( C ) Percentage of substitutions in TR -cDNA determined by sequencing. ( D ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity with the 580 nt DGR RNA as template for 2 h (left) or 12 h (right). Either standard dNTPs (dATP, dGTP, dCTP, TTP), as indicated by ‘+’,were present in the reaction, or standard dNTPs excluding dATP (-A), dGTP (–G), or TTP (-T) were present. Products were treated with RNase, and resolved by denaturing PAGE. ( E ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying TTP (top) or dUTP (bottom) concentrations. Products were treated with RNase, and resolved by denaturing PAGE. ( F ) Radiolabeled 120 and 90 nt cDNA products, indicated by arrowheads, resulting from bRT-Avd activity for 2 h with the 580 nt DGR RNA as template with varying dUTP concentrations. Products were either RNase-treated (top), or both RNase- and UDG-treated (bottom), and resolved by denaturing PAGE.

    Article Snippet: Reactions were carried out in 20 μl containing 1.8 μM bRT-Avd, bRT or Avd, 100 ng/μl RNA template, 100 μM dNTPs (Promega) (or varying concentrations of certain dNTPs), 0.5 μCi/μl [α-32 P]dCTP, 20 units RNase inhibitor (NEB) in 75 mM KCl, 3 mM MgCl2 , 10 mM DTT, 50 mM HEPES, pH 7.5, 10% glycerol for 2 h at 37°C.

    Techniques: Mutagenesis, Reverse Transcription Polymerase Chain Reaction, Agarose Gel Electrophoresis, Staining, Amplification, Produced, Sequencing, Activity Assay, Polyacrylamide Gel Electrophoresis

    Primer extension with deoxynucleotide triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.

    Journal: Nucleic Acids Research

    Article Title: Single base extension (SBE) with proofreading polymerases and phosphorothioate primers: improved fidelity in single-substrate assays

    doi:

    Figure Lengend Snippet: Primer extension with deoxynucleotide triphosphates. The positions of unextended (inverted open triangle), singly extended (inverted black triangle) and doubly extended primers (inverted grey triangle) are indicated. All spectra contain one additional equivalent of primer as a post-reaction standard. (A–D) Incorporation of dGTP for the following primer/template/polymerase combinations. ( A ) P1/LTT/exo – KF. ( B ) SP1/LTT/exo – KF. ( C ) SP1/LTT/exo + KF. ( D ) SP1/LTC/exo + KF. (E–H) Incorporation of dATP for the following primer/template/polymerase combinations. ( E ) P1/LTT/exo – KF. ( F ) SP1/LTT/exo – KF. ( G ) SP1/LTT/exo + KF. ( H ) SP1/LTT/T4 DNAP. All spectra include a signal from primer added post-reaction as an internal standard.

    Article Snippet: Deoxynucleotide triphosphates (Promega), dideoxynucleotide triphosphates (MBI) and acyclonucleotide triphosphates (NEB) were purchased commercially.

    Techniques: