Journal: Nucleic Acids Research

Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

doi: 10.1093/nar/gks316

Figure Lengend Snippet: Sensitive detection of purified DNA polymerase using DPE-PCR. ( A ) A commercial source of DNA polymerase I was assayed in duplicate at 10-fold increments starting at 2 × 10 −5 U down to 2 × 10 −11 U per reaction. A representative DPE-PCR curve is shown for each polymerase input level and NIC. ( B ) A plot was constructed from n = 4 data points per polymerase input level, taken from two independent experiments and linear regression analysis was performed. ( C ) Triplicate reactions containing 2 × 10 −7 U of DNA polymerase I, Klenow, Klenow (exo−) and E. coli DNA Ligase were assayed in comparison to an NIC. A representative DPE-PCR curve is presented for each of the assayed enzymes and NIC. ( D ) Triplicate DPE-PCR curves are shown from corresponding DPE reactions containing a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with 50 µM of either dCTP or ddCTP. A schematic representing some of the first available sites for dCTP or ddCTP incorporation within the DNA substrate is presented adjacent to the DPE-PCR curves.

Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

Techniques: Purification, Polymerase Chain Reaction, Construct

Journal: Nucleic Acids Research

Article Title: Characterization of a novel DNA polymerase activity assay enabling sensitive, quantitative and universal detection of viable microbes

doi: 10.1093/nar/gks316

Figure Lengend Snippet: Detection of bacteria by DPE-PCR is blocked by ddCTP and rescued with dCTP. ( A ) E. coli suspensions were added to bead lysis-coupled DNA polymerase assays composed of a 50 µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM ddCTP. DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the upper left region of the qPCR graph ( B ) E. coli suspensions were added to bead lysis tubes containing 50 µl reaction buffer with 50-µM (dATP, dGTP, dTTP, ddCTP). Prior to lysis, 1 µl of dCTP (2.5, 0.25, 0.025 and 0.0025 mM) was added to selected ddCTP-containing reactions. Reactions containing 50 µM (dATP, dGTP, dTTP, dCTP) alone or 50 µM (dATP, dGTP, dTTP, ddCTP) alone were run in parallel as ‘non-terminated’ and ‘terminated’ comparators. The resultant DPE-PCR curves representing E. coli -derived DNA polymerase activity is presented. Approximate colony forming unit input as determined by plating is presented in the lower left region of the qPCR graph. ( C ) Escherichia coli gene-specific PCR was also performed on the same lysates used for DNA polymerase detection presented in Figure 2 B. Linear plots of dCTP-dependent rescue of bacterial DNA polymerase detection versus gsPCR of genomic DNA are shown. Plots were generated using the average qPCR C t values from triplicate reactions at the indicated conditions. ( D–F ) ddCTP termination and dCTP rescue experiments were performed for S. aureus exactly as described above for E. coli .

Article Snippet: Termination of purified DPE activity with dideoxyCTP DPE reactions were prepared as described above with a 50 -µM (dATP, dGTP, dTTP) mixture supplemented with either 50 µM dCTP or 50 µM dideoxyCTP (ddCTP) (Affymetrix cat# 77332).

Techniques: Polymerase Chain Reaction, Lysis, Derivative Assay, Activity Assay, Real-time Polymerase Chain Reaction, Generated

Journal: Biochemistry

Article Title: Following DNA chain extension and protein conformational changes in crystals of a Y-family DNA polymerase by Raman crystallography

doi: 10.1021/bi400524h

Figure Lengend Snippet: The Raman spectra of deoxycytidine triphosphate. A) Raman spectrum of 100 mM 2′-deoxycytidine- 13 C 9 , 15 N 3 , (labeled deoxycytidine) 5′-triphosphate (d*CTP) in solution, pH 7.0, in a hanging drop. B) Raman spectrum of 100 mM 2′-deoxycytidine

Article Snippet: 2′-Deoxycytidine 5′-triphosphate (dCTP), thymidine 5′-triphosphate, and 15 N and 13 C labeled dCTP (d*CTP) were purchased from Sigma-Aldrich.

Techniques: Labeling

Journal: Biochemistry

Article Title: The Mechanism and Dynamics of Translesion DNA Synthesis Catalyzed by the Escherichia coli Klenow fragment

doi: 10.1021/bi800324r

Figure Lengend Snippet: (A) Structures of 2' deoxynucleoside triphosphates used or referred to in this study are dATP, dCTP, dGTP, dTTP, Ind-TP, 5-FITP, 5-AITP, 5-NITP, 5-PhITP, 5-CE-ITP, 5-CH-ITP, and 5-NapITP. For convenience, dR is used to represent the 2'-deoxyribose 5'-triphosphate portion of the nucleotides. (B) Defined DNA substrates used for kinetic analysis. “X” in the template strand denotes T or the presence of a tetrahydrofuran moiety that mimics an abasic site.

Article Snippet: Oligonucleotides, including those containing a tetrahydrofuran moiety mimicking an abasic site, were synthesized by Operon Technologies (Alameda, CA). dATP, dCTP, dGTP, and dTTP were obtained from Sigma in greater than 99% purity.

Techniques:

Journal: Nucleic Acids Research

Article Title: A strategically located serine residue is critical for the mutator activity of DNA polymerase IV from Escherichia coli

doi: 10.1093/nar/gkt146

Figure Lengend Snippet: Fidelity profile of wt PolIV and the mutant Ser42Ala. The results for primer extension assay wherein wtPolIV ( A ) or the Ser42Ala mutant ( B ) was incubated with DNA duplexes (T A P*, T T P*, T G P* and T C P*) and different dNTPs are displayed. (B) 3D plot exhibiting the misincorporation profile of wtPolIV and the mutant. Incorporation was quantitated in the form of percentage of primer extended. The level of incorporation of the incorrect nucleotide is plotted on the z -axis for different template nucleotides (dA, dT, dG and dC; y -axis), for all four incoming nucleotides (dATP, dTTP, dGTP and dCTP; x -axis) for the two proteins (wtPolIV and Ser42Ala; x -axis).

Article Snippet: In the case of chain terminated DNA, dCTP (GE Amersham) was added to a final concentration of 5 mM.

Techniques: Mutagenesis, Primer Extension Assay, Incubation

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Infectivity analysis of SLCMV-[SeM] partial dimers in N. benthamiana plants. ( a ) Symptoms displayed by N. benthamiana plants agroinoculated with the SLCMV-[SeM] partial dimers. Bottom half shows individual leaves of the tested plants. ( b ) Southern blot analysis using SLCMV-[SeM] DNA A (without common region, ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-SLCMV-Ma-A digested with PstI (50 pg and 250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled SLCMV-[SeM] DNA B (ΔCR) as the probe. The plasmid pBS-SLCMV-Ma-B digested with BamHI (50 pg and 250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected plant (C), plant mock infected with the Agrobacterium tumefaciens strain Ach5 (Mock), plant agroinoculated with partial dimers of DNA A alone (A), DNA B alone (B) and two plants independently co-agroinoculated with the partial dimers of DNA A + DNA B (A + B) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Infection, Southern Blot, Labeling, Plasmid Preparation, Positive Control, Staining, Molecular Weight

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Sri Lankan cassava mosaic virus (SLCMV) DNA A and small interfering RNA (siRNA) analysis in symptomatic and symptom-recovered cassava plants. ( a ) Southern blot analysis of field-infected cassava plants maintained in the greenhouse. DNA (1 μg) samples from a virus-free, axenic (tissue culture-grown) cassava plant (C), and field-infected cassava (cultivar Sengutchi) plants collected from Malappuram (M) and Thiruvananthapuram (T) were analyzed. The field-infected cassava plants were established in the Madurai Kamaraj University (MKU) greenhouse and the DNA was extracted during first year (M1 and T1), second year (M2 and T2) and third year (M3 and T3) of establishment in the greenhouse ( Figure 1 ). pBS-SLCMV-Ma-A plasmid (50 pg and 250 pg) digested with PstI was used as a positive control. [α- 32 P]dCTP-labeled full-length SLCMV-[SeM] DNA A was used as the probe. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom panel. ( b ) Diagnostic multiplex PCR using SLCMV- and ICMV-specific primers to analyze mixed infection in the plant sample T1 (field-infected SLCMV-SeT1, symptomatic plant) and TR (plant which initially recovered and subsequently showed re-emergence of CMD symptoms in the Basel greenhouse) ( Figure 1 ). Genomic DNA and RCA-amplified DNA from T1 and TR were used as templates for polymerase chain reaction (PCR) analysis. Amplified fragments of SLCMV (615bp) and Indian cassava mosaic virus (ICMV) (386bp) are marked. M, Molecular weight marker. ( c ) Small RNA Northern blot analysis of field-infected cassava plants. RNA (10 μg) from field-infected cassava plants collected from Malappuram (M3) and Thiruvananthapuram (T3) were analyzed. The field-infected cassava plants were established in the MKU greenhouse and RNA was extracted after 25 months of initial establishment in the greenhouse (M3 and T3). [α- 32 P]dCTP-labeled SLCMV-[SeM] AC1 (1 kb) gene fragment was used as the probe. The position of siRNA (21 nt) is marked. The bottom panel shows ethidium bromide stained tRNA, as a loading control. ( d ) The proportion of the plant genome (cassava)- and the viral genome (DNA A + DNA B)-derived small RNAs in a total population of 20–25nt small RNAs accumulating in the Sengutchi cassava plants “SeM-SLCMV”, “SeT-recovered” and “SeT-ICMV” ( Figure 1 ).

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Small Interfering RNA, Southern Blot, Infection, Plasmid Preparation, Positive Control, Labeling, Staining, Molecular Weight, Diagnostic Assay, Multiplex Assay, Polymerase Chain Reaction, Amplification, Marker, Northern Blot, Derivative Assay

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Pseudo-recombination in N. benthamiana with SLCMV-[SeM] DNA A and ICMV-[SeT4] DNA B. ( a ) Symptoms in N. benthamiana mock inoculated plants (Mock) and plants agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + DNA B, ICMV-[SeT4] DNA A + DNA B and SLCMV-[SeM] DNA A + ICMV-[SeT4] DNA B. ( b ) Southern blot analysis using SLCMV-[SeM] DNA A (ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-SLCMV-Ma-A digested with PstI (50 pg and 250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled SLCMV-[SeM] DNA B (ΔCR) as the probe. The plasmid pBS-SLCMV-Ma-B digested with BamHI (250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected N. benthamiana plant (C), plant mock infected with the A. tumefaciens strain Ach5 (Mock), plant co-agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + DNA B (SA + SB), plant co-agroinoculated with the partial dimers of ICMV-[SeT4] DNA A + DNA B (IA + IB) and plants co-agroinoculated with the partial dimers of SLCMV-[SeM] DNA A + ICMV-[SeT4] DNA B (SA + IB) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Southern Blot, Labeling, Plasmid Preparation, Positive Control, Infection, IA, Staining, Molecular Weight

Journal: Viruses

Article Title: Emergence of a Latent Indian Cassava Mosaic Virus from Cassava Which Recovered from Infection by a Non-Persistent Sri Lankan Cassava Mosaic Virus

doi: 10.3390/v8100264

Figure Lengend Snippet: Infectivity analysis of ICMV-[SeT4] in N. benthamiana plants. ( a ) Symptoms displayed by N. benthamiana plants agroinoculated with the partial dimers of ICMV-[SeT4]. Bottom half shows individual leaves of the tested plants. ( b ) Southern blot analysis using ICMV-[SeT4] DNA A (ΔCR) labeled with [α- 32 P]dCTP as the probe. The plasmid pBS-ICMV-Tv-A digested with PstI (250 pg) was used as the positive control. ( c ) Southern blot analysis using [α- 32 P]dCTP-labeled ICMV-[SeT4] DNA B (ΔCR) as the probe. The plasmid pBS-ICMV-Tv-B digested with BamHI (50 pg and 250 pg) was used as the positive control. ( b , c ) DNA (1 μg) from uninfected plant (C), plant mock infected with the A. tumefaciens strain Ach5 (Mock), plant agroinoculated with the partial dimers of DNA A alone (A), DNA B alone (B) and two plants independently co-agroinoculated with the partial dimers of DNA A + DNA B (A + B) were loaded in the respective lanes. Positions of different forms of viral DNA, single stranded ( ss ), super-coiled ( sc ), open circular ( oc ) and linear ( lin ), are marked. Ethidium bromide stained high molecular weight plant DNA is shown as loading control at the bottom.

Article Snippet: The probe DNA was labeled with [α-32 P] deoxycytidine triphosphate (dCTP) using the Megaprime DNA labeling system (GE Healthcare UK Ltd.).

Techniques: Infection, Southern Blot, Labeling, Plasmid Preparation, Positive Control, Staining, Molecular Weight

Journal: Leukemia

Article Title: AZA-MS: a novel multiparameter mass spectrometry method to determine the intracellular dynamics of azacitidine therapy in vivo

doi: 10.1038/leu.2017.340

Figure Lengend Snippet: Development of a quantitative high-resolution mass spectrometry to directly measure intracellular 5-AZA-CdR and AZA. ( a ) Chemical structures of cytidine, deoxycytidine, AZA and 5-AZA-CdR. ( b ) Schematic depicting intracellular metabolism of AZA. Following cellular uptake and phosphorylation, ~80% of AZA gets incorporated into RNA by RNA polymerases. The remaining fraction is converted into 5-AZA-CdR by ribonucleotide reductase and incorporated into DNA by DNA polymerases. ( c ) Representative high-resolution Orbitrap mass spectra at RT of 0.98 min, showing clear baseline separation between 5-AZA-CdR, 15N-dC and 13C-dC (left, with respective m/z values) despite their identical chromatographic retention times (right). ( d ) Schematic depicting the optimised method incorporating steps to improve sensitivity of detection of intracellular 5-AZA-CdR and AZA by LC–MS.

Article Snippet: Calibration standard mixes containing all analytes (AZA, 5-AZA-CdR, C, dC, mC and mdC) were prepared by dilution in CE buffer of the following chemicals: 5-azacytidine and decitabine (Selleckchem, Houston, TX, USA), cytidine triphosphate and deoxycytidine triphosphate (Promega, Madison, WI, USA) and 5-methylcytidine and 5-methyl-2′-deoxycytidine (MP Biomedicals, Irvine, CA, USA).

Techniques: Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy

Journal: BMC Genomics

Article Title: A streamlined tethered chromosome conformation capture protocol

doi: 10.1186/s12864-016-2596-3

Figure Lengend Snippet: Overview of RTCC protocol. a Diagram shows a schematic description of steps from a crude tissue homogenate to a proximity sequencing library (details provided in the Methods section). For our studies (using C. elegans ), animals flash-frozen in liquid nitrogen were finely ground using either mortar and pestle or using an electric drill with “Cellcrusher” drill-bit and “Cellcrusher” base held at liquid nitrogen temperature and treated with formaldehyde to covalently cross-link proteins to each other and to DNA (red and purple strands, threaded through the blue amorphous complex, representing proteins). (1) Chromatin is solubilized with detergent and proteins were non-specifically biotinylated (orange balls on sticks). (2) DNA was digested with a restriction enzyme that generates 5’ overhangs. (3) Cross-linked complexes were immobilized at a very low density on the surface of streptavidin-coated magnetic beads (grey color arc) through the biotinylated proteins, while the non-cross-linked DNA fragments were removed. (4) 5′ overhangs were filled in using DNA polymerase and a nucleotide mixture containing biotin-14-dCTP (orange balls on sticks) to generate blunt ends. (5) Blunt DNA ends were ligated. (6) Cross-linking was reversed and DNA was purified. (7) The DNA was fragmented and tagged (light blue strands) using Nextera tagmentase. (8) DNA fragments containing biotinylated CTP were selected on streptavidin-coated beads. This selects for ligation junctions and DNA molecules biotinylated at their terminus. (9) A Sequencing library was generated via PCR using the Nextera [ http://www.illumina.com/products/nextera_dna_library_prep_kit.html ] adaptors introduced at step 7. This amplification step should provide a substantial enrichment for ligation junctions, since molecules that were biotinylated solely on their termini would carry a Nextera adaptor only on one side. b RTCC protocol timeline

Article Snippet: Restriction enzyme (DpnII or AvaII) generated 5’ overhangs were filled in by adding 63 μl water, 1 μl 1 M MgCl, 10 μl 10× NEBuffer 2, 0.7 μl 10 mM dATP, 0.7 μl 10 mM dTTP, 0.7 μl 10 mM 2’-Deoxyguanosine-5’-O-(1-thiotriphosphate), sodium salt, Sp-isomer (Axxora, San Diego, CA, USA), 15 μl 0.4 mM Biotin-14-dCTP (Life Technologies), 4 μl 10 % Triton X-100 and 5 μl 5 U/μl DNA Polymerase I, Large (Klenow) Fragment (NEB).

Techniques: Sequencing, Magnetic Beads, Purification, Ligation, Generated, Polymerase Chain Reaction, Amplification

Journal: Nucleic Acids Research

Article Title: Parallel competition analysis of Saccharomyces cerevisiae strains differing by a single base using polymerase colonies

doi:

Figure Lengend Snippet: Representative polony images. Lys131Glu mutant time series from left to right: ( A ) 0 h, ( B ) 51.75 h and ( C ) 97 h. Polonies were initially extended with cyanine-5-dCTP (A and C) or cyanine-5-dUTP (B). The slides were then imaged and the extended primers were removed. The procedure was then repeated with cyanine-5-dCTP used to extend (B) and cyanine-5-dUTP used to extend (A) and (C). A positive extension with cyanine-5-dUTP indicates that the wild-type nucleotide is present at position 391 of PGK1 (red), while positive extension with cyanine-5-dCTP indicates the mutant nucleotide (green) at this position.

Article Snippet: Slides were then covered with 55 µl of an extension solution consisting of 50.67 µl Klenow Extension Buffer, 1 µl Klenow fragment (50 000 U/ml; New England Biolabs), 0.83 µl single-stranded DNA binding protein (1–5 µg/µl; USB) and 2.5 µl of either cyanine-5-dATP, cyanine-5-dUTP, cyanine-5-dCTP or cyanine-5-dGTP (10 µM; PerkinElmer Life Sciences) depending upon the desired extension.

Techniques: Mutagenesis

Journal: Nucleic Acids Research

Article Title: Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

doi:

Figure Lengend Snippet: Dynamics of association of the ori-β to the nuclear matrix during the cell cycle of CHO cells. ( A ) Schematic representation of the position of the ori-β probe in the non-transcribed spacer 3′ of the DHFR gene in CHO cells. A 479 bp DNA fragment (filled box) was amplified by PCR and used as a probe. ( B ) Relative abundance of the DHFR ori-β in the matrix-attached DNA in CHO cells. Aliquots of matrix-attached DNA isolated from CHO cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probe in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. The results are means of five independent experiments and the standard deviations are shown with error bars.

Article Snippet: DNA probes were labeled in vitro with [32 P]dCTP (3000 Ci/mmol; DuPont) using the RadPrime DNA Labeling System (Gibco BRL).

Techniques: Amplification, Polymerase Chain Reaction, Isolation, In Vitro, Labeling

Journal: Nucleic Acids Research

Article Title: Dynamics of association of origins of DNA replication with the nuclear matrix during the cell cycle

doi:

Figure Lengend Snippet: Dynamics of association of the β-globin origin of replication with the nuclear matrix during the cell cycle of HeLa cells. ( A ) Schematic representation of the positions of the probes from the human β-globin locus (filled boxes). ( B ) Aliquots of matrix-attached DNA isolated from HeLa cells synchronized at the indicated stages of the cell cycle were dot-blotted and hybridized with DNA probes in vitro labeled with [ 32 P]dCTP. For determination of relative abundance the blots were rehybridized with genomic DNA. The autoradiographs were scanned and quantified and the ratios of probes to genomic DNA were calculated and presented in arbitrary units, assuming the early G 1 ratio as 1.0. Shaded columns, relative abundance of the control probe C1; filled columns, relative abundance of the β-globin replicator probe; white columns, relative abundance of the control probe C2. The results are means of five independent experiments and the standard deviations are shown with error bars.

Article Snippet: DNA probes were labeled in vitro with [32 P]dCTP (3000 Ci/mmol; DuPont) using the RadPrime DNA Labeling System (Gibco BRL).

Techniques: Isolation, In Vitro, Labeling