Structured Review

Thermo Fisher dntp
Effects of the next complementary <t>dNTP</t> on DNase I protection and stable complex formation by <t>HIV-1</t> RT
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Images

1) Product Images from "Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet"

Article Title: Stable Complexes Formed by HIV-1 Reverse Transcriptase at Distinct Positions on the Primer-Template Controlled by Binding Deoxynucleoside Triphosphates or Foscarnet

Journal:

doi: 10.1016/j.jmb.2007.03.006

Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT
Figure Legend Snippet: Effects of the next complementary dNTP on DNase I protection and stable complex formation by HIV-1 RT

Techniques Used:

2) 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

3) Product Images from "Efficient in situ barcode sequencing using padlock probe-based BaristaSeq"

Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkx1206

Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).
Figure Legend Snippet: Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

Techniques Used: In Situ, Sequencing, Amplification, Infection, In Vitro

4) Product Images from "Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism"

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism

Journal: BMC Microbiology

doi: 10.1186/1471-2180-4-21

Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.
Figure Legend Snippet: Construction of DNA control plasmid designed for the 4 CDC Category A DNA agents (Smallpox virus [seq1], Bacillus anthracis [seq2], Francisella tularensis [seq3], and Yersinia pestis [seq4]). Assembling of the smallpox virus and B. anthracis sequences is presented as an example. Successive steps are indicated by framed numbers. 1, PCR amplification of the two matrix sequences by primers consisting of the stabilization and the restriction site sequences (italics). PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C. PCR products were electrophorezed in 3% TAE-agarose gel containing ethidium bromide and visualized under UV transillumination. Column purification of the PCR products. PCR products of the expected size were column-purified by using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, and eluted in 50 μl of RNase free distillated water. When two bands or more were observed by gel analysis, the band of expected size was excised from the gel and purified by glass milk extraction with the GenClean III Kit (Q-Bio-Gene, Carlsbad CA, USA). 2, assemblage was conducted by pair, seq1 with seq2 (resulting in seq1-2). Equal volumes (10 μl) of purified seq1- and seq2-dsDNA were incubated at 37°C in the presence of Sac I . Sac I site is located at the 3' and 5' ends of seq1 and seq2, respectively. 3, the reaction product was column purified using the protocol aforementioned to discard the 15-nt DNA fragments corresponding to the 5' and 3' ends to avoid their re-ligation to their respective complementary sequences at step 5. 4, Overnight incubation at 4°C in the presence of T4 DNA ligase. Ten μl of the reaction was incubated with T4 DNA ligase (Roche, Basel, Switzerland) according to the manufaturer's instructions. 5, PCR amplification by using the external primers (italics) was performed according to the protocol described at step 1. Then column purification using the protocol detailed at step 2 of the resulting PCR product. At this step the seq1-2 PCR product may be cloned into PGEM-T for storage. The same procedure was performed for seq3 and seq4. Ultimately, seq1-2 and seq3-4 were assembled by using the same protocol (sections 1–9). The final product cloned into PGEM-T plasmid includes seq1-2-3-4 flanked by the two Sseq and restriction sites.

Techniques Used: Plasmid Preparation, Polymerase Chain Reaction, Amplification, Size-exclusion Chromatography, Agarose Gel Electrophoresis, Purification, Incubation, Ligation, Clone Assay

5) Product Images from "Mechanical properties of DNA-like polymers"

Article Title: Mechanical properties of DNA-like polymers

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkt808

Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

6) Product Images from "Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline"

Article Title: Molecular detection and species identification ofAlexandrium (Dinophyceae) causing harmful algal blooms along the Chilean coastline

Journal: AoB Plants

doi: 10.1093/aobpla/pls033

Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Amplification of local Alexandrium strains ACC01, ACC02 and ACC07 using species - specific primers; A. tamarense (lanes 1, 2 and 3) and A. catenella (lanes 4, 5 and 6). M = 100-bp DNA size marker. Species-specific amplification in the rDNA region using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller™ 100-bp DNA ladder was used for size estimation of amplified fragments.

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

Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.
Figure Legend Snippet: Alexandrium tamarense microsatellite amplifications of the three local strains with (A) specific primers ATB8 (lanes 1, 2 and 3) and ATD8 (lanes 4, 5 and 6). Lanes 7 and 8 correspond to controls with primers ATB8 without DNA. (B) Specific amplification with primers ATB1 (lanes 1, 2 and 3) and ATF11 (lanes 6, 7 and 8). Lanes 4–5 and 9–10 are controls without DNA for primer sets ATB1 and ATF11, respectively. M = 100-bp DNA size marker. Species-specific microsatellite amplifications using A. catenella and A. tamarense primers were carried out in a MaxiGene Gradiente thermocycler (Axygen) in 1× PCR buffer, 20–50 ng of genomic DNA template, 3 mM MgCl 2, 100 µM each dNTP, 0.1 µM each primer and 0.4 U of TopTaq DNA polymerase (Fermentas) in a 10-µL reaction volume. Five microlitres of each PCR product were analysed in a 2 % agarose gel. A Fermentas GeneRuller TM 100-bp DNA ladder was used for size estimation of amplified fragments.

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

7) Product Images from "Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *"

Article Title: Conformational Changes during Nucleotide Selection by Sulfolobus solfataricus DNA Polymerase Dpo4 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M109.009506

The F-helix exhibits reduced HDX rates during ternary complex formation that are independent of dNTP identity. A , the kinetic profile for HDX in residues 151–170 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP
Figure Legend Snippet: The F-helix exhibits reduced HDX rates during ternary complex formation that are independent of dNTP identity. A , the kinetic profile for HDX in residues 151–170 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP

Techniques Used:

Changes in HDX kinetics for the thumb domain are sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 180–202 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles
Figure Legend Snippet: Changes in HDX kinetics for the thumb domain are sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 180–202 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles

Techniques Used:

The kinetics of HDX in the finger domain is sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 48–63 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles ), +DNA/Mg
Figure Legend Snippet: The kinetics of HDX in the finger domain is sensitive to the identity of the incoming dNTP. A , the kinetic profile for HDX in residues 48–63 is plotted for Dpo4 alone ( black circles ), +DNA/Mg 2+ ( blue squares ), +DNA/Mg 2+ /dCTP ( red circles ), +DNA/Mg

Techniques Used:

Overview of Changes in HDX as a Function of Primer/Template DNA, MgCl2 , and Incoming dNTP
Figure Legend Snippet: Overview of Changes in HDX as a Function of Primer/Template DNA, MgCl2 , and Incoming dNTP

Techniques Used:

8) Product Images from "Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa"

Article Title: Non-cognate DNA damage prevents formation of active conformation of Y-family DNA polymerases DinB and pol kappa

Journal: The FEBS journal

doi: 10.1111/febs.13304

Proposed reaction pathway for DinB and pol κ. The free pol binds DNA which results in a relatively closed and stable binary complex. In the case of correct dNTP incorporation opposite preferred DNA bases, there is a conserved conformational change
Figure Legend Snippet: Proposed reaction pathway for DinB and pol κ. The free pol binds DNA which results in a relatively closed and stable binary complex. In the case of correct dNTP incorporation opposite preferred DNA bases, there is a conserved conformational change

Techniques Used:

DinB and pol κ are protected from exchange in the presence of undamaged DNA and the correct dNTP. (a) Minimal model for nucleotide incorporation. The free pol is denoted E, the binary pol-DNA complex ED, and the ternary pol-DNA-dNTP complex EDN.
Figure Legend Snippet: DinB and pol κ are protected from exchange in the presence of undamaged DNA and the correct dNTP. (a) Minimal model for nucleotide incorporation. The free pol is denoted E, the binary pol-DNA complex ED, and the ternary pol-DNA-dNTP complex EDN.

Techniques Used:

9) Product Images from "Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions"

Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions

Journal: The American Journal of Pathology

doi:

AmpliSeq. A: Theoretical AmpliSeq reaction. Because of supplemental dNTPs, PCR amplification is supported during initial cycles of the reaction. As PCR occurs, dNTPs are consumed resulting in an increase in the ddNTP/dNTP ratio. This causes the reaction to convert itself to cycle sequencing in latter cycles. B: Anticipated PCR product generated during AmpliSeq. Forward and reverse primer sequences are shown in dark blue and the rest of the PCR product in light blue. In the reverse primer, dots denote the abasic region and stripes , the nontemplated thymidines. Yellow highlight designates the mutation site ( asterisk ). C: Bidirectional AmpliSeq results of a factor V wild-type homozygote. D: Unidirectional AmpliSeq of a factor V wild-type homozygote. Yellow highlighting indicates the potential mutation site in sequencing products.
Figure Legend Snippet: AmpliSeq. A: Theoretical AmpliSeq reaction. Because of supplemental dNTPs, PCR amplification is supported during initial cycles of the reaction. As PCR occurs, dNTPs are consumed resulting in an increase in the ddNTP/dNTP ratio. This causes the reaction to convert itself to cycle sequencing in latter cycles. B: Anticipated PCR product generated during AmpliSeq. Forward and reverse primer sequences are shown in dark blue and the rest of the PCR product in light blue. In the reverse primer, dots denote the abasic region and stripes , the nontemplated thymidines. Yellow highlight designates the mutation site ( asterisk ). C: Bidirectional AmpliSeq results of a factor V wild-type homozygote. D: Unidirectional AmpliSeq of a factor V wild-type homozygote. Yellow highlighting indicates the potential mutation site in sequencing products.

Techniques Used: Polymerase Chain Reaction, Amplification, Sequencing, Generated, Mutagenesis

Related Articles

Amplification:

Article Title: Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods ▿ Gene Expression in Foods ▿ †
Article Snippet: .. The 50-μl PCR mixture (10 ng DNA, 0.5 μM each primer, 1.5 mM MgCl2 , 0.4 mM each deoxynucleoside triphosphate [dNTP], 1.25 U ThermoStart Taq polymerase [all from ABgene]) was activated (95°C for 15 min), followed by 30 amplification cycles (95°C for 30 s, 60°C for 45 s, and 72°C for 1 min) and an elongation step (72°C for 5 min). .. Total DNA was isolated as described previously , and plasmid DNA was prepared using standard procedures.

Spectrophotometry:

Article Title: A novel dengue virus detection method that couples DNAzyme and gold nanoparticle approaches
Article Snippet: .. The stability of DDZ-M-AuNP was tested against increasing concentrations of MgCl2 (0 mM to 20 mM) at room temperature every 5 minutes for up to 30 minutes (Figure ), and absorbencies were measured with a NanoDrop spectrophotometer at 520 nm. ..

SYBR Green Assay:

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
Article Snippet: .. Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl. ..

Concentration Assay:

Article Title: Association of COX-2 Promoter Polymorphisms -765G/C and -1195A/G with Migraine
Article Snippet: .. Therefore, the final volume of 25 μl PCR reactions in 0.2 ml tubes containing 500 ng/μl of DNA template, 2 mM of MgCl2 concentration, 2 mM dNTPs, 10 pmol/μl of each primer, 5 μl of 10X PCR buffer and 1 U of Taq DNA polymerase (Fermentas, Germany) was performed. .. Thermal PCR conditions consisted of denaturation phase for 5 min at 95 °C, followed by 30 cycles of 94 °C for 1 min, temperature of 59 °C for COX-2-1195A>G (rs689466) primer and 56 °C for COX-2-765G>C (rs20417) primer for 1 min, and 72 °C for 1 min, with a final extension for 5 min at 72 °C.

Incubation:

Article Title: A unique insertion in STARD9's motor domain regulates its stability
Article Snippet: .. After 3 h, a master mix containing all the ubiquitination components (ubiquitin [Enzo Life Sciences], ROC1, E1, E2, Skp1, with or without β-TrCP, with or without CUL1, in a buffer containing 20 mM HEPES, 5 mM NaCl, 5 mM MgCl2 , DTT, MG132, and protease and phosphatase inhibitor cocktail (Thermo Scientific) and ATP regeneration system was added to the tubes and further incubated for 90 min at 30°C. ..

Polymerase Chain Reaction:

Article Title: Identification of the Main Promoter Directing Cereulide Biosynthesis in Emetic Bacillus cereus and Its Application for Real-Time Monitoring of ces Gene Expression in Foods ▿ Gene Expression in Foods ▿ †
Article Snippet: .. The 50-μl PCR mixture (10 ng DNA, 0.5 μM each primer, 1.5 mM MgCl2 , 0.4 mM each deoxynucleoside triphosphate [dNTP], 1.25 U ThermoStart Taq polymerase [all from ABgene]) was activated (95°C for 15 min), followed by 30 amplification cycles (95°C for 30 s, 60°C for 45 s, and 72°C for 1 min) and an elongation step (72°C for 5 min). .. Total DNA was isolated as described previously , and plasmid DNA was prepared using standard procedures.

Article Title: Association of COX-2 Promoter Polymorphisms -765G/C and -1195A/G with Migraine
Article Snippet: .. Therefore, the final volume of 25 μl PCR reactions in 0.2 ml tubes containing 500 ng/μl of DNA template, 2 mM of MgCl2 concentration, 2 mM dNTPs, 10 pmol/μl of each primer, 5 μl of 10X PCR buffer and 1 U of Taq DNA polymerase (Fermentas, Germany) was performed. .. Thermal PCR conditions consisted of denaturation phase for 5 min at 95 °C, followed by 30 cycles of 94 °C for 1 min, temperature of 59 °C for COX-2-1195A>G (rs689466) primer and 56 °C for COX-2-765G>C (rs20417) primer for 1 min, and 72 °C for 1 min, with a final extension for 5 min at 72 °C.

Article Title: Rapid and simple comparison of messenger RNA levels using real-time PCR
Article Snippet: .. Each sample consisted of: 50 ng cDNA, 3 mM MgCl2 , 200 μM dNTP, 500 nM of primers, 2 μl of 10X PCR buffer, 0.1 unit of Taq polymerase and SYBR® Green (Molecular Probe, Eugene, OR; 1/30 000 dilution), in a reaction volume of 20 μl. ..

Article Title: Rapid, Quantitative PCR Monitoring of Growth of Clostridium botulinum Type E in Modified-Atmosphere-Packaged Fish
Article Snippet: .. Reaction volumes (50 μl) for the PCR consisted of 5 μl of template DNA; 5.0 mM MgCl2 ; 5 μl of 10×TaqMan buffer A; 200 nM each primer; 200 μM each dATP, dCTP, and dGTP; 400 μM dUTP; 1.25 U of AmpliTaq Gold DNA polymerase (Applied Biosystems); 0.5 U of uracil- N -glycosidase (AmpErase UNG; Applied Biosystems); and 100 nM TaqMan probe. .. Amplification and detection were performed with the ABI PRISM 7700 sequence detector (Applied Biosystems).

Article Title: Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism
Article Snippet: .. PCR reactions were carried out in a volume of 50 μl that included 10 mM Tris-HCl [pH 9.0], 1.5 mM MgCl2, 50 mM KCl, 0.1% Triton X-100, 200 μM each dNTP, 0.4 μM of each oligonucleotide primer, 0.4 μM of the single stranded DNA, and 1.5 U of Taq DNA polymerase (Invitrogen, Cergy-Pontoise, France). .. The thermocycler (Biometra, Göttingen, Germany) profile was 5 min at 95°C, followed by 35 cycles of 30 sec at 95°C, 30 sec at 55°C, and 1 min at 72°C, and terminated by a final extension for 7 min at 72°C.

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  • 92
    Thermo Fisher dntp
    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ <t>cDNA)</t> or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated <t>dNTP</t> concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).
    Dntp, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 5312 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 92 stars, based on 5312 article reviews
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    dntp - by Bioz Stars, 2020-08
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    90
    Thermo Fisher fluorescein labelled dntp s
    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of <t>dNTP’s</t> on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .
    Fluorescein Labelled Dntp S, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fluorescein labelled dntp s/product/Thermo Fisher
    Average 90 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    fluorescein labelled dntp s - by Bioz Stars, 2020-08
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    Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Journal: Nucleic Acids Research

    Article Title: Efficient in situ barcode sequencing using padlock probe-based BaristaSeq

    doi: 10.1093/nar/gkx1206

    Figure Lengend Snippet: Optimization of gap-filling in in situ barcode sequencing. ( A ) Illustrations of normal (top) and aberrant gap-filling products caused by insufficient gap-filling (middle) and overextension (bottom). ( B ) In situ barcode amplification in infected BHK cells with padlock probes that require (gap) or do not require (no gap) gap-filling using the indicated polymerases. Scale bars = 20 μm. ( C ) In vitro gap-filling assay performed on padlocks that require (+ gap) or do not require (– gap) gap-filling using no polymerase (–), the Stoffel fragment (S) or Phusion DNA polymerase (P) in the presence (+ cDNA) or absence (– cDNA) of a cDNA template. Arrows on the left, from top to bottom, indicate positions for strand displaced gap-filling product, correct gap-filling product for ‘+ Gap’ padlock, pre-gapfilling padlock, and the cDNA template. ( D ) The means and SEMs of the fraction of the correct gap-filling product using the gap padlock (top, black bars) and the fraction of over-extended products using the no-gap padlock (bottom, white bars) with the indicated polymerases. N = 3 for all enzymes. ( E – G ) The means and SEMs of the faction of correct gap-filling product using the gap padlock (top) and the fraction of over-extended products (bottom) using either the gap padlock (solid lines) or the no-gap padlock (dashed lines) with either Phusion DNA polymerase (black dots) or the Stoffel fragment (white dots). The reactions were performed with the indicated dNTP concentrations (E), with the indicated enzyme concentrations (F), or at the indicated temperature (G). N = 3 for each condition in (E) and (F) and N = 4 for each condition in (G).

    Article Snippet: In vitro gap-filling assay In vitro gap-filling assays were performed in 1X Ampligase buffer (Epicentre) with 10 nM padlock probes (XC1149 and XC1151) and 10 nM cDNA template (XC1498), 20 μM dNTP, 0.012 U/μl Phusion High-Fidelity DNA Polymerase (Thermo Fisher Scientific) or Stoffel fragment (DNA Gdansk), additional 50 mM KCl, 20% formamide, and glycerol to a final concentration of 10%.

    Techniques: In Situ, Sequencing, Amplification, Infection, In Vitro

    Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Journal: Scientific Reports

    Article Title: Phosphoproteomic comparison of Pik3ca and Pten signalling identifies the nucleotidase NT5C as a novel AKT substrate

    doi: 10.1038/srep39985

    Figure Lengend Snippet: Impact of S184 phosphorylation of NT5C catalytic activity. ( a ) S184 phosphorylation does not regulate NT5C nucleotidase activity in vitro . (left) Immunoprecipitates of Flag-NT5C ectopically expressed in HEK293 cells were incubated with 5 mM of the indicated nucleotides. Phosphate release was measured using a malachite green colorimetric assay and expressed as a percent of total nucleotide. The experiment was performed in duplicate and repeated 3 times independently. Error bars are sem. (right) Representative immunoblot from experimental cells. ( b ) Cells expressing Pik3ca H1047R have elevated dNTP levels. (left, middle) Nucleotides were extracted from primary MEFs and analysed by UPLC-MS/MS. The experiment was performed in triplicate and repeated 4 times independently. Error bars are sem, *p

    Article Snippet: dNTP Quantification by UPLC-MS/MS Chromatography and Mass Spectrometry Method Analytes were resolved using an ultra-performance liquid chromatography system (Accela UPLC, Thermo Scientific, UK) equipped with a Biobasic AX 5 μm, 100 × 2.1 mm column (Thermo Electron Corporation, Murrieta, CA, USA) and a mobile phase consisting of a mixture of 10 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 6.0 (buffer A), and 1 mM NH4 Ac in ACN/H2 O (30:70 v/v), pH 10.5 (buffer B).

    Techniques: Activity Assay, In Vitro, Incubation, Colorimetric Assay, Expressing, Mass Spectrometry

    Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: Determination of the dNTP and NTP concentrations in actively dividing and quiescent mouse Balb/3T3 fibroblasts. ( A ) Flow cytometry histograms of actively dividing and quiescent cells. The percent of cells in each cell cycle phase is shown above the peaks. ( B ) Amounts of dNTPs in actively dividing and quiescent cells presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( C ) Amounts of NTPs in actively dividing and quiescent cells, presented as the mean ± SEM measured in three independent Balb/3T3 cell extracts. ( D ) Cell volumes of actively dividing and quiescent cells. The horizontal lines indicate the mean ± SEM.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: Flow Cytometry, Cytometry

    ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Journal: Nucleic Acids Research

    Article Title: Simultaneous determination of ribonucleoside and deoxyribonucleoside triphosphates in biological samples by hydrophilic interaction liquid chromatography coupled with tandem mass spectrometry

    doi: 10.1093/nar/gky203

    Figure Lengend Snippet: ZIC-cHILIC-HPLC multiple reaction monitoring (MRM) chromatograms of all dNTP and NTP analytes in a standard mixture solution and in extracts from Balb/3T3 fibroblasts and their 13 C 15 N-labeled internal standards. The MRM transitions are shown for each of the target analytes.

    Article Snippet: Chemicals dNTP and NTP standards, including 2′-deoxyadenosine 5′-triphosphate (dATP); 2′-deoxythymidine 5′-triphosphate (dTTP); 2′-deoxyguanosine 5′-triphosphate (dGTP); 2′-deoxycytidine 5′-triphosphate (dCTP); adenosine 5′-triphosphate (ATP); uridine 5′-triphosphate (UTP); guanosine 5′- triphosphate (GTP); and cytidine 5′-triphosphate (CTP) were from Thermo Fisher Scientific.

    Techniques: High Performance Liquid Chromatography, Labeling

    R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: R18 is crucial for nucleotide recruitment, reverse transcription and infectivity. a , Superposed monomers of R18G (light-pink) and wild-type (light-green) CA Hexamer . b , Binding of capsid variants to dCTP as measured by fluorescence anisotropy. c , DSF stability measurements expressed as T m for WT and R18G ± DTT. d , DSF measurements of the effect of dNTP’s on the stability of WT and R18G expressed as ΔT m relative to unbound. e , Fluorescence anisotropy titrations of dTTP-binding by chimeric CA Hexamers with different R:G ratios at position 18. f , Comparison of infectivity and reverse transcription of chimeric viruses. g , h , Correlation between HIV-1 capsid dTTP affinity, viral infectivity g and reverse transcription h .

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Infection, Binding Assay, Fluorescence

    The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: The HIV-1 capsid pore is strongly electropositive and recruits dNTP’s with rapid association and dissociation kinetics. a , Model of an HIV-1 virion with hexamers in an open conformation reveals that the capsid is porous. Surface electrostatic potential shows that the pores are highly electropositive. b , Cross sections through the closed (β-hairpin green) and open (β-hairpin pink) CA Hexamer showing a central chamber that is accessible in the open state. R18 (cyan) creates a bottleneck at the base of the chamber underneath the β-hairpin. c , Fluorescence anisotropy measurements of dNTP’s binding to CA Hexamer . d , Example of pre-steady state association kinetics of dCTP with CA Hexamer . e , Apparent rate constant (k app ) at increasing CA Hexamer concentrations. f , Dissociation of unlabeled dCTP:CA Hexamer by excess fluorescent-dCTP. g , R18 co-ordinates the phosphates in a dATP-bound CA Hexamer structure.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Fluorescence, Binding Assay

    ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Journal: Nature

    Article Title: HIV-1 uses dynamic capsid pores to import nucleotides and fuel encapsidated DNA synthesis

    doi: 10.1038/nature19098

    Figure Lengend Snippet: ERT assay. a , HIV-1 cores were prepared by ultracentrifugation through a Triton X-100 layer over a sucrose gradient. Resulting fractions were subjected to ELISA for p24 and fractions 3 – 7 were pooled for further experiments. b , Endogenous RT activity for strong stop in the presence of DNase I using HIV-1 fractions that were prepared with or without the Triton X-100 spin-through layer. Input levels of p24 were normalized between reactions. c , dNTP’s were added to HIV-1 cores prepared by Triton X-100 spin-through in the presence of DNase I. Reactions were stopped at the indicated time point by shifting to -80° C and levels of strong stop were quantified. d , Levels of strong-stop (RU5), first-strand transfer (1ST) and second-strand transfer (2ST) DNA after overnight incubation of HIV-1 cores with or without dNTP’s in the presence of DNase I. e , Levels of naked HIV-1 DNA genomes untreated or incubated overnight with DNase I or Benzonase. f , Effect of carboxybenzene compounds on recombinant reverse transcriptase activity.

    Article Snippet: It was found that the triphosphate was not stable over the timescale of the competition binding experiments; so fluorescein-labelled dNTP’s were substituted for a non-hydrolysable BODIPY-labelled GTP-γ-S (ThermoFisher Scientific).

    Techniques: Enzyme-linked Immunosorbent Assay, Activity Assay, Incubation, Recombinant