uracil dna glycosylase  (Thermo Fisher)


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  • 99
    Name:
    Uracil-DNA Glycosylase
    Description:
    Thermo Scientific Uracil-DNA Glycosylase (UDG, UNG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, leaving an apyrimidinic site in uracil-containing single or double-stranded DNA (see Figure 1 in Supporting Data). The enzyme shows no activity on RNA.Highlights• Active in Thermo Scientific buffers for restriction enzymes and thermophilic polymerasesApplications• Control of carry-over contamination in PCR• Glycosylase mediated single nucleotide polymorphism detection (GMPD)• Site-directed mutagenesis• As a probe for protein-DNA interaction studies• SNP genotyping• Cloning of PCR products• Generation of single strand overhangs of PCR products and cDNANoteThe abasic sites formed in DNA by Uracil-DNA Glycosylase may be subsequently cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites. UDG (UNG) is active in the presence or absence of divalent cations.Use of this enzyme in certain applications may be covered by patents and may require a license.
    Catalog Number:
    EN0361
    Price:
    None
    Applications:
    PCR|PCR & Real-Time PCR|Real Time PCR (qPCR)
    Size:
    200 units
    Category:
    Proteins, Enzymes, & Peptides, PCR & Cloning Enzymes, DNA⁄RNA Modifying Enzymes
    Score:
    85
    Buy from Supplier


    Structured Review

    Thermo Fisher uracil dna glycosylase
    Pol D does not cut uracil-containing <t>DNA.</t> ( A ) Hexachlorofluorescein-labelled oligodeoxynucleotides used in these experiments. ( B ) Results seen when Pfu-Pol D exo + or exo − (80 nM) was incubated with the oligodeoxynucleotides (20 nM) at 50°C for 30 min followed by heating at 95°C for 5 min in the presence of NaOH. Control = no Pfu-Pol D added; UDG = addition of uracil-DNA <t>glycosylase</t> (positive control for strand cleavage at uracil). The starting oligodeoxynucleotides (22 bases) and the 13 base products expected for cleavage at uracil are shown arrowed. The ladders of products slightly reduced in length (seen with Pfu-Pol D exo + , most prominently when uracil is present) arise from 3′ to 5′ proof-reading exonuclease activity.
    Thermo Scientific Uracil-DNA Glycosylase (UDG, UNG) catalyzes the hydrolysis of the N-glycosylic bond between uracil and sugar, leaving an apyrimidinic site in uracil-containing single or double-stranded DNA (see Figure 1 in Supporting Data). The enzyme shows no activity on RNA.Highlights• Active in Thermo Scientific buffers for restriction enzymes and thermophilic polymerasesApplications• Control of carry-over contamination in PCR• Glycosylase mediated single nucleotide polymorphism detection (GMPD)• Site-directed mutagenesis• As a probe for protein-DNA interaction studies• SNP genotyping• Cloning of PCR products• Generation of single strand overhangs of PCR products and cDNANoteThe abasic sites formed in DNA by Uracil-DNA Glycosylase may be subsequently cleaved by heat, alkali-treatment or endonucleases that cleave specifically at abasic sites. UDG (UNG) is active in the presence or absence of divalent cations.Use of this enzyme in certain applications may be covered by patents and may require a license.
    https://www.bioz.com/result/uracil dna glycosylase/product/Thermo Fisher
    Average 99 stars, based on 26 article reviews
    Price from $9.99 to $1999.99
    uracil dna glycosylase - by Bioz Stars, 2019-10
    99/100 stars

    Images

    1) Product Images from "Novel inhibition of archaeal family-D DNA polymerase by uracil"

    Article Title: Novel inhibition of archaeal family-D DNA polymerase by uracil

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt083

    Pol D does not cut uracil-containing DNA. ( A ) Hexachlorofluorescein-labelled oligodeoxynucleotides used in these experiments. ( B ) Results seen when Pfu-Pol D exo + or exo − (80 nM) was incubated with the oligodeoxynucleotides (20 nM) at 50°C for 30 min followed by heating at 95°C for 5 min in the presence of NaOH. Control = no Pfu-Pol D added; UDG = addition of uracil-DNA glycosylase (positive control for strand cleavage at uracil). The starting oligodeoxynucleotides (22 bases) and the 13 base products expected for cleavage at uracil are shown arrowed. The ladders of products slightly reduced in length (seen with Pfu-Pol D exo + , most prominently when uracil is present) arise from 3′ to 5′ proof-reading exonuclease activity.
    Figure Legend Snippet: Pol D does not cut uracil-containing DNA. ( A ) Hexachlorofluorescein-labelled oligodeoxynucleotides used in these experiments. ( B ) Results seen when Pfu-Pol D exo + or exo − (80 nM) was incubated with the oligodeoxynucleotides (20 nM) at 50°C for 30 min followed by heating at 95°C for 5 min in the presence of NaOH. Control = no Pfu-Pol D added; UDG = addition of uracil-DNA glycosylase (positive control for strand cleavage at uracil). The starting oligodeoxynucleotides (22 bases) and the 13 base products expected for cleavage at uracil are shown arrowed. The ladders of products slightly reduced in length (seen with Pfu-Pol D exo + , most prominently when uracil is present) arise from 3′ to 5′ proof-reading exonuclease activity.

    Techniques Used: Incubation, Positive Control, Activity Assay

    Related Articles

    Clone Assay:

    Article Title: Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene
    Article Snippet: The assay was used: (1) to confirm the Sequenom results; (2) to identify the presence and incidence of minor tumour clones undetectable by the standard methods; (3) to determine the real nature of the subclonal mutations. .. In brief, 30 ng of DNA/reaction were added to fast COLD-PCR reagents and preliminary submitted to treatment at 37 °C for 30′ in the presence or absence of 0.5 U of Uracil-DNA-Glycosylase (UDG, Thermo Fisher) ( ).

    Amplification:

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Sequencing libraries were prepared from fragmented cDNA using NEBNext DNA sample preparation kit (New England Biolabs) and Illumina PE adapters (PE-102–1003). .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems). .. Sequencing was performed on Illumina HiSeq 2000 sequencer in NIDDK Genomics core in 2×50 bp paired-end mode.

    Article Title: Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene
    Article Snippet: In brief, 30 ng of DNA/reaction were added to fast COLD-PCR reagents and preliminary submitted to treatment at 37 °C for 30′ in the presence or absence of 0.5 U of Uracil-DNA-Glycosylase (UDG, Thermo Fisher) ( ). .. The protocol of the fast COLD-PCR analysis was derived from Mancini et al ( ) in absence of fluorophores in the reagent mix, under conditions modified as follows: 20 cycles of standard PCR (95.0 °C 8'', 60.0 °C 30'', 72.0 °C 30'') followed by 35 cycles of COLD-PCR (82.5 °C 8'', 58.0 °C 30'', 72.0 °C 30'').

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: Briefly, 100 ηg of total DNA extracted from liver samples were amplified in a final volume of 50 μL containing, 1 unit of Biolase DNA polymerase, 1 mM of MgCl2 , 50 pmol of Alu primer and 10–100 pmol of primer HB1 ( ). .. The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C.

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: Products were separated by agarose gel electrophoresis, and fragments between 300 and 350 bp were excised and eluted. .. Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified. .. Library quantification and quality control was performed using the Agilent 2100 Bio-analyzer and the ABI StepOnePlus Real-Time PCR System.

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers for amplification of rat TRPA1 (Trpa1, Ref NM_207608.1) were as follows: TRPA1 forward (5′-GCC CCT GTC TCT GTA AAT AAC C-3′, TM = 55°C) and TRPA1 reverse (5′-CTT GTG TCG CTG ATG TCT TG-3′, TM = 54°C). .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Positive Control:

    Article Title: Novel inhibition of archaeal family-D DNA polymerase by uracil
    Article Snippet: Pfu-Pol D exo+ or exo− (80 nM) was incubated with fluorescent oligodeoxynucleotides (single and double stranded; +/− uracil) (20 nM) at 50°C for 30 min in the buffer given above. .. As a positive control for strand cutting, a reaction was carried out with uracil-DNA glycosylase (0.5 units, Fermentas). .. After 30 min, the reactions were quenched by heating with stop buffer.

    Synthesized:

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: All oligonucleotides were synthesized and purified by Oligo’s Etc. (Wilsonville, OR). .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes.

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: First-strand complementary DNA (cDNA) was synthesized using the Superscript cDNA Synthesis kit (Life Technologies) with random hexamer primers and 1 μg μl−1 Actinomycin D. The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol, but substituting dTTP for dUTP. .. The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity.

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: The 2nd strand was synthesized with a dNTP mix containing dUTP. .. After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA).

    Construct:

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: Illumina strand-specific RNA-seq libraries were constructed with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per manufacturer’s protocol. .. After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA).

    Real-time Polymerase Chain Reaction:

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains
    Article Snippet: MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). .. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA).

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: Total RNA (500 ng) was quantitated at 260 nm and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's protocol, at 37°C for 15 min and 85°C for 30 sec. qPCR was performed using the SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.) according to the manufacturer's protocol in the ABI PRISM 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Microarray:

    Article Title: Lifelong Football Training: Effects on Autophagy and Healthy Longevity Promotion
    Article Snippet: The RNA samples were prepared using the WT PLUS Reagent kit, followed by hybridization on HTA 2.0 microarray chips. .. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Affymetrix) in conjunction with incorporated dUTPs.

    Random Hexamer Labeling:

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: First-strand complementary DNA (cDNA) was synthesized using the Superscript cDNA Synthesis kit (Life Technologies) with random hexamer primers and 1 μg μl−1 Actinomycin D. The second strand cDNA was synthesized following the Superscript cDNA Synthesis protocol, but substituting dTTP for dUTP. .. The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity.

    In Silico:

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA). .. In order to achieve sufficient levels of RNA sequencing for bacterial transcripts in the presence of an abundance of mouse transcripts (dual RNA-seq), libraries were loaded on 150nt paired-end runs of the Illumina HiSeq4000 sequencing platform as follows: half of a channel for each of the BIP and HIP libraries, a quarter of a channel for each of the biofilm and planktonic libraries, and an eighth of a channel for each of the three-control uninfected mouse heart libraries.

    Expressing:

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified. .. Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified.

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains
    Article Snippet: One µg of total RNA was used to generate cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon sur Yvette, France) with Multiscribe Reverse Transcriptase and random primers. .. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). .. To quantify total MAPT mRNA levels, we used the Hs00902194_m1 probe located at the junction between exons 12 and 13 (i.e. within a region present in all coding transcripts).

    Article Title: Lifelong Football Training: Effects on Autophagy and Healthy Longevity Promotion
    Article Snippet: 100 ng of total RNA were subjected to two cycles of cDNA synthesis with the Affymetrix WT PLUS expression Kit. .. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Affymetrix) in conjunction with incorporated dUTPs.

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA). .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: FAP-α expression levels were normalized to GAPDH expression using the 2−ΔΔCq method ( ). .. The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Human HLA-F adjacent transcript 10 promotes the formation of cancer initiating cells and cisplatin resistance in bladder cancer
    Article Snippet: The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Hybridization:

    Article Title: A microfluidic platform towards automated multiplexed in situ sequencing
    Article Snippet: Secure-Seal hybridization chambers were purchased from Invitrogen. .. Superfrost Plus slides and Uracil-DNA glycosylase were purchased from Thermo Fischer Scientific.

    Article Title: Lifelong Football Training: Effects on Autophagy and Healthy Longevity Promotion
    Article Snippet: The RNA samples were prepared using the WT PLUS Reagent kit, followed by hybridization on HTA 2.0 microarray chips. .. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Affymetrix) in conjunction with incorporated dUTPs.

    Ligation:

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: Adapters containing 6 nucleotide indexes were ligated to the double-stranded cDNA. .. After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA). .. Size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA).

    Cell Culture:

    Article Title: A microfluidic platform towards automated multiplexed in situ sequencing
    Article Snippet: DMEM cell culture medium was purchased from Gibco. .. Superfrost Plus slides and Uracil-DNA glycosylase were purchased from Thermo Fischer Scientific.

    Generated:

    Article Title: Novel inhibition of archaeal family-D DNA polymerase by uracil
    Article Snippet: As a positive control for strand cutting, a reaction was carried out with uracil-DNA glycosylase (0.5 units, Fermentas). .. As a positive control for strand cutting, a reaction was carried out with uracil-DNA glycosylase (0.5 units, Fermentas).

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems). .. After initial processing with Illumina pipeline, quality-filtered sequencing reads were aligned to monDom5 version of opossum genome assembly using BWA38 .

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: First-strand cDNA was generated using random hexamer-primed reverse transcription, with First Strand Master Mix and the SuperScript II Reverse Transcriptase kit (Invitrogen), with dUTP used during second-strand synthesis. .. Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified.

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: Quantitative reverse transcriptase PCR (qRT-PCR) primers and probes were designed with the RealTimeDesign software (Biosearch Technologies, Inc.) and were generated by Life Technologies. .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Translesion Synthesis:

    Article Title:
    Article Snippet: Paragraph title: Analysis of Translesion Synthesis Products from Primer-extension Reactions with Pol ϵ ... The uracil was removed by uracil- N -glycosylase (Fermentas) to create a natural abasic site.

    Sequencing:

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Sequencing libraries were prepared from fragmented cDNA using NEBNext DNA sample preparation kit (New England Biolabs) and Illumina PE adapters (PE-102–1003). .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems).

    Article Title: Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene
    Article Snippet: Paragraph title: Fast COLD-PCR followed by Sanger Sequencing for codons 12 and 13 of KRAS ... In brief, 30 ng of DNA/reaction were added to fast COLD-PCR reagents and preliminary submitted to treatment at 37 °C for 30′ in the presence or absence of 0.5 U of Uracil-DNA-Glycosylase (UDG, Thermo Fisher) ( ).

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Finally, 5 μl of the amplified product was used for a second round of amplification using the HB2 and Tag5 primers under the follow conditions: 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min and one final step at 72°C for 8 min.

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA). .. Size selection of the library was performed with AMPure XT beads (Beckman Coulter Genomics, Danvers, MA).

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified. .. Library quantification and quality control was performed using the Agilent 2100 Bio-analyzer and the ABI StepOnePlus Real-Time PCR System.

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: PCR specificity was determined by agarose gel electrophoresis and sequencing of products. .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers span exons 4 and 5, align with nucleotides 345–365 and 473–492 and yield a 148 bp product. .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. The specificity of these amplifications was verified by melt curve analysis with detection of only a single peak.

    TaqMan Assay:

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains
    Article Snippet: MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). .. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA).

    Cellular Antioxidant Activity Assay:

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers for β-actin (Actb, Ref NM_031144.2) were as follows: β-Actin forward (5′-CAC TTT CTA CAA TGA GCT GCG-3′, TM = 54°C) and β-actin reverse (5′-CTG GAT GGC TAC GTA CAT GG-3′, TM = 55°C). .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Nucleic Acid Electrophoresis:

    Article Title: Novel inhibition of archaeal family-D DNA polymerase by uracil
    Article Snippet: As a positive control for strand cutting, a reaction was carried out with uracil-DNA glycosylase (0.5 units, Fermentas). .. This buffer contains NaOH resulting in an alkaline pH and heating at 95°C for 5 min is expected to cut DNA at any abasic sites generated by the polymerase.

    RNA Sequencing Assay:

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Paragraph title: RNA-Seq ... Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems).

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: Paragraph title: RNA-seq and comparative transcriptomic analyses ... After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA).

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA). .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Fluorescence:

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. Real-time qPCR data were analyzed with the ΔΔCT method as described by Livak and Schmittgen.28 Transcript levels were compared in whole TG or nasal or dural cell populations of room air (control) and acrolein exposed animals, values normalized to β-actin and calibrated to control data using the ΔΔCT method. β-actin was used as a reference gene, and its level was not altered across these experimental conditions.

    Isolation:

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA).

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA).

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: Paragraph title: RNA isolation and quantitative RT-PCR ... A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Size-exclusion Chromatography:

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Double-stranded cDNA was fragmented using Bioruptor (Diagenode) (15 min, Low power, 30 sec ON, 30 sec OFF) in 50 µl volume. .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems).

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: Total RNA (500 ng) was quantitated at 260 nm and reverse-transcribed into cDNA using the PrimeScript RT reagent kit (Takara Biotechnology, Co., Ltd., Dalian, China) according to the manufacturer's protocol, at 37°C for 15 min and 85°C for 30 sec. qPCR was performed using the SYBR Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.) according to the manufacturer's protocol in the ABI PRISM 7900HT system (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Labeling:

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: Real-time primers were labeled at the 5′ end with 6-FAM (6-fluorescein amidite) fluorescent dye and were coupled with BHQ-1 quencher (black hole quencher 1). .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Purification:

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: All oligonucleotides were synthesized and purified by Oligo’s Etc. (Wilsonville, OR). .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes.

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Reaction mixture was purified using QIAquick PCR purification kit (Qiagen) and second strand synthesis was performed using SuperScript double-stranded cDNA synthesis kit (Invitrogen) as recommended by the manufacturer with the exception that 200 µM dTTP was replaced with 400 µM dUTP. .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems).

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Finally, 5 μl of the amplified product was used for a second round of amplification using the HB2 and Tag5 primers under the follow conditions: 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min and one final step at 72°C for 8 min.

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: Purified cDNA was subjected to end repair and phosphorylation by T4 DNA polymerase (NEB), Klenow DNA polymerase (NEB) and T4 polynucleotide kinase (NEB) in a single reaction, followed by 3′ A-tailing by Klenow fragment (3′–5′ exo minus, NEB). .. The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity.

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: The resulting cDNA was purified with Agencourt AMPure XP Beads (Beckman Coulter) then end-repaired and 3′ adenylated and adaptors were ligated. .. Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified.

    Article Title: Lifelong Football Training: Effects on Autophagy and Healthy Longevity Promotion
    Article Snippet: This cRNA is subjected to a second cycle – first strand synthesis in the presence of dUTP in a fixed ratio relative to dTTP. .. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Affymetrix) in conjunction with incorporated dUTPs. .. DNA fragments are then terminally labeled by terminal deoxynucleotidyl transferase (Affymetrix) with biotin.

    Polymerase Chain Reaction:

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: All oligonucleotides were synthesized and purified by Oligo’s Etc. (Wilsonville, OR). .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes. .. Cycle sequencing was performed using the BigDye version 2.0 terminator cycle-sequencing kit according to manufacturer’s instructions (Applied Biosystems).

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Sequencing libraries were prepared from fragmented cDNA using NEBNext DNA sample preparation kit (New England Biolabs) and Illumina PE adapters (PE-102–1003). .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems). .. Sequencing was performed on Illumina HiSeq 2000 sequencer in NIDDK Genomics core in 2×50 bp paired-end mode.

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The cycling conditions included an initial denaturation step at 94°C for 3 min and 10 cycles of 94°C for 30 s, 59°C for 30 s, and 70°C for 3 min. .. The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Subsequently, 1 unit of Biolase DNA polymerase and 10 mmol of HB2 and Tag5 primers ( ) were added and a Touch Down PCR was carried out.

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity. .. After purification, products were PCR-amplified using Phusion DNA Polymerase (Thermo Fisher Scientific) and Illumina's PE primer set, with cycle conditions of 98 °C for 30 s followed by 10–15 cycles of 98 °C for 10 s, 65 °C for 30 s and 72 °C for 30 s, and then 72 °C for 5 min. Purified PCR products were analysed using a LabChip GX (PerkinElmer).

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains
    Article Snippet: One µg of total RNA was used to generate cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon sur Yvette, France) with Multiscribe Reverse Transcriptase and random primers. .. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). .. To quantify total MAPT mRNA levels, we used the Hs00902194_m1 probe located at the junction between exons 12 and 13 (i.e. within a region present in all coding transcripts).

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA).

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: PCR specificity was determined by agarose gel electrophoresis and sequencing of products. .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA). .. The following cycling program was used: 52°C for 2 min (1 cycle), 95°C for 10 min (1 cycle), and 95°C for 10 s and 58°C for 1 min (45 cycles).

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: DNA was isolated from 200 μL of GEB using the High Pure PCR Template preparation Kit (Roche, Mannheim, Germany), according to the manufacturers’ protocol. .. Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA).

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers span exons 4 and 5, align with nucleotides 345–365 and 473–492 and yield a 148 bp product. .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. The specificity of these amplifications was verified by melt curve analysis with detection of only a single peak.

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Human HLA-F adjacent transcript 10 promotes the formation of cancer initiating cells and cisplatin resistance in bladder cancer
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for microRNAs ... The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Quantitative RT-PCR:

    Article Title: Alternative promoter usage generates novel shorter MAPT mRNA transcripts in Alzheimer’s disease and progressive supranuclear palsy brains
    Article Snippet: One µg of total RNA was used to generate cDNA by using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Villebon sur Yvette, France) with Multiscribe Reverse Transcriptase and random primers. .. MAPT expression levels were determined by qRT-PCR, using TaqMan® Gene Expression Assays, TaqMan® Universal PCR Master Mix II, and uracil-N glycosylase (Life Technologies, Carlsbad, CA, USA). .. To quantify total MAPT mRNA levels, we used the Hs00902194_m1 probe located at the junction between exons 12 and 13 (i.e. within a region present in all coding transcripts).

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: Paragraph title: qRT-PCR. ... For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: Paragraph title: RNA isolation and quantitative RT-PCR ... A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) ... The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Human HLA-F adjacent transcript 10 promotes the formation of cancer initiating cells and cisplatin resistance in bladder cancer
    Article Snippet: Paragraph title: Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) for microRNAs ... The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Polyacrylamide Gel Electrophoresis:

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: The PCR products were identified on 10% polyacrylamide gel electrophoresis and then purified using the QIAquick PCR Purification kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes.

    Gel Extraction:

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Finally, 5 μl of the amplified product was used for a second round of amplification using the HB2 and Tag5 primers under the follow conditions: 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min and one final step at 72°C for 8 min.

    Sample Prep:

    Article Title: Rsx, a metatherian RNA with Xist-like properties
    Article Snippet: Sequencing libraries were prepared from fragmented cDNA using NEBNext DNA sample preparation kit (New England Biolabs) and Illumina PE adapters (PE-102–1003). .. Before final PCR amplification samples were treated with Uracil-N-Glycosylase (Applied Biosystems).

    Article Title: Streptococcus pneumoniae in the heart subvert the host response through biofilm-mediated resident macrophage killing
    Article Snippet: Illumina strand-specific RNA-seq libraries were constructed with the TruSeq RNA Sample Prep kit (Illumina, San Diego, CA) per manufacturer’s protocol. .. After adapter ligation, the 2nd strand cDNA was digested with 2 units of Uracil-N-Glycosylase (Applied Biosystems, Carlsbad, CA).

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers for β-actin (Actb, Ref NM_031144.2) were as follows: β-Actin forward (5′-CAC TTT CTA CAA TGA GCT GCG-3′, TM = 54°C) and β-actin reverse (5′-CTG GAT GGC TAC GTA CAT GG-3′, TM = 55°C). .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Mouse Assay:

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Article Title: Phenotypic diversity and drug susceptibility of Trypanosoma cruzi TcV clinical isolates
    Article Snippet: Parasite DNA was amplified using a T . cruzi satellite DNA sequence of 140 bp flanked by the Sat Fw and Sat Rv oligonucleotides [ ] and SYBR GreenER qPCR SuperMix Universal Kit with integrated uracil DNA glycosylase (UDG; Invitrogen, Life Technologies, Grand Island, NY, USA). .. DNA amplification was performed in an ABI 7500 thermocycler (Applied Biosystems, Carlsbad, CA, USA) in duplicates using 5 μL of extracted DNA as template (~100 ng) in a final volume of 20 μL.

    Software:

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: Quantitative reverse transcriptase PCR (qRT-PCR) primers and probes were designed with the RealTimeDesign software (Biosearch Technologies, Inc.) and were generated by Life Technologies. .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers span exons 4 and 5, align with nucleotides 345–365 and 473–492 and yield a 148 bp product. .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. The specificity of these amplifications was verified by melt curve analysis with detection of only a single peak.

    Article Title: miR-204 reverses temozolomide resistance and inhibits cancer initiating cells phenotypes by degrading FAP-α in glioblastoma
    Article Snippet: Cycle threshold (Cq) values were determined using SDS version 2.4 software (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted at a ratio of 1:40 and mixed with 1 µl miR-204 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    Article Title: Human HLA-F adjacent transcript 10 promotes the formation of cancer initiating cells and cisplatin resistance in bladder cancer
    Article Snippet: The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.). .. The resulting cDNA was diluted in the ratio 1:40 and mixed with 1 µl miR-200 or U6 TaqMan primers in triplicate wells using TaqMan Universal Master Mix II without Uracil DNA glycosylase (Applied Biosystems; Thermo Fisher Scientific, Inc.).

    SYBR Green Assay:

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers span exons 4 and 5, align with nucleotides 345–365 and 473–492 and yield a 148 bp product. .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software. .. The specificity of these amplifications was verified by melt curve analysis with detection of only a single peak.

    Selection:

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity. .. After purification, products were PCR-amplified using Phusion DNA Polymerase (Thermo Fisher Scientific) and Illumina's PE primer set, with cycle conditions of 98 °C for 30 s followed by 10–15 cycles of 98 °C for 10 s, 65 °C for 30 s and 72 °C for 30 s, and then 72 °C for 5 min. Purified PCR products were analysed using a LabChip GX (PerkinElmer).

    Agarose Gel Electrophoresis:

    Article Title: The RNA-binding protein LARP1 is a post-transcriptional regulator of survival and tumorigenesis in ovarian cancer
    Article Snippet: Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified. .. Uracil-N-Glycosylase (UNG, Applied Biosystems) was used to degrade the second-strand cDNA, and products were amplified and re-purified.

    Article Title: Genomic, Transcriptomic, and Phenotypic Analyses of Neisseria meningitidis Isolates from Disease Patients and Their Household Contacts
    Article Snippet: PCR specificity was determined by agarose gel electrophoresis and sequencing of products. .. For simplicity, all PCR assays were carried out using 250 nM probe and 200 nM primer , using TaqMan universal master mix II with uracil-N -glycosylase (UNG; Thermo Fisher, USA).

    In Vitro:

    Article Title: Lifelong Football Training: Effects on Autophagy and Healthy Longevity Promotion
    Article Snippet: After second-strand synthesis, the resulting cDNA is in vitro transcribed with the T7 RNA polymerase to generate a cRNA. .. Single strand cDNA is then purified and fragmented with a mixture of uracil DNA glycosylase and apurinic/apyrimidinic endonuclease 1 (Affymetrix) in conjunction with incorporated dUTPs.

    Incubation:

    Article Title: Novel inhibition of archaeal family-D DNA polymerase by uracil
    Article Snippet: Pfu-Pol D exo+ or exo− (80 nM) was incubated with fluorescent oligodeoxynucleotides (single and double stranded; +/− uracil) (20 nM) at 50°C for 30 min in the buffer given above. .. As a positive control for strand cutting, a reaction was carried out with uracil-DNA glycosylase (0.5 units, Fermentas).

    Article Title:
    Article Snippet: The uracil was removed by uracil- N -glycosylase (Fermentas) to create a natural abasic site. .. The uracil was removed by uracil- N -glycosylase (Fermentas) to create a natural abasic site.

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: All oligonucleotides were synthesized and purified by Oligo’s Etc. (Wilsonville, OR). .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes. .. Cycle sequencing was performed using the BigDye version 2.0 terminator cycle-sequencing kit according to manufacturer’s instructions (Applied Biosystems).

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The cycling conditions included an initial denaturation step at 94°C for 3 min and 10 cycles of 94°C for 30 s, 59°C for 30 s, and 70°C for 3 min. .. The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Subsequently, 1 unit of Biolase DNA polymerase and 10 mmol of HB2 and Tag5 primers ( ) were added and a Touch Down PCR was carried out.

    Concentration Assay:

    Article Title: Simultaneous Sequencing of Multiple Polymerase Chain Reaction Products and Combined Polymerase Chain Reaction with Cycle Sequencing in Single Reactions
    Article Snippet: PCR was performed in a 50-μl reactions containing a final concentration of 1× PCR Buffer (Applied Biosystems, Foster City, CA), 50 μmol/L each of dNTP, 1.25 U Taq Gold (Applied Biosystems), 0.01% gelatin, and 0.2 μmol/L each forward and reverse primer. .. After PCR for prothrombin , to some samples 3 μl (1 U/μl) of uracil- N -glycosylase (Life Technologies, Carlsbad, CA) was added to the 50 μl PCR product, and incubated at 37°C for 30 minutes.

    Ii Assay:

    Article Title: MEF2B mutations in non-Hodgkin lymphoma dysregulate cell migration by decreasing MEF2B target gene activation
    Article Snippet: The first strand was then digested using uracil-N -glycosylase (Life Technologies), thus achieving strand specificity. .. PCR products with a desired size range were purified using a 96-channel size selection robot developed at the BCGSC.

    CTG Assay:

    Article Title: Role of intraganglionic transmission in the trigeminovascular pathway
    Article Snippet: The primers for amplification of rat TRPA1 (Trpa1, Ref NM_207608.1) were as follows: TRPA1 forward (5′-GCC CCT GTC TCT GTA AAT AAC C-3′, TM = 55°C) and TRPA1 reverse (5′-CTT GTG TCG CTG ATG TCT TG-3′, TM = 54°C). .. A mixture of cDNA template, SYBR Green Master mix and forward and reverse primers was treated with uracil N-glycosylase (Life Technologies) before undergoing the following protocol: 50°C for 2 min, 95°C for 10 min, then 45 cycles of 95°C for 15 s, 60°C for 1 min, followed by one cycle of 95°C for 15 s, 60°C for 15 s, and 95°C for 15 s. The PCR products were analyzed with ABI PRISM sequence detection software.

    Staining:

    Article Title: Molecular characterization of occult hepatitis B virus infection in patients with end-stage liver disease in Colombia
    Article Snippet: The PCR products were then incubated with 1 U of uracil-DNA glycosylase (Life Technologies, USA) for 30 min at 37°C followed by 10 min at 94°C. .. Finally, 5 μl of the amplified product was used for a second round of amplification using the HB2 and Tag5 primers under the follow conditions: 20 cycles at 94°C for 30 s, 55°C for 30 s, and 70°C for 3 min and one final step at 72°C for 8 min.

    Co-amplification at Lower Denaturation temperature PCR:

    Article Title: Extreme assay sensitivity in molecular diagnostics further unveils intratumour heterogeneity in metastatic colorectal cancer as well as artifactual low-frequency mutations in the KRAS gene
    Article Snippet: The assay was used: (1) to confirm the Sequenom results; (2) to identify the presence and incidence of minor tumour clones undetectable by the standard methods; (3) to determine the real nature of the subclonal mutations. .. In brief, 30 ng of DNA/reaction were added to fast COLD-PCR reagents and preliminary submitted to treatment at 37 °C for 30′ in the presence or absence of 0.5 U of Uracil-DNA-Glycosylase (UDG, Thermo Fisher) ( ). .. The protocol of the fast COLD-PCR analysis was derived from Mancini et al ( ) in absence of fluorophores in the reagent mix, under conditions modified as follows: 20 cycles of standard PCR (95.0 °C 8'', 60.0 °C 30'', 72.0 °C 30'') followed by 35 cycles of COLD-PCR (82.5 °C 8'', 58.0 °C 30'', 72.0 °C 30'').

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    Thermo Fisher gene exp mpg hs00357983 g1
    Overexpression of the BER protein Polβ, but not, APE1 reverses the MX-induced potentiation of TMZ in <t>LN428/MPG</t> cells. (A) Overexpression of WT Polβ or the 5′dRP lyase null mutant (K72A) of Polβ in LN428/MPG cells as determined
    Gene Exp Mpg Hs00357983 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 84/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher gene exp smug1 mm00452897 m1
    Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , <t>Smug1</t> , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.
    Gene Exp Smug1 Mm00452897 M1, 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
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    Price from $9.99 to $1999.99
    gene exp smug1 mm00452897 m1 - by Bioz Stars, 2019-10
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    Thermo Fisher gene exp ogg1 mm01184571 g1
    <t>Ogg1</t> silencing in rat hepatoma cells. ( A ) Semiquantitative mRNA evaluation of rat hepatoma cells transfected with 8-oxoguanine DNA glycosylase (OGG1) small interfering (si)RNA demonstrating decreased OGG1 mRNA levels compared with a scrambled siRNA control.
    Gene Exp Ogg1 Mm01184571 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/gene exp ogg1 mm01184571 g1/product/Thermo Fisher
    Average 85 stars, based on 2 article reviews
    Price from $9.99 to $1999.99
    gene exp ogg1 mm01184571 g1 - by Bioz Stars, 2019-10
    85/100 stars
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    Image Search Results


    Overexpression of the BER protein Polβ, but not, APE1 reverses the MX-induced potentiation of TMZ in LN428/MPG cells. (A) Overexpression of WT Polβ or the 5′dRP lyase null mutant (K72A) of Polβ in LN428/MPG cells as determined

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Overexpression of the BER protein Polβ, but not, APE1 reverses the MX-induced potentiation of TMZ in LN428/MPG cells. (A) Overexpression of WT Polβ or the 5′dRP lyase null mutant (K72A) of Polβ in LN428/MPG cells as determined

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Over Expression, Mutagenesis

    Relative mRNA expression levels of MPG, PARP1, and Polβ in GBM tumors as compared with normal brain. (A–C) The relative expression of mRNA for (A) MPG, (B) Polβ, and (C) PARP1 in GBM tumors (tissue #4–16), as indicated

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Relative mRNA expression levels of MPG, PARP1, and Polβ in GBM tumors as compared with normal brain. (A–C) The relative expression of mRNA for (A) MPG, (B) Polβ, and (C) PARP1 in GBM tumors (tissue #4–16), as indicated

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Expressing

    Expression profile of MPG, PARP1, and Polβ in established glioma cell lines. (A–C) The relative expression of mRNA for (A) MPG, (B) Polβ, and (C) PARP1 in GBM cell lines, as indicated in the figure, was measured by quantitative

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Expression profile of MPG, PARP1, and Polβ in established glioma cell lines. (A–C) The relative expression of mRNA for (A) MPG, (B) Polβ, and (C) PARP1 in GBM cell lines, as indicated in the figure, was measured by quantitative

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Expressing

    Over-expression of MPG increases the PARG KD–induced potentiation of TMZ in LN428/MGMT cells. (A) MGMT expression, as determined by immunoblot analysis of nuclear protein extracted from LN428 cells (lane 1) and T98G cells (lane 2; used as a positive

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Over-expression of MPG increases the PARG KD–induced potentiation of TMZ in LN428/MGMT cells. (A) MGMT expression, as determined by immunoblot analysis of nuclear protein extracted from LN428 cells (lane 1) and T98G cells (lane 2; used as a positive

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Over Expression, Expressing

    Overexpression of MPG in LN428 cells dramatically increases MX-induced potentiation of TMZ. (A) MPG overexpression as determined by immunoblot analysis of nuclear proteins isolated from the LN428 cells (lane 1) or LN428/MPG cells (lane 2). Expression

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Overexpression of MPG in LN428 cells dramatically increases MX-induced potentiation of TMZ. (A) MPG overexpression as determined by immunoblot analysis of nuclear proteins isolated from the LN428 cells (lane 1) or LN428/MPG cells (lane 2). Expression

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Over Expression, Isolation, Expressing

    Overexpression of MPG increases the PARP inhibitor-induced potentiation of TMZ in glioma cells with elevated levels of MGMT. (A) PJ34 significantly sensitized LN428/MGMT/MPG cells, but not the LN428/MGMT cells, to TMZ, as measured by long-term cell survival

    Journal:

    Article Title: N-methylpurine DNA glycosylase and DNA polymerase ? modulate BER inhibitor potentiation of glioma cells to temozolomide

    doi: 10.1093/neuonc/nor011

    Figure Lengend Snippet: Overexpression of MPG increases the PARP inhibitor-induced potentiation of TMZ in glioma cells with elevated levels of MGMT. (A) PJ34 significantly sensitized LN428/MGMT/MPG cells, but not the LN428/MGMT cells, to TMZ, as measured by long-term cell survival

    Article Snippet: The preamplified cDNA was next analyzed using validated Applied Biosystems TaqMan Gene Expression Assays (human MPG: Hs00357983-G1; human Polβ: Hs01099715-M1; and human PARP1: Hs00911369-G1) and normalized to the expression of human β-actin (part #4333762T).

    Techniques: Over Expression

    Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , Smug1 , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: Impact of uracil glycosylase silencing on the mutation profile of Pms2 −/− Ung −/− GC B cells. (A) Analysis of mutations in J H 4 intronic sequences from Pms2 −/− Ung −/− splenic GC B cells (B220 + GL7 + CD95 + ) silenced for Mbd4 , Tdg , Smug1 , or both Tdg and Smug1 , as well as Tdg -silenced WT cells. NT represents control cells isolated from bone marrow of Pms2 −/− Ung −/− mice that were cultivated in the same conditions but not transduced before injection in Rag2 −/− recipient mice. KD, knockdown. (B) Pattern of nucleotide substitution for all the genotypes, corrected for base composition. No significant differences for frequencies of GYW/WR(C) or WA/TW hotspot mutations were observed for comparisons of values from individual mice with NT controls (two-tailed Mann Whitney U test). (C) Mutagenesis at A-T base pairs (percentage). Efficiency of knockdown in the analyzed GFP + GC B cells compared with GFP − GC B cells is represented by a color code. Silencing of Tdg was achieved with two different shRNA sequences, represented by dots or triangles. **, P = 0.0012 (TDG KD vs. NT) or 0.0061 (TGD/SMUG KD vs. TDG KD ); two-tailed Mann-Whitney U test. All restored mice (three to seven for each antisense transduction), analyzed individually for glycosylase inhibition and J H 4 mutations, are represented.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Mutagenesis, Gas Chromatography, Isolation, Mouse Assay, Injection, Two Tailed Test, MANN-WHITNEY, shRNA, Transduction, Inhibition

    Analysis of uracil glycosylase expression levels by qRT-PCR. (A) Relative expression levels of Mbd4 , Tdg , and Smug1 in naive B cells (B220 + GL7 − CD95 − ) from spleen of immunized WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice, compared with β-2 microglobulin (b2m). (B) Relative expression of Mbd4 , Tdg , and Smug1 in GC B cells (B220 + GL7 + CD95 + ) compared with naive B cells from spleens of WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice. The dotted line represents a ratio of 1 between GC and naive B cells. Quantification was performed in triplicates for RNA samples from three to four different mice. Error bars represent SD.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: Analysis of uracil glycosylase expression levels by qRT-PCR. (A) Relative expression levels of Mbd4 , Tdg , and Smug1 in naive B cells (B220 + GL7 − CD95 − ) from spleen of immunized WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice, compared with β-2 microglobulin (b2m). (B) Relative expression of Mbd4 , Tdg , and Smug1 in GC B cells (B220 + GL7 + CD95 + ) compared with naive B cells from spleens of WT ( n = 4) and Ung −/− Pms2 −/− ( n = 3) mice. The dotted line represents a ratio of 1 between GC and naive B cells. Quantification was performed in triplicates for RNA samples from three to four different mice. Error bars represent SD.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Expressing, Quantitative RT-PCR, Mouse Assay, Gas Chromatography

    A model whereby both uracil glycosylases and the Pms2/Mlh1 complex enable A-T base pair mutagenesis with a distinct strand bias. (A) Overlapping enzymatic activities generate entry sites for ExoI in MMR-mediated A-T mutagenesis. (Right) Recognition of AID deamination products by Msh2–Msh6 stimulates the endonuclease activity of the Pms2–Mlh1 complex, which acts at distance on either DNA strands. (Left) In a situation of processive deamination, nicks introduced by uracil glycosylase–mediated base excision (UNG, SMUG1, and TDG) and APEX2 strand incision can participate in MMR-mediated error-prone repair. Glycosylases may also act in the context of two independent deamination events on both DNA strands or, possibly, in the context of a single uracil (see Discussion). (B) Pms2 versus uracil glycosylase contribution estimated from A/T mutation ratios. The 1.8- to twofold A over T ratio observed in hypermutation was estimated to correspond to a 3:1 strand bias in error-prone DNA synthesis, taking into account the intrinsic T over A mutation preference of Pol η (a 4:1 higher mutation frequency opposite T than opposite A; Zivojnovic et al., 2014 ). This strand bias is represented by arrows of unequal length, representing the relative A:T mutagenic patch on each strand, without aiming at indicating whether the patch is shorter or less frequent on the transcribed strand or differently located on the locus. If one considers, from mutations observed in Pms2 −/− mice, that uracil glycosylases operate mainly on the coding, nontranscribed strand and that PMS2 endonuclease operates without strand bias and is responsible for most mutations introduced in the transcribed strand, both pathways appear to contribute equally to the generation of Msh2/6-driven A-T mutations.

    Journal: The Journal of Experimental Medicine

    Article Title: Pms2 and uracil-DNA glycosylases act jointly in the mismatch repair pathway to generate Ig gene mutations at A-T base pairs

    doi: 10.1084/jem.20161576

    Figure Lengend Snippet: A model whereby both uracil glycosylases and the Pms2/Mlh1 complex enable A-T base pair mutagenesis with a distinct strand bias. (A) Overlapping enzymatic activities generate entry sites for ExoI in MMR-mediated A-T mutagenesis. (Right) Recognition of AID deamination products by Msh2–Msh6 stimulates the endonuclease activity of the Pms2–Mlh1 complex, which acts at distance on either DNA strands. (Left) In a situation of processive deamination, nicks introduced by uracil glycosylase–mediated base excision (UNG, SMUG1, and TDG) and APEX2 strand incision can participate in MMR-mediated error-prone repair. Glycosylases may also act in the context of two independent deamination events on both DNA strands or, possibly, in the context of a single uracil (see Discussion). (B) Pms2 versus uracil glycosylase contribution estimated from A/T mutation ratios. The 1.8- to twofold A over T ratio observed in hypermutation was estimated to correspond to a 3:1 strand bias in error-prone DNA synthesis, taking into account the intrinsic T over A mutation preference of Pol η (a 4:1 higher mutation frequency opposite T than opposite A; Zivojnovic et al., 2014 ). This strand bias is represented by arrows of unequal length, representing the relative A:T mutagenic patch on each strand, without aiming at indicating whether the patch is shorter or less frequent on the transcribed strand or differently located on the locus. If one considers, from mutations observed in Pms2 −/− mice, that uracil glycosylases operate mainly on the coding, nontranscribed strand and that PMS2 endonuclease operates without strand bias and is responsible for most mutations introduced in the transcribed strand, both pathways appear to contribute equally to the generation of Msh2/6-driven A-T mutations.

    Article Snippet: Relative transcript abundance was assessed by real-time PCR on a PCR machine (7500 Fast System Real-Time; Applied Biosystems; 2 min at 50°C, 10 min at 95°C, and 40 cycles of 10 s at 95°C and 1 min at 60°C) in universal PCR master mix (No AmpErase UNG; Applied Biosystems), with the following validated TaqMan assays: Mm00437762_m1 (β2-microglobulin), Mm01225357_g1 (Tdg), Mm01184338_m1 (Mbd4), and Mm00452897_m1 (Smug1).

    Techniques: Mutagenesis, Activity Assay, Activated Clotting Time Assay, DNA Synthesis, Mouse Assay

    Ogg1 silencing in rat hepatoma cells. ( A ) Semiquantitative mRNA evaluation of rat hepatoma cells transfected with 8-oxoguanine DNA glycosylase (OGG1) small interfering (si)RNA demonstrating decreased OGG1 mRNA levels compared with a scrambled siRNA control.

    Journal:

    Article Title: Staphylococcus aureus Sepsis and Mitochondrial Accrual of the 8-Oxoguanine DNA Glycosylase DNA Repair Enzyme in Mice

    doi: 10.1164/rccm.200911-1709OC

    Figure Lengend Snippet: Ogg1 silencing in rat hepatoma cells. ( A ) Semiquantitative mRNA evaluation of rat hepatoma cells transfected with 8-oxoguanine DNA glycosylase (OGG1) small interfering (si)RNA demonstrating decreased OGG1 mRNA levels compared with a scrambled siRNA control.

    Article Snippet: qRT-PCR was performed using an ABI Prism 7000 Sequence Detection System with TaqMan gene expression and premix assays for IL-6, Mm00446190_m1; tumor necrosis factor (TNF), Mm00443258_m1; Tfam, Mm01148667_m1; OGG1, Mm01184571_g1; NRF-1, Mm01135607_m1; NRF-2, Mm00484597_m1 (Applied Biosystems).

    Techniques: Transfection

    Analysis of hepatic Ogg1 in control and S. aureus –infected mice. ( A ) Pooled analysis of three mice at each time point of real-time reverse transcriptase–polymerase chain reaction for Ogg1 expression in liver tissues and indicates that

    Journal:

    Article Title: Staphylococcus aureus Sepsis and Mitochondrial Accrual of the 8-Oxoguanine DNA Glycosylase DNA Repair Enzyme in Mice

    doi: 10.1164/rccm.200911-1709OC

    Figure Lengend Snippet: Analysis of hepatic Ogg1 in control and S. aureus –infected mice. ( A ) Pooled analysis of three mice at each time point of real-time reverse transcriptase–polymerase chain reaction for Ogg1 expression in liver tissues and indicates that

    Article Snippet: qRT-PCR was performed using an ABI Prism 7000 Sequence Detection System with TaqMan gene expression and premix assays for IL-6, Mm00446190_m1; tumor necrosis factor (TNF), Mm00443258_m1; Tfam, Mm01148667_m1; OGG1, Mm01184571_g1; NRF-1, Mm01135607_m1; NRF-2, Mm00484597_m1 (Applied Biosystems).

    Techniques: Infection, Mouse Assay, Polymerase Chain Reaction, Expressing

    In silico and chromatin immunoprecipitation analysis of Ogg1 promoter site for nuclear respiratory factor (NRF)-1 and NRF-2α. NRF-1 and NRF-2α consensus site binding motifs were found for Ogg1 at 3 kb from the transcription start site.

    Journal:

    Article Title: Staphylococcus aureus Sepsis and Mitochondrial Accrual of the 8-Oxoguanine DNA Glycosylase DNA Repair Enzyme in Mice

    doi: 10.1164/rccm.200911-1709OC

    Figure Lengend Snippet: In silico and chromatin immunoprecipitation analysis of Ogg1 promoter site for nuclear respiratory factor (NRF)-1 and NRF-2α. NRF-1 and NRF-2α consensus site binding motifs were found for Ogg1 at 3 kb from the transcription start site.

    Article Snippet: qRT-PCR was performed using an ABI Prism 7000 Sequence Detection System with TaqMan gene expression and premix assays for IL-6, Mm00446190_m1; tumor necrosis factor (TNF), Mm00443258_m1; Tfam, Mm01148667_m1; OGG1, Mm01184571_g1; NRF-1, Mm01135607_m1; NRF-2, Mm00484597_m1 (Applied Biosystems).

    Techniques: In Silico, Chromatin Immunoprecipitation, Binding Assay