Structured Review

Millipore dntps
Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no <t>NTPs.</t> Positive control: <t>dNTPs</t> or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.
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Images

1) Product Images from "A general strategy for expanding polymerase function by droplet microfluidics"

Article Title: A general strategy for expanding polymerase function by droplet microfluidics

Journal: Nature Communications

doi: 10.1038/ncomms11235

Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.
Figure Legend Snippet: Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

Techniques Used: Activity Assay, Fluorescence, Polyacrylamide Gel Electrophoresis, Negative Control, Positive Control, Functional Assay, Flow Cytometry, Cytometry

2) Product Images from "Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ? *"

Article Title: Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ? *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M805476200

Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”
Figure Legend Snippet: Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”

Techniques Used: Concentration Assay, Activity Assay

3) Product Images from "Direct Cell Lysis for Single-Cell Gene Expression Profiling"

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

Journal: Frontiers in Oncology

doi: 10.3389/fonc.2013.00274

Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.
Figure Legend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

Techniques Used: Lysis, Polymerase Chain Reaction

4) Product Images from "Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence"

Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

Journal: BMC Biochemistry

doi: 10.1186/1471-2091-12-47

Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.
Figure Legend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

Techniques Used: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling

5) Product Images from "RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication"

Article Title: RNA primer–primase complexes serve as the signal for polymerase recycling and Okazaki fragment initiation in T4 phage DNA replication

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

doi: 10.1073/pnas.1620459114

Extension of the primer in pppRNA–primase complex by holoenzyme or polymerase. ( A ) Denaturing polyacrylamide gel analysis testing the ability of the holoenzyme or polymerase to use the primer of a pppRNA–primase complex to initiate Okazaki fragment synthesis. The pppRNA–primase complex was first bound to a ssDNA substrate containing a primer recognition site. The reactions were initiated by the addition of holoenzyme components or polymerase and dNTPs; the reactions were quenched at the indicated time points. The resulting products were separated on a 7.5 M urea–14% polyacrylamide gel; the migration positions of the starting primer, 40-mer product, and 70-mer template are indicated. The amount of each product was quantitated using a Persona Molecular Imager (Bio Rad), and the final concentration of product in each reaction was calculated using a standard curve of dCTP [α- 32 P]. ( B ) The amount of primer in pppRNA–primase complexes extended by holoenzyme (red high–low bars) or polymerase (blue squares) is plotted over time. Linear least squares regression of the data yields a primer extension rate of 0.22 nM primer per second for holoenzyme and 0.0024 nM primer per second for polymerase under these conditions of 200 nM pppRNA–primase complex and 300 nM holoenzyme or polymerase. During the approximate 25-s half-life of the pppRNA–primase complex, holoenzyme was able to extend only 2.8% of the primers, indicating the unlikelihood that primers synthesized by the disassembly mechanism are used to initiate Okazaki fragment synthesis.
Figure Legend Snippet: Extension of the primer in pppRNA–primase complex by holoenzyme or polymerase. ( A ) Denaturing polyacrylamide gel analysis testing the ability of the holoenzyme or polymerase to use the primer of a pppRNA–primase complex to initiate Okazaki fragment synthesis. The pppRNA–primase complex was first bound to a ssDNA substrate containing a primer recognition site. The reactions were initiated by the addition of holoenzyme components or polymerase and dNTPs; the reactions were quenched at the indicated time points. The resulting products were separated on a 7.5 M urea–14% polyacrylamide gel; the migration positions of the starting primer, 40-mer product, and 70-mer template are indicated. The amount of each product was quantitated using a Persona Molecular Imager (Bio Rad), and the final concentration of product in each reaction was calculated using a standard curve of dCTP [α- 32 P]. ( B ) The amount of primer in pppRNA–primase complexes extended by holoenzyme (red high–low bars) or polymerase (blue squares) is plotted over time. Linear least squares regression of the data yields a primer extension rate of 0.22 nM primer per second for holoenzyme and 0.0024 nM primer per second for polymerase under these conditions of 200 nM pppRNA–primase complex and 300 nM holoenzyme or polymerase. During the approximate 25-s half-life of the pppRNA–primase complex, holoenzyme was able to extend only 2.8% of the primers, indicating the unlikelihood that primers synthesized by the disassembly mechanism are used to initiate Okazaki fragment synthesis.

Techniques Used: Migration, Concentration Assay, Synthesized

6) Product Images from "A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels"

Article Title: A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels

Journal: Pharmaceutical research

doi: 10.1007/s11095-009-9863-9

Determination of dNTPs and NTPs Levels in Bone Marrow Samples from a Leukemia Patient
Figure Legend Snippet: Determination of dNTPs and NTPs Levels in Bone Marrow Samples from a Leukemia Patient

Techniques Used:

Product ion mass spectra of the deprotonated molecular ions of dNTPs and NTPs.
Figure Legend Snippet: Product ion mass spectra of the deprotonated molecular ions of dNTPs and NTPs.

Techniques Used:

A The extract ion chromatograms ( XIC ) of dNTPs and NTPs, 50 nM each, spiked into acid phosphatase-treated blank K562 cell extracts. B The XICs of dNTPs and NTPs in blank acid phosphatase-treated K562 cell extracts. No significant interference peaks were
Figure Legend Snippet: A The extract ion chromatograms ( XIC ) of dNTPs and NTPs, 50 nM each, spiked into acid phosphatase-treated blank K562 cell extracts. B The XICs of dNTPs and NTPs in blank acid phosphatase-treated K562 cell extracts. No significant interference peaks were

Techniques Used:

Standard curves of dNTPs and NTPs in K562 cell matrices.
Figure Legend Snippet: Standard curves of dNTPs and NTPs in K562 cell matrices.

Techniques Used:

Stability study of dNTPs and NTPs in K562 cell matrices.
Figure Legend Snippet: Stability study of dNTPs and NTPs in K562 cell matrices.

Techniques Used:

Analysis of dNTPs and NTPs in Different Cell Lines
Figure Legend Snippet: Analysis of dNTPs and NTPs in Different Cell Lines

Techniques Used:

7) Product Images from "Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ? *"

Article Title: Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ? *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M805476200

Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”
Figure Legend Snippet: Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”

Techniques Used: Concentration Assay, Activity Assay

8) Product Images from "Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability"

Article Title: Oxidized dNTPs and the OGG1 and MUTYH DNA glycosylases combine to induce CAG/CTG repeat instability

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw170

Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.
Figure Legend Snippet: Incorporation of 8-oxodGMP and 2-OH-dAMP in TNR sequences. ( A and B ) Incorporation of dGMP and 8-oxodGMP opposite cytosine. ( A ) The primer/template sequence and representative gels are shown. Primer/template substrate (160 nM) (S1/T1) was incubated with POL β and increasing concentration of 8-oxodGTP or dGTP at 37°C for 1 h (0–30 μM and 0–3 μM, respectively). M is the DNA substrate without enzyme. Reaction products were separated by 20% denaturing PAGE at 500 V for 2.5 h. Bands were visualized by fluorescence emission by Typhoon scanner and the analysis of the band intensities was performed by ImageJ software. ( B ) Percentage of incorporated dNMP plotted as a function of added dNTPs. Data were fitted by Kaleidagraph software to evaluate kinetics parameters. ( C and D ) Incorporation of 8-oxodGMP and dTMP opposite adenine. Primer/template sequences is S2/T1 (Supplementary Table S1). Experimental conditions and analyses were as described above. The concentration range of 8-oxodGTP and dTTP was 0–2 μM and 0–1 μM, respectively. ( E – F ) Incorporation of 2-OH-dAMP and dAMP in CAG/CTG repeat sequence. ( E ) Primer/template sequence is S3/T2 (Supplementary Table S1); ( F ) Primer/template duplex (160 nM) was incubated with POL β (0.1U) in 10 μl reaction buffer in the absence of dNTP (lane 1), after addition of 2-OH-dATP (lane 2), dGTP and 2-OH-dATP (lane 3); 2-OH-dATP, dGTP and dCTP (lane 4), dATP (lane 5); dATP and dGTP (lane 6), dATP, dGTP and dCTP (lane 7). All nucleotide triphosphates were at 10 μM final concentration. Reaction products were separated by 15% denaturing PAGE and image acquisition and analysis was performed as described before.

Techniques Used: Sequencing, Incubation, Concentration Assay, Polyacrylamide Gel Electrophoresis, Fluorescence, Software, CTG Assay

Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.
Figure Legend Snippet: Incorporation and extension of 8-oxodGMP by POL β. Primer/template (160 nM) (Supplementary Table S1, S3/T2 panel A; S1/T1 panel B) were used. Both substrates were incubated with POL β (0.1 U) and 8-oxodGTP or dGTP and others dNTPs (50 μM final concentration each). ( A ) Lane 1, primer; lane 2, 8-oxodGTP; lane 3, as lane 2 plus dCTP; lane 4, as lane 3 plus dATP; lane 5, as lane 4 plus dTTP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 7 plus dATP; lane 9, as lane 8 plus dTTP. ( B ) Lane 1, primer; lane 2, primer plus 8- oxodGTP; lane 3, as lane 2 plus dCTP; lane 4 as lane 3 plus dTTP; lane 5, as lane 4 plus dATP; lane 6, primer plus dGTP; lane 7, as lane 6 plus dCTP; lane 8 as lane 6 plus dTTP and dATP. ( C ) The DNA substrate (160 nM) was built by annealing three oligomers of 22, 77 and 100 bases respectively indicated as S1, S4 and T1 in Supplementary Table S1, in order to produce a preformed nicked duplex containing CTG/CAG repeats. Incorporation of dGTP and 8-oxodGTP (lanes 1 and 4) and elongation (lanes 2 and 5) was obtained by incubating the substrate (lane 3) with POL β (0.1 U) at 37°C for 1 h.

Techniques Used: Incubation, Concentration Assay, CTG Assay

Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).
Figure Legend Snippet: Novel contributors in TNR expansion process. Following an initial incision event mediated by OGG1 and APE1 at an 8-oxodG site in the top strand (red, step 1), POL drives repair synthesis by LP BER. Long flaps might eventually fold in stable secondary structures (step 2). A faulty removal by FEN1 depending on flap conformations might leave hairpins with unligatable dRP ends (step 3). Removal of dRP by POL β allows ligation by LIG1 (step 4). If 8-oxodGTP is present in the dNTPs pool, 8-oxodGMP can be incorporated opposite A in the complementary strand creating a substrate for MUTYH (step 5). MUTYH activity on the bottom strand (blue) allows the initiation of a new repair event, as well as an elongation process on this side (step 6). Realignements of the strands will result in TNR expansion (step 7). Newly synthesized tracts are represented by full rectangles. The proposed model has been modified from refs. ( 12 , 16 , 40 ).

Techniques Used: Ligation, Activity Assay, Synthesized, Modification

Related Articles

Polymerase Chain Reaction:

Article Title: Mechanical properties of DNA-like polymers
Article Snippet: For analogs 3 , 4 , 5 , 6 and 9 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, PrimeSTAR GC buffer (Takara), 0.2 mM each dNTP (again with dTTP completely replaced with analog, except for variant 9 , which completely replaced dATP), 2 M betaine (Sigma-Aldrich) and 5 U PrimeSTAR HS DNA polymerase (Takara). .. For analogs 7 and 8 , PCR reactions (100 µl) included 20 ng template, 0.4 mM forward and reverse primers, Pwo PCR buffer (Roche), GC-rich solution (Roche), 0.2 mM each dNTP (dTTP completely replaced with analog), 2 M betaine (Sigma-Aldrich) and 5 U Pwo SuperYield DNA Polymerase (Roche). .. To generate DNA where only one strand contained analog 8 , the previous conditions were modified so that the template was replaced with the desired amount of unmodified PCR product and the number of cycles was reduced from 30 to a single extension cycle.

Incubation:

Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling
Article Snippet: Reverse transcription SuperScript™ III Reverse Transcriptase (Invitrogen) was used for reverse transcription. .. Directly lysed cells were incubated in 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo-dT (Metabion), and 2.5 μM random hexamers (Metabion) at 65°C for 5 min and then chilled on ice. .. 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2 , 5 mM dithiothreitol, 10 U RNaseOut, and 50 U SuperScript III were added to a final volume of 10 μl (all Invitrogen).

Article Title: A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels
Article Snippet: .. Dephosphorylation of intracellular dNTPs and NTPs was achieved by addition of 16 U of acid phosphatase (type XA, Sigma; St. Louis, MO, USA) and 50 μL of 1 M sodium acetate, pH 4.0, followed by a 1-h incubation at 37°C. ..

De-Phosphorylation Assay:

Article Title: A LC-MS/MS Method for the Analysis of Intracellular Nucleoside Triphosphate Levels
Article Snippet: .. Dephosphorylation of intracellular dNTPs and NTPs was achieved by addition of 16 U of acid phosphatase (type XA, Sigma; St. Louis, MO, USA) and 50 μL of 1 M sodium acetate, pH 4.0, followed by a 1-h incubation at 37°C. ..

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    Millipore dntps
    Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no <t>NTPs.</t> Positive control: <t>dNTPs</t> or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.
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    Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

    Journal: Nature Communications

    Article Title: A general strategy for expanding polymerase function by droplet microfluidics

    doi: 10.1038/ncomms11235

    Figure Lengend Snippet: Droplet-based optical polymerase sorting. ( a ) We have developed a fluorescent reporter system that produces an optical signal when a primer–template complex is extended to full-length product. The reporter consists of a primer–template complex (pink and green) containing a downstream fluorophore that is quenched when a DNA-quencher (black) anneals to the unextended region. ( b ) The assay was designed with a metastable probe to allow dissociation at elevated temperatures, where thermophilic polymerases function with optimal activity. Red arrow marks the maximium fluorescence observed in the absence of the quencher probe. ( c ) Flourophore (F)/quencher (Q) pairs were screened to identify a dye pair with the maximum signal-to-noise ratio. ( d ) Primer-extension analysis by denaturing PAGE (top) and fluorescence (bottom) for 9n and 9n-GLK polymerases using dNTP and NTP substrates. Negative control: no NTPs. Positive control: dNTPs or no DNA-quencher probe. ( e ) Single-emulsion droplets containing a functional 9n-GLK polymerase that extends a primer–template complex with RNA (top) and non-functional (bottom) wild-type 9n polymerase. The panel shows a cartoon depiction of the droplet, a bright-field micrograph of encapsulated E. coli (arrow), a fluorescence micrograph of the same field of view and an overlay of the two images. Scale bars, 10 μm. ( f ) Flow cytometry analysis of 9n and 9n-GLK polymerases following NTP extension in water-in-oil-in-water (w/o/w) droplets.

    Article Snippet: NTPs and dNTPs were purchased from Sigma (St Louis, MO). tNTPs were obtained by chemical synthesis as previously described .

    Techniques: Activity Assay, Fluorescence, Polyacrylamide Gel Electrophoresis, Negative Control, Positive Control, Functional Assay, Flow Cytometry, Cytometry

    Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”

    Journal: The Journal of Biological Chemistry

    Article Title: Initiation of New DNA Strands by the Herpes Simplex Virus-1 Primase-Helicase Complex and Either Herpes DNA Polymerase or Human DNA Polymerase ? *

    doi: 10.1074/jbc.M805476200

    Figure Lengend Snippet: Effects of increasing the dNTP concentration on primase-coupled polymerase activity. Assays contained primase-helicase, polymerase (UL30-UL42), 3′-d(T 20 GTCCT 36 )-5′, and either [α- 32 P]NTPs or NTPs and [α- 32 P]dNTPs to measure primase activity and primase-coupled polymerase activity, respectively. Coupled activity was measured in terms of pmol of dATP incorporated. The fraction of primers elongated was determined as described under “Experimental Procedures.”

    Article Snippet: Unlabeled NTPs and dNTPs were from Sigma, and radiolabeled NTPs and dNTPs were from PerkinElmer Life Sciences.

    Techniques: Concentration Assay, Activity Assay

    Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols on RT-qPCR . (A) The RT-qPCR yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike using 17 lysis conditions. Five nanograms of purified RNA was used in all RT reactions. Relative RT yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage relative to the water control for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Lysis conditions with Cq-values below that of the water control are RT enhancing agents, while conditions with higher Cq-values are inhibitory. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are shown in Table S4 in Supplementary Material. (B) Mean RT yield for Gapdh , Vim , Dll , and Jag1 . The relative transcript yield of each transcript was averaged and compared to the optimal RT-qPCR condition (RT mix). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: Directly lysed cells were incubated in 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo-dT (Metabion), and 2.5 μM random hexamers (Metabion) at 65°C for 5 min and then chilled on ice.

    Techniques: Lysis, Quantitative RT-PCR, Purification, Polymerase Chain Reaction

    Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Journal: Frontiers in Oncology

    Article Title: Direct Cell Lysis for Single-Cell Gene Expression Profiling

    doi: 10.3389/fonc.2013.00274

    Figure Lengend Snippet: Evaluation of direct cell lysis protocols . (A) The lysis yields of Gapdh , Vim , Dll1 , Jag1 , DNA, and RNA spike compared at 17 lysis conditions. Thirty-two astrocytes were sorted for each condition. Relative cDNA yields are presented as Cq-values on the left y -axis and relative transcript numbers on the right y -axis. The relative transcript number is expressed in percentage compared to the optimal lysis condition for each gene, assuming 100% RT efficiency and 100% PCR efficiency. Data are shown as mean ± SD ( n = 4). Missing data were excluded and are listed in Table S3 in Supplementary Material. (B) Mean cDNA yield of the transcripts. Expressions of Gapdh , Vim , Dll , and Jag1 were averaged and are compared to the overall optimal lysis condition (1 mg/ml BSA). Data are shown as mean ± SD ( n = 4). 7-deaz GTP, 7-deaza-2′ deoxyguanosine 5′ triphosphate lithium salt; GTC, guanidine thiocyanate; LPA, linear polyacrylamide; polyI, polyinosinic acid potassium salt; 2× RT buffer, 2× reverse transcription buffer; RT mix, 2× RT buffer, 5 μM random hexamers, 5 μM oligo-dT, and 1 mM dNTP.

    Article Snippet: Directly lysed cells were incubated in 0.5 mM dNTP (Sigma-Aldrich), 2.5 μM oligo-dT (Metabion), and 2.5 μM random hexamers (Metabion) at 65°C for 5 min and then chilled on ice.

    Techniques: Lysis, Polymerase Chain Reaction

    Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Journal: BMC Biochemistry

    Article Title: Enzymatic synthesis of long double-stranded DNA labeled with haloderivatives of nucleobases in a precisely pre-determined sequence

    doi: 10.1186/1471-2091-12-47

    Figure Lengend Snippet: Incorporation of double and single BrdU residues by Bst exo - DNA Polymerase into the 466 bp hybrid molecule . Incorporation reactions using BrdUTP alone or in combination with dTTP were carried out with Bst exo - DNA Polymerase. Lanes M, Perfect 100 bp Ladder (selected bands marked). Enzyme purity and reaction steps controls: lane 1, uncut 437 bp PCR fragment amplified from pGCN1 plasmid; lane 2, uncut 480 bp PCR fragment amplified from pGCN2 plasmid; lane 3, BsaI-cut 437 bp fragment; lane 4, BsaI-cut 480 bp fragment; lane 5, BsaI restriction fragment I (191 bp) filled in with BrdUTP isolated from agarose gel; lane 6, BsaI restriction fragment III (270 bp) filled in with BrdUTP isolated from agarose gel; lane 7, BsaI-cut 437 bp fragment, purified and back-ligated; lane 8, BsaI-cut 437 bp fragment, purified, incubated with Bst exo- DNA Pol without dNTPs and back-ligated. Incorporation reaction: lane 9, fragment I (191 bp) filled in with dTTP, ligated to BrdU-labeled fragment III (270 bp); lane 10, fragment I (191 bp) filled in with BrdUTP, ligated to BrdU-labeled fragment III (270 bp). I, III BsaI restriction fragments numbered as in Figure 1.

    Article Snippet: BrdUTP and dNTPs were from Sigma-Aldrich (St. Louis, MO, USA).

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Isolation, Agarose Gel Electrophoresis, Purification, Incubation, Labeling