t4 polynucleotide kinase  (New England Biolabs)


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    Name:
    T4 Polynucleotide Kinase 3 phosphatase minus
    Description:
    T4 Polynucleotide Kinase 3 phosphatase minus 1 000 units
    Catalog Number:
    M0236L
    Price:
    428
    Size:
    1 000 units
    Category:
    Polynucleotide Kinases
    Score:
    85
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    New England Biolabs t4 polynucleotide kinase
    T4 Polynucleotide Kinase 3 phosphatase minus
    T4 Polynucleotide Kinase 3 phosphatase minus 1 000 units
    https://www.bioz.com/result/t4 polynucleotide kinase/product/New England Biolabs
    Average 99 stars, based on 817 article reviews
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    t4 polynucleotide kinase - by Bioz Stars, 2019-10
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    1) Product Images from "Catalysts from synthetic genetic polymers"

    Article Title: Catalysts from synthetic genetic polymers

    Journal: Nature

    doi: 10.1038/nature13982

    Analysis of RNA endonuclease XNAzyme cleavage products a , 5′ cleavage product of FANAzyme FR17_6 reaction shows expected mass for a 2′,3′ cyclic phosphate ( > p) using Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-ToF).  b , Hydrolysis of 5′ FR17_6 cleavage product  > p in low pH and dephosphosphorylation with calf intestinal phosphatase (removes 2′p or 3′p, but not  > p).  c , Phosphorylation of 3′ FR17_6 cleavage product with T4 polynucleotide kinase (adds 5′p). Mass spectra and dephosphorylation assays of 5′ cleavage products of  d , ANAzyme AR17_5,  e , HNAzyme HR16_1 and  f , CeNAzyme CeR16_3 reveal all RNA endonuclease XNAzymes yield products with 2′,3′ cyclic phosphates. (RNase T1) and ( − OH) indicate partial hydrolysis reactions of the RNA substrates used.  g , Bivalent metal ion requirements and titration of,  h , pH or  i , MgC l2 , of FANAzyme FR17_6min reaction with NucS R _min.  j , Reaction catalyzed by RNA endonuclease XNAzymes.
    Figure Legend Snippet: Analysis of RNA endonuclease XNAzyme cleavage products a , 5′ cleavage product of FANAzyme FR17_6 reaction shows expected mass for a 2′,3′ cyclic phosphate ( > p) using Matrix-Assisted Laser Desorption/Ionization-Time of Flight mass spectrometry (MALDI-ToF). b , Hydrolysis of 5′ FR17_6 cleavage product > p in low pH and dephosphosphorylation with calf intestinal phosphatase (removes 2′p or 3′p, but not > p). c , Phosphorylation of 3′ FR17_6 cleavage product with T4 polynucleotide kinase (adds 5′p). Mass spectra and dephosphorylation assays of 5′ cleavage products of d , ANAzyme AR17_5, e , HNAzyme HR16_1 and f , CeNAzyme CeR16_3 reveal all RNA endonuclease XNAzymes yield products with 2′,3′ cyclic phosphates. (RNase T1) and ( − OH) indicate partial hydrolysis reactions of the RNA substrates used. g , Bivalent metal ion requirements and titration of, h , pH or i , MgC l2 , of FANAzyme FR17_6min reaction with NucS R _min. j , Reaction catalyzed by RNA endonuclease XNAzymes.

    Techniques Used: Mass Spectrometry, De-Phosphorylation Assay, Titration

    2) Product Images from "Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity"

    Article Title: Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-6-37

    Genetic requirement for the DNase activity present in unfertilized oocytes.  Genomic DNA extracted from purified oocytes ( 'fer-1  oocytes’) or from liquid-nitrogen-flash-frozen samples (N2 (wild type) embryos and adults of various genotypes) was radioactively-labeled using T4 polynucleotide kinase and gamma-32P-ATP, and resolved on a denaturing 12% polyacrylamide gel (8 M urea). A total of 2 μg of DNA were used for labeling and PAGE analysis for all samples except DNA from isolated  fer-1(b232)  oocytes (
    Figure Legend Snippet: Genetic requirement for the DNase activity present in unfertilized oocytes. Genomic DNA extracted from purified oocytes ( 'fer-1 oocytes’) or from liquid-nitrogen-flash-frozen samples (N2 (wild type) embryos and adults of various genotypes) was radioactively-labeled using T4 polynucleotide kinase and gamma-32P-ATP, and resolved on a denaturing 12% polyacrylamide gel (8 M urea). A total of 2 μg of DNA were used for labeling and PAGE analysis for all samples except DNA from isolated fer-1(b232) oocytes (

    Techniques Used: Activity Assay, Purification, Labeling, Polyacrylamide Gel Electrophoresis, Isolation

    3) Product Images from "Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation"

    Article Title: Synthetic Circular RNA Functions as a miR-21 Sponge to Suppress Gastric Carcinoma Cell Proliferation

    Journal: Molecular Therapy. Nucleic Acids

    doi: 10.1016/j.omtn.2018.09.010

    Workflow for Producing Synthetic miR-21 circRNA Sponge Each binding site sequence is perfectly complementary to the miR-21 seed region, but contains a bulge at positions 9–12 to prevent RNAi-type cleavage and degradation. One PCR cycle facilitates synthesis to generate a double-stranded DNA PCR fragment, which is then cloned into the TOPO PCR cloning vector (Invitrogen). The T7 RNA polymerase binding site located just 5′ to the PCR insert is used to generate, via T7 RNA polymerase, large quantities (150 μg) of linear RNA containing the miR sponge sequence described above. Calf intestinal phosphatase dephosphorylates the 5′ end of the RNA transcript, and T4 polynucleotide kinase (in the presence of ATP) generates RNA molecules suitable for ligation. Incubation with T4 RNA ligase results in RNA circularization.
    Figure Legend Snippet: Workflow for Producing Synthetic miR-21 circRNA Sponge Each binding site sequence is perfectly complementary to the miR-21 seed region, but contains a bulge at positions 9–12 to prevent RNAi-type cleavage and degradation. One PCR cycle facilitates synthesis to generate a double-stranded DNA PCR fragment, which is then cloned into the TOPO PCR cloning vector (Invitrogen). The T7 RNA polymerase binding site located just 5′ to the PCR insert is used to generate, via T7 RNA polymerase, large quantities (150 μg) of linear RNA containing the miR sponge sequence described above. Calf intestinal phosphatase dephosphorylates the 5′ end of the RNA transcript, and T4 polynucleotide kinase (in the presence of ATP) generates RNA molecules suitable for ligation. Incubation with T4 RNA ligase results in RNA circularization.

    Techniques Used: Binding Assay, Sequencing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation, Ligation, Incubation

    4) Product Images from "Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication"

    Article Title: Phosphorylation of Large T Antigen Regulates Merkel Cell Polyomavirus Replication

    Journal: Cancers

    doi: 10.3390/cancers6031464

    MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [  14 ]. This origin was used for replication assays (  Figure 3  and   Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun  et al.  [  31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.
    Figure Legend Snippet: MCPyV LT phospho-mutants bind the viral Ori with different affinities. ( A ) Schematic of the MCPyV Ori and the EMSA Probe. Only one strand of DNA is shown for clarity. The MCPyV Ori sequence was cloned from the R17a isolate of MCPyV into a pcDNA4c vector [ 14 ]. This origin was used for replication assays ( Figure 3 and Figure 4 ). Consensus GAGGC pentanucleotide repeats which are recognized by the OBD of LT are marked with arrows and numbered as was reported by Kwun et al. [ 31 ]. Arrows with dashed lines indicate imperfect pentanucleotides. The EMSA Probe was generated by PCR amplification of the indicated region of the MCPyV Ori. This PCR product was 5' end-labeled with [ 32 P-γ] ATP using T4 polynucleotide kinase (indicated by red asterisk); ( B ) Western blot of purified MCPyV proteins (0.25 µg) used in EMSA. The buffer control contained residual TEV protease (also in LT samples); ( C ) Electromobility shift assays were performed with the EMSA probe in ( A ) and increasing amounts of MCPyV wild type or phospho-mutant LT affinity purified from HEK 293 cells. Reactions with buffer and residual TEV protease served as a negative control (first lane). Positions of free probe and LT bound probe are indicated. Data in ( B , C ) are representative of at least three experiments.

    Techniques Used: Sequencing, Clone Assay, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Labeling, Western Blot, Purification, Mutagenesis, Affinity Purification, Negative Control

    5) Product Images from "Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity"

    Article Title: Unusual DNA packaging characteristics in endoreduplicated Caenorhabditis elegans oocytes defined by in vivo accessibility to an endogenous nuclease activity

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-6-37

    Genetic requirement for the DNase activity present in unfertilized oocytes.  Genomic DNA extracted from purified oocytes ( 'fer-1  oocytes’) or from liquid-nitrogen-flash-frozen samples (N2 (wild type) embryos and adults of various genotypes) was radioactively-labeled using T4 polynucleotide kinase and gamma-32P-ATP, and resolved on a denaturing 12% polyacrylamide gel (8 M urea). A total of 2 μg of DNA were used for labeling and PAGE analysis for all samples except DNA from isolated  fer-1(b232)  oocytes (
    Figure Legend Snippet: Genetic requirement for the DNase activity present in unfertilized oocytes. Genomic DNA extracted from purified oocytes ( 'fer-1 oocytes’) or from liquid-nitrogen-flash-frozen samples (N2 (wild type) embryos and adults of various genotypes) was radioactively-labeled using T4 polynucleotide kinase and gamma-32P-ATP, and resolved on a denaturing 12% polyacrylamide gel (8 M urea). A total of 2 μg of DNA were used for labeling and PAGE analysis for all samples except DNA from isolated fer-1(b232) oocytes (

    Techniques Used: Activity Assay, Purification, Labeling, Polyacrylamide Gel Electrophoresis, Isolation

    6) Product Images from "Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique"

    Article Title: Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-888

    Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b).  Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library  (a)  the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol  (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.
    Figure Legend Snippet: Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b). Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library (a) the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.

    Techniques Used: Isolation, Ligation, Amplification, Sequencing, Polymerase Chain Reaction

    7) Product Images from "Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period"

    Article Title: Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks779

    Top1 binds to intermediate region between ROREs in  Bmal1  promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30  and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.
    Figure Legend Snippet: Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.

    Techniques Used: Cleavage Assay, Labeling, Purification, Cycling Probe Technology

    8) Product Images from "Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles"

    Article Title: Deep small RNA sequencing from the nematode Ascaris reveals conservation, functional diversification, and novel developmental profiles

    Journal:

    doi: 10.1101/gr.121426.111

    Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in
    Figure Legend Snippet: Characteristics of Ascaris small RNAs. ( A ) 5′ end-labeled Ascaris small RNAs. Low-molecular-weight (LMW) enriched RNAs were treated with calf alkaline phosphatase and then 5′ end labeled with 32 P using T4 polynucleotide kinase. RNAs in

    Techniques Used: Labeling, Molecular Weight

    9) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL "

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL

    Journal:

    doi: 10.1128/JVI.05479-11

    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.
    Figure Legend Snippet: Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.

    Techniques Used: Purification, Binding Assay, Footprinting, Labeling

    10) Product Images from "High-throughput screening of soluble recombinant proteins"

    Article Title: High-throughput screening of soluble recombinant proteins

    Journal:

    doi:

    Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.
    Figure Legend Snippet: Moleclular cloning strategy. Four PCR primers and reactions were used in two separate tubes. An equal amount of the two PCR products were mixed, and then the 5` ends were phosphorylated with T4 polynucleotide kinase. After denaturing (95°C for 5 min) and renaturing (65°C for 10 min), ∼25% of the final products carry EcoRI (5`) and XhoI (3`) cohesive ends and are ready for ligation with the vectors.

    Techniques Used: Clone Assay, Polymerase Chain Reaction, Ligation

    11) Product Images from "Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique"

    Article Title: Comprehensive analysis of the Corynebacterium glutamicum transcriptome using an improved RNAseq technique

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-14-888

    Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b).  Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library  (a)  the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol  (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.
    Figure Legend Snippet: Experimental workflow for the preparation of a whole transcriptome library (a) and of a library enriched for primary 5′-transcript ends (b). Both protocols start with isolated total RNA. Stable RNA is then depleted using the Ribo-Zero rRNA removal kit and the obtained RNA is fragmented my metal hydrolysis to a size of 200 - 500 nt. For the whole transcriptome library (a) the 5′-triphosphate ends are processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase, unphosphorylated 5′-ends are phosphorylated, and phosphorylated 3′-ends are then dephosphorylated using T4 polynucleotide kinase. For the native 5′-end protocol (b) , all fragments containing a 5′-monophosphate are degraded by treatment with a 5′-phosphate dependent exonuclease and the 5′-triphosphate ends of native transcripts are then processed to 5′-monophosphate ends by a RNA 5′-polyphosphatase. Next, for both libraries RNA adapters are ligated to the 5′-ends carrying a 5′-monophosphate group. The tagging of the 3′-end of the RNA with flanking sequences necessary for reverse transcription is performed in a ligation-free approach with a loop DNA adapter containing seven unpaired wobble bases at its 3′-end. After reverse transcription of the RNA fragments into cDNA fragments, the cDNA fragments are amplified, tagged with sequencing linkers at their ends by PCR and finally sequenced. Stable RNA species (rRNA, tRNA) are depicted in red, other RNAs are given in green, and DNA in blue.

    Techniques Used: Isolation, Ligation, Amplification, Sequencing, Polymerase Chain Reaction

    12) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with  Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography, Modification

    13) Product Images from "Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period"

    Article Title: Rhythmic binding of Topoisomerase I impacts on the transcription of Bmal1 and circadian period

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gks779

    Top1 binds to intermediate region between ROREs in  Bmal1  promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30  and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.
    Figure Legend Snippet: Top1 binds to intermediate region between ROREs in Bmal1 promoter region. Top1-mediated cleavage assay ( A ). DNA fragment around the ROREs (nucleotides −88 to −22: 5′-GATTGGTCGGAAAGTAGGTTAGTGGTGCGACATTTAGGGAAGGCAGAAAGTAGGTCAGGGACGGAGG-3′) was end-labeled with [γ- 32 P]ATP using T4 polynucleotide kinase. DNA fragment was reacted with either 50 units of Top1 alone or with 2–50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels. CPT, camptothecin. EMSA using probe, nucleotides −88 to −22, 15 units of Top1 protein and a 100-fold molar excess of the following competitors (Comp): AT, control oligonucleotides, (dA) 30 and (dT) 30 ; unlabeled probe, nucleotides −88 to −22; −67 to −43, nucleotides −67 to −4 ( B ). Arrowhead, shifted band.

    Techniques Used: Cleavage Assay, Labeling, Purification, Cycling Probe Technology

    14) Product Images from "A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions"

    Article Title: A library-based method to rapidly analyse chromatin accessibility at multiple genomic regions

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp037

    Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro  controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short  and Adaptor-A long  or Adaptor-B short  and Adaptor-B long , where adaptor oligonucleotide B long  is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by  Bst  polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.
    Figure Legend Snippet: Overview of the experimental steps required to create and analyse a chromatin accessibility library. ( A ) Step 1: fungal mycelia pre-grown under specific conditions or isolated DNA ( in vitro controls) are processed as described in Materials and methods section and digested with MNase or restriction enzymes of choice. Step 2: digested DNA is blunt-ended and phosphorylated by subsequent treatment of the chromatin with Klenow fragment polymerase, T4 polynucleotide kinase. This step produces blunt-ended DNA fragments for ligation with adaptors. Step 3: DNA fragments are ligated with double-stranded adaptors A and B, originating from oligonucleotides Adaptor-A short and Adaptor-A long or Adaptor-B short and Adaptor-B long , where adaptor oligonucleotide B long is biotinylated for later retention on the streptavidin beads. In this step, fragments containing all adaptor combinations (A-A, A-B and B-B) are generated. Step 4: the ligation step leaves nicks at the 3′-terminus that are repaired by Bst polymerase treatment. Step 5: all fragments containing biotinylated adaptor B are captured on streptavidin-coated magnetic beads. At this step, adaptor A-A fragments are lost. Step 6: after a washing step, the retained fragments (adaptors A-B and B-B fragments) are denatured at 95°C. The denaturation step results in the release of single strands which exclusively carry A-B adaptor fragments. Step 7: the single-stranded A-B adaptor fragment library is amplified by a nested PCR approach to give the final A-B fragment library. The input and output fragment libraries are quality controlled by amplification with single A and B, as well as mixed A-B primers. Only the A-B primer mix should result in the amplification of fragments in the range of 200–1000 bp (see Panel B). Step 8: the resulting A-B adaptor fragment library is diluted and aliquots are used for analytical PCR amplifications for fragment size analysis of specific loci of interest. In the final analytical PCR step, either gene-specific or adaptor-specific primers can be labelled for subsequent capillary sequencer analysis. The chromatograms are finally analysed by image analysis software. ( B ) Example of quality control of A-B adaptor fragment libraries. Two input chromatin fragment libraries without adaptor ligation (lanes 1 and 2) are compared to two output libraries with adaptor ligation as described in Materials and methods section (lanes 3 and 4). Libraries originating from nitrate-grown cells (lanes 1 and 3) as well as from ammonium-grown cells (lanes 2 and 4) are shown as an example. M, DNA size marker.

    Techniques Used: Isolation, In Vitro, Ligation, Generated, Magnetic Beads, Amplification, Nested PCR, Polymerase Chain Reaction, Software, Marker

    15) Product Images from "Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL"

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus Rta Tetramers Make High-Affinity Interactions with Repetitive DNA Elements in the Mta Promoter To Stimulate DNA Binding of RBP-Jk/CSL

    Journal:

    doi: 10.1128/JVI.05479-11

    Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.
    Figure Legend Snippet: Purified Rta makes extended contacts to the Mta promoter that flank the RBP-Jk binding site. (A) Footprinting of the indicated proteins to the top strand of the Mta promoter was performed using 3 × 10 3 cpm DNA labeled with T4 polynucleotide kinase.

    Techniques Used: Purification, Binding Assay, Footprinting, Labeling

    16) Product Images from "RISC is a 5? phosphomonoester-producing RNA endonuclease"

    Article Title: RISC is a 5? phosphomonoester-producing RNA endonuclease

    Journal:

    doi: 10.1101/gad.1187904

    Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′-32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′-32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′-32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.
    Figure Legend Snippet: Target RNA is cleaved endonucleolytically producing 5′-phosphate and 3′-hydroxyl termini. ( A ) Preparation of site-specifically labeled substrates and cleavage assay. 5′-32 P-labeled and 3′ aminolinker (L) protected 12-nt oligoribonucleotide was ligated to nonphosphorylated 9-nt oligoribonucleotide using T4 RNA ligase. An aliquot of the ligation product was further 5′-32 P-labeled using T4 polynucleotide kinase. The purified substrates were incubated with affinity-purified RISC programmed with single-stranded guide siRNA. ( B ) PhosphorImaging of cleavage reactions incubated for 2 h at 30°C, and resolved on a 20% denaturing polyacrylamide gel. 5′-32 P-labeled 9- and 12-nt oligoribonucleotides were loaded as marker in lanes 1 and 2 , respectively. The cleavage reactions with single- and double-labeled 21-nt substrate are loaded in lanes 4 and 5 , respectively. Lane 3 contains the 12-nt cleavage product isolated from a prior cleavage reaction. ( C ) Two-dimensional thin layer chromatography analysis of the ribonuclease T2-digested RISC-cleavage product. The oval depicts the unlabeled pAp as detected by UV shadowing. The radioactive signal comigrates with the 5′ 32 pAp released by ribonuclease T2 digestion from the gel-purified 12-nt cleavage product.

    Techniques Used: Labeling, Cleavage Assay, Ligation, Purification, Incubation, Affinity Purification, Marker, Isolation, Thin Layer Chromatography

    17) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with  Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography, Modification

    18) Product Images from "A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA"

    Article Title: A novel method for the efficient and selective identification of 5-hydroxymethylcytosine in genomic DNA

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkr051

    The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with  Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.
    Figure Legend Snippet: The β-gt can specifically modify 5hmC residues at a high efficiency. ( a ) Oligonucleotides that were either incubated in the presence or absence of the β-gt were digested with Taq I, treated with alkaline phosphatase, 5′-end labeled using T4 polynucleotide kinase and digested to 5′-mononucleotides using DNase I and Snake Venom Phosphodiesterase. Radiolabeled mononucleotides were analyzed by two-dimensional TLC. C, 3′-deoxyribocytosine-5′-monophosphate; T, 3′-deoxyribothymidine-5′-monophosphate; 5meC, 3′-deoxyribo-N5-methylcytosine-5′-monophosphate; 5hmC, 3′-deoxyribo-N5-hydroxymethylcytosine-5′-monophosphate. ( b ) HPLC coupled to tandem mass spectrometry was used to measure the efficiency of the β-gt reaction. Substrates analyzed were 2.7 kb linear PCR products of pUC18: the dC substrate contained only cytosine residues; the 5meC substrate was created by methylating the CpG dinucleotide of the cytosine substrate; the 5hmC substrate was created by using d5hmC in place of dCTP in the PCR reactions; the β-glu-5hmC substrate was created by incubating the 5hmC substrate with the β-gt in the presence of UDP-glucose. Control DNA was prepared from salmon sperm. LC/MS/MS chromatograms of the cytosine residues from each of the substrates are presented. Abbreviations: dC, 3′-deoxyribocytosine; 5me(dC), 3′-deoxyribo-N5-methylcytosine; 5hm(dC), 3′-deoxyribo-N5-hydroxymethylcytosine; 5-glu-hm(dC), 3′-deoxyribo-N5-(β- d -glucosyl(hydroxymethyl))cytosine. Asterisks indictes that cytosines are only 5meC modified at CpG sequences.

    Techniques Used: Incubation, Labeling, Thin Layer Chromatography, High Performance Liquid Chromatography, Mass Spectrometry, Polymerase Chain Reaction, Liquid Chromatography, Modification

    19) Product Images from "Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease"

    Article Title: Crystal structure and assembly of the functional Nanoarchaeum equitans tRNA splicing endonuclease

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp537

    Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the  N. equitans  tRNA Glu  precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of  N. equitans  tRNA Glu  precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) (  13 ,  27 ,  28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB (  28 ), a bulge–helix–loop (BHL) (  29 ) and a  trans -spliced BHL formed by two split half tRNA genes (  13 ,  29 ).
    Figure Legend Snippet: Splicing activity and specificity of the NEQ RNA splicing endonuclease. ( A ) A 3′-radiolabeled RNA transcript of the N. equitans tRNA Glu precursor was incubated without enzyme (–) or with either 1 μM NEQ205-NEQ261 splicing endonuclease at 65°C for 20 min alone or followed by incubation with T4 polynucleotide kinase (PNK) and T4 RNA ligase. AFU splicing endonuclease was incubated with a substrate as a positive control. ( B ) Secondary structure of the relaxed BHB motif of N. equitans tRNA Glu precursor. The predicted cleavage sites are indicated by arrows and the CUC anticodon is indicated by a line. ( C ) The mature tRNA product was excised, amplified by RT-PCR and sequenced. The anticodon loop was correctly assembled and the anticodon is underlined. ( D ) Examples of the RNA substrates cleaved by the tRNA splicing endonuclease that have been confirmed biochemically, i.e. canonical bulge–helix–bulge (BHB) RNA substrate (left panel) ( 13 , 27 , 28 ) and non-canonical BHB substrates (right panel). For non-canonical substrates, from the left: a synthetic 4–3–3 and 2–3–3 BHB ( 28 ), a bulge–helix–loop (BHL) ( 29 ) and a trans -spliced BHL formed by two split half tRNA genes ( 13 , 29 ).

    Techniques Used: Activity Assay, Incubation, Positive Control, Amplification, Reverse Transcription Polymerase Chain Reaction

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    Chloramphenicol Acetyltransferase Assay:

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: For the ssDNA substrate, the following high-performance liquid chromatography (HPLC)-purified oligonucleotide was used: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. The 5′-end of the oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP.

    Hybridization:

    Article Title: Irradiation-Induced Deinococcus radiodurans Genome Fragmentation Triggers Transposition of a Single Resident Insertion SequenceA Major Role of the RecFOR Pathway in DNA Double-Strand-Break Repair through ESDSA in Deinococcus radioduransRising from the Ashes: DNA Repair in Deinococcus radiodurans
    Article Snippet: 10 pmol of oligonucleotide was mixed with 16 pmol of [γ-32 P] ATP (5000 Ci/mmol, Amersham Inc.) and 1 unit of T4 kinase (NEB Inc.) in T4 kinase buffer (70 mM Tris–HCl pH 7.6, 10 mM MgCl2 , 5 mM DTT). .. Labelled oligonucleotides were purified by filtration through Sephadex G25.

    High Performance Liquid Chromatography:

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: For the ssDNA substrate, the following high-performance liquid chromatography (HPLC)-purified oligonucleotide was used: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. The 5′-end of the oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP.

    Ligation:

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C. .. Y-tailed Illumina adapters were used, that allow amplification from 2 different primers complementary to single strand tails of the adapter (Genomic DNA/ChIP-Seq oligonucleotide sequences at http://intron.ccam.uchc.edu/groups/tgcore/wiki/013c0/Solexa_Library_Primer_Sequences.html ).

    Northern Blot:

    Article Title: Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing
    Article Snippet: Paragraph title: Northern blot hybridizations ... DNA oligonucleotides with the exact reverse-complementary sequence to miRNAs were end-labeled with 32 P-γ-ATP and T4 polynucleotide kinase (New England Biolabs) to generate high specific activity probes.

    Generated:

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP. .. The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP.

    other:

    Article Title: Binding of the baculovirus very late expression factor 1 (VLF-1) to different DNA structures
    Article Snippet: Radiolabeled nucleotide [γ-32 P]ATP was from Perkin-Elmer, and T4 polynucleotide kinase was from New England Biolabs, bacteriophage T4 endonuclease VII was the kind gift of Dr. B. Kemper [ ].

    Article Title: Altered Order of Substrate Binding by DNA Polymerase X from African Swine Fever Virus
    Article Snippet: T4 polynucleotide kinase was obtained from New England BioLabs.

    Article Title: Formation of DNA-Protein Cross-Links Between ?-Hydroxypropanodeoxyguanosine and EcoRI
    Article Snippet: T4 polynucleotide kinase and PvuII were obtained from New England Biolabs (Beverly, MA).

    Article Title: Repair of mitomycin C mono- and interstrand cross-linked DNA adducts by UvrABC: a new model
    Article Snippet: The DS 61-bp fragments containing a site-specific ICL dG-MC-dG were 5′-end-labeled with γ-32 P-ATP by T4 polynucleotide kinase at both ends; the DNA fragments were then digested with SmaI to generate single 5′-end 32 P-labeled fragments.

    Sequencing:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: An electrophoretic mobility shift assay using hPOT1 was performed on the consensus TTA-22mer telomeric sequence (5′-AGGGTTAGGGTTAGGGTTAGGG-3′), on the CTA-22mer variant-repeat sequence (5′-AGGGCTAGGGCTAGGGCTAGGG-3′) or on the TCA-22mer variant-repeat sequence (5′-AGGGTCAGGGTCAGGGTCAGGG-3′). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling
    Article Snippet: Nuclear extracts (5 μg) were incubated with a 32 P-labelled 22-mer double-stranded oligonucleotide (5′-AGTT GAGGGGACTTTCC CAGGC-3′) containing the NF-κB consensus sequence (underlined) and 1 μg of poly(dI·dC) (Amersham Biosciences) in binding buffer (10 mM Tris–HCl, 100 mM NaCl, 2 mM EDTA, 4% (w/v) Ficoll, 1 mM DTT), for 30 min at 30°C. .. DNA probes were prepared by end-labelling bo th strands of the oligonucleotide using [γ-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

    Article Title: Molecular Evolution of a Viral Non-Coding Sequence under the Selective Pressure of amiRNA-Mediated Silencing
    Article Snippet: Ten µg of total RNA was resolved in a 15% polyacryamide/1× TBE (8.9 mM Tris, 8.9 mM boric acid, 20 mM EDTA)/8M urea gel and blotted to a Hybond-N+ membrane (Amersham). .. DNA oligonucleotides with the exact reverse-complementary sequence to miRNAs were end-labeled with 32 P-γ-ATP and T4 polynucleotide kinase (New England Biolabs) to generate high specific activity probes. .. Hybridization was carried out using the ULTRAHyb-Oligo solution according to the manufacturer's directions (Ambion) and signals were detected by autoradiography.

    Article Title: Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites
    Article Snippet: The fragment length distribution of the DNase I-treated naked DNA sample was similar to that of the DNase I-treated chromatin-specific DNA. .. After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB). .. The ligated products were purified on a MicroSpin S-400 spin column (GE Healthcare), sonicated to obtain fragments with a median length of 500 bp, purified with biotin-streptavidin interaction magnetic beads (Dynal), phosphorylated and blunted-end as above, and ligated to Adaptor-II (sequence available in Additional file ).

    Article Title: Expression of HNF4alpha in the human and rat choroid plexus - Implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier
    Article Snippet: Protein concentrations were determined according to the method of Smith et al [ ]. .. Forward and reverse oligonucleotides (for sequence information see Table ) were purchased from MWG Biotech (Ebersberg/Muenchen, Germany), annealed and 32 P-labeled using 32 PγATP and T4-kinase (New England Biolabs, Frankfurt, Germany). .. 2,5 μg Caco-2 cell nuclear extract and 105 cpm (0.027 ng) radiolabeled probe were incubated in binding buffer consisted of 25 mM HEPES, pH 7.6, 5 mM MgCl2 , 34 mM KCl, 2 mM DTT, 2 mM Pefabloc, 2% Aprotinin, 40 ng of poly (dI-dC)/μl and 100 ng of bovine serum albumin/μl for 20 minutes on ice.

    Sonication:

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: 20 ug of DNA in 500 ul H2 O was sonicated on ice using a Branson 450 sonifier (10% amplitude, 150 seconds, using pulses of 0.5 sec on, 0.5 sec off). .. End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C.

    Recombinant:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: Purified recombinant hPOT1 and TRF2 prepared from baculovirus expression were a generous gift from Dr D. Gomez (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Nucleic Acid Electrophoresis:

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: All oligonucleotides used for preparation of DNA constructs were purchased and purified by denaturing gel electrophoresis. .. The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP.

    Isolation:

    Article Title: In silico identification of conserved microRNAs in large number of diverse plant species
    Article Snippet: Low-molecular-weight RNA was isolated from total RNA by use of PEG precipitation. .. DNA oligonucleotides complementary to miRNA sequences were end labeled with γ-32P-ATP by use of T4 polynucleotide kinase (New England Biolabs).

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: Genomic DNA isolated from a lymphoblastoid cell line of HapMap individual NA12872 was used in this study. .. End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C.

    Size-exclusion Chromatography:

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: 20 ug of DNA in 500 ul H2 O was sonicated on ice using a Branson 450 sonifier (10% amplitude, 150 seconds, using pulses of 0.5 sec on, 0.5 sec off). .. End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C.

    Electrophoretic Mobility Shift Assay:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: An electrophoretic mobility shift assay using hPOT1 was performed on the consensus TTA-22mer telomeric sequence (5′-AGGGTTAGGGTTAGGGTTAGGG-3′), on the CTA-22mer variant-repeat sequence (5′-AGGGCTAGGGCTAGGGCTAGGG-3′) or on the TCA-22mer variant-repeat sequence (5′-AGGGTCAGGGTCAGGGTCAGGG-3′). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: Paragraph title: Electrophoretic mobility shift assay ... The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP.

    Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling
    Article Snippet: Paragraph title: Preparation of nuclear extracts and gel shift to confirm NF-κB downregulation ... DNA probes were prepared by end-labelling bo th strands of the oligonucleotide using [γ-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

    Article Title: Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites
    Article Snippet: After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB). .. Adapter-ligated DNase I-treated fragments attached to Dynal beads were amplified by PCR with Platinum Taq DNA Polymerase (Invitrogen).

    Article Title: Expression of HNF4alpha in the human and rat choroid plexus - Implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier
    Article Snippet: Paragraph title: Electrophoretic mobility shift assays ... Forward and reverse oligonucleotides (for sequence information see Table ) were purchased from MWG Biotech (Ebersberg/Muenchen, Germany), annealed and 32 P-labeled using 32 PγATP and T4-kinase (New England Biolabs, Frankfurt, Germany).

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer
    Article Snippet: Paragraph title: Electrophoretic mobility shift assay (EMSA) ... Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs).

    Purification:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: Purified recombinant hPOT1 and TRF2 prepared from baculovirus expression were a generous gift from Dr D. Gomez (Institut de Pharmacologie et de Biologie Structurale, Toulouse, France). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: All oligonucleotides used for preparation of DNA constructs were purchased and purified by denaturing gel electrophoresis. .. The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP.

    Article Title: Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites
    Article Snippet: The fragment length distribution of the DNase I-treated naked DNA sample was similar to that of the DNase I-treated chromatin-specific DNA. .. After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB). .. The ligated products were purified on a MicroSpin S-400 spin column (GE Healthcare), sonicated to obtain fragments with a median length of 500 bp, purified with biotin-streptavidin interaction magnetic beads (Dynal), phosphorylated and blunted-end as above, and ligated to Adaptor-II (sequence available in Additional file ).

    Article Title: Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope
    Article Snippet: Reactions were stopped by adding SDS sample buffer and processed for immunoblotting. .. Double-stranded oligonucleotides containing the AP-1 (5′-CGCTTGA TGAGTCA G-3′; AP-1 site underlined) and the Sp1 (5′-ATTCGATCG GGGCGG GGCGAGC-3′; Sp1 site underlined) consensus sites were labeled with γ[32 P]dATP using polynucleotide kinase (New England Biolabs, Inc.) and purified on a Sephadex G-50 column. .. EMSA was performed using the SNF of NIH-3T3 cells (5 μg total protein) and wild-type and lamin A/C–null mice MEFs (15 μg total protein) as previously described ( ).

    Polymerase Chain Reaction:

    Article Title: Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites
    Article Snippet: After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB). .. After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB).

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C. .. DNA was purified with phenol∶chloroform∶isoamyl alcohol (25∶24∶1) and precipitated with salt/isopropanol.

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer
    Article Snippet: EMSA was performed as previously described . .. Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs). .. Protein–DNA complexes were incubated for 90 min at room temperature in binding buffer containing 25 mM Tris pH 7.4, 0.1 mM ZnSO4, 5 mM MgCl2, 5% Nonidet P-40 in PBS, 0.25 mM Mercaptoethanol, 10% glycerol and 0.5 µg of poly dI-dC.

    Labeling:

    Article Title: In silico identification of conserved microRNAs in large number of diverse plant species
    Article Snippet: Membranes were UV cross-linked and baked for 2 h at 80°C. .. DNA oligonucleotides complementary to miRNA sequences were end labeled with γ-32P-ATP by use of T4 polynucleotide kinase (New England Biolabs). .. Membranes were prehybridized for at least 1 h and hybridized overnight with use of Perfect hybridization buffer (Sigma) at 38°C.

    Article Title: Characterization of global microRNA expression reveals oncogenic potential of miR-145 in metastatic colorectal cancer
    Article Snippet: Sequences of Starfire probes used are shown in Additional File . .. The U6 snRNA oligonucleotide probe (5' AAC GCT TCA CGA ATT TGC GT 3') was end labeled using 20 pmole oligonucleotide probe, 1× T4 polynucleotide buffer (NEB), 50 μCi γ-32 P-dATP (10 mCi/mL, 6000 Ci/mmol) (Perkin Elmer) and 10 U T4 polynucleotide kinase (NEB), in a final volume of 20 μL. .. The reaction was stopped by the addition of 40 μL 10 mM EDTA.

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: A common 37-nt oligonucleotide was used to generate the different constructs. .. The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP. .. The sequences of duplex as well as DNA with nick, gap, loops and overhangs were as described in ( ) or presented in Supplementary Table S1 .

    Article Title: Genome-scale identification of Caenorhabditis elegans regulatory elements by tiling-array mapping of DNase I hypersensitive sites
    Article Snippet: After treatment with T4 Polynucleotide Kinase (NEB) the DNase I-treated fragments were blunt-ended with Klenow DNA Polymerase (NEB), purified with the QIAquick Nucleotide Removal Kit (Qiagen), and ligated to the biotinylated Adaptor-I (sequence available in Additional file ) with T4 DNA ligase (NEB). .. Adapter-ligated DNase I-treated fragments attached to Dynal beads were amplified by PCR with Platinum Taq DNA Polymerase (Invitrogen).

    Article Title: Fast regulation of AP-1 activity through interaction of lamin A/C, ERK1/2, and c-Fos at the nuclear envelope
    Article Snippet: Reactions were stopped by adding SDS sample buffer and processed for immunoblotting. .. Double-stranded oligonucleotides containing the AP-1 (5′-CGCTTGA TGAGTCA G-3′; AP-1 site underlined) and the Sp1 (5′-ATTCGATCG GGGCGG GGCGAGC-3′; Sp1 site underlined) consensus sites were labeled with γ[32 P]dATP using polynucleotide kinase (New England Biolabs, Inc.) and purified on a Sephadex G-50 column. .. EMSA was performed using the SNF of NIH-3T3 cells (5 μg total protein) and wild-type and lamin A/C–null mice MEFs (15 μg total protein) as previously described ( ).

    Article Title: The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study
    Article Snippet: Diverging lines (lines crossing at the right of the y-axis) indicate antagonism [ ]. .. DNA primers D19, D25, and D26 were 5'-end labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and [γ-32 P]ATP as previously described [ ]. .. All DNA/DNA primer/templates were annealed by incubating a 1:1.3 molar ratio of primer to template in 50 mM Tris-HCl buffer containing 50 mM NaCl at 90°C for 3 min, 50°C for 10 min, and left on ice for 10 min.

    Article Title: Human PSF binds to RAD51 and modulates its homologous-pairing and strand-exchange activities
    Article Snippet: For the strand exchange assay with olignucleotides, the following HPLC-purified DNA and RNA oligonucleotides were purchased from Nihon Gene Research Laboratory and Gene Design: 63-mer 5′-TCC TTT TGA TAA GAG GTC ATT TTT GCGGAT GGC TTA GAG CTT AAT TGC TGA ATC TGG TGCTGT-3′, 32-mer top strand, 5′-CCA TCC GCA AAA ATG ACC TCT TAT CAA AAG GA-3′, 32-mer bottom strand, 5′-TCC TTT TGA TAA GAG GTC ATT TTT GCG GAT GG-3′, 32-mer RNA, 5′-UCC UUU UGA UAA GAG GUC AUU UUU GCG GAU GG-3′. .. The 5′ ends of the oligonucleotide (32-mer top strand) were labeled with T4 polynucleotide kinase in the presence of [γ-32 P]ATP. .. For the pull-down assay with the ssDNA beads, the following HPLC-purified 5′ biotinylated DNA oligonucleotide was purchased from Nihon Gene Research Laboratory: 80-mer 5′-CTG CTT TAT CAA GAT AAT TTT TCG ACT CAT CAG AAA TAT CCG AAA GTG TTA ACT TCT GCG TCA TGG AAG CGA TAA AAC TC-3′.

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: For the ssDNA substrate, the following high-performance liquid chromatography (HPLC)-purified oligonucleotide was used: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. The 5′-end of the oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP. .. RAD51 (0.1 μM) was incubated with the indicated amount of DIDS at 37°C for 5 min, in 7 μl of reaction buffer, containing 22 mM HEPES–NaOH (pH 7.5), 15 mM NaCl, 0.01 mM EDTA, 0.2 mM 2-mercaptoethanol, 1% glycerol, 1 mM MgCl2 , 1 mM DTT, 2 mM AMPPNP and 0.1 mg/ml BSA.

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer
    Article Snippet: EMSA was performed as previously described . .. Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs). .. Protein–DNA complexes were incubated for 90 min at room temperature in binding buffer containing 25 mM Tris pH 7.4, 0.1 mM ZnSO4, 5 mM MgCl2, 5% Nonidet P-40 in PBS, 0.25 mM Mercaptoethanol, 10% glycerol and 0.5 µg of poly dI-dC.

    Sample Prep:

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: Paragraph title: Sample preparation ... End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C.

    Activated Clotting Time Assay:

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: For the ssDNA substrate, the following high-performance liquid chromatography (HPLC)-purified oligonucleotide was used: 50-mer, 5′-GGA ATT CGG TAT TCC CAG GCG GTC TCC CAT CCA AGT ACT AAC CGA GCC CT-3′. .. The 5′-end of the oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP.

    Plasmid Preparation:

    Article Title: DIDS, a chemical compound that inhibits RAD51-mediated homologous pairing and strand exchange
    Article Snippet: To prevent the dsDNA substrates from undergoing irreversible denaturation, superhelical dsDNA (pB5Sarray DNA), which contained 11 repeats of a sea urchin 5S rRNA gene (207-bp fragment) within the pBlueScript II SK(+) vector, was prepared by a method avoiding alkaline treatment of the cells harboring the plasmid DNA ( , ). .. The 5′-end of the oligonucleotide was labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA, USA) in the presence of [γ-32 P]ATP.

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer
    Article Snippet: Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs). .. Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs).

    Software:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Irradiation:

    Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling
    Article Snippet: Nuclei were prepared from the irradiated cells by washing the collected cells twice in 500 μl hypotonic buffer, followed by lysis in hypotonic buffer (10 mM Tris–HCl (pH 7.8), 5 mM KCl, 2 mM MgCl2 , 1 mM DTT, complete protease inhibitors (Roche Diagnostics)), supplemented with 0.25% IGEPAL CA-630 (Sigma), for 5 min on ice. .. DNA probes were prepared by end-labelling bo th strands of the oligonucleotide using [γ-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

    Binding Assay:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: Paragraph title: TRF2 and POT1 binding assays ... Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

    Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling
    Article Snippet: Nuclear extracts (5 μg) were incubated with a 32 P-labelled 22-mer double-stranded oligonucleotide (5′-AGTT GAGGGGACTTTCC CAGGC-3′) containing the NF-κB consensus sequence (underlined) and 1 μg of poly(dI·dC) (Amersham Biosciences) in binding buffer (10 mM Tris–HCl, 100 mM NaCl, 2 mM EDTA, 4% (w/v) Ficoll, 1 mM DTT), for 30 min at 30°C. .. DNA probes were prepared by end-labelling bo th strands of the oligonucleotide using [γ-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

    Article Title: Expression of HNF4alpha in the human and rat choroid plexus - Implications for drug transport across the blood-cerebrospinal-fluid (CSF) barrier
    Article Snippet: Forward and reverse oligonucleotides (for sequence information see Table ) were purchased from MWG Biotech (Ebersberg/Muenchen, Germany), annealed and 32 P-labeled using 32 PγATP and T4-kinase (New England Biolabs, Frankfurt, Germany). .. Forward and reverse oligonucleotides (for sequence information see Table ) were purchased from MWG Biotech (Ebersberg/Muenchen, Germany), annealed and 32 P-labeled using 32 PγATP and T4-kinase (New England Biolabs, Frankfurt, Germany).

    Agarose Gel Electrophoresis:

    Article Title: Detection of Genomic Variation by Selection of a 9 Mb DNA Region and High Throughput Sequencing
    Article Snippet: End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C. .. End filling of DNA breaks was performed in 200 ul (18U T4 DNA Polymerase, 60U T4 Polynucleotide Kinase, NEB T4 DNA Polymerase Buffer with BSA, 1 mM dNTP, 0.5 mM ATP (New England Biolabs)) for 20 minutes at 12°C, 20 minutes at 25°C, and 20 minutes at 75°C.

    In Vitro:

    Article Title: The Structural Complexity of the Human BORIS Gene in Gametogenesis and Cancer
    Article Snippet: Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs). .. Briefly, PCR fragments were labeled using 32 P-γ-ATP with T4 polynucleotide kinase (New England Biolabs).

    Modification:

    Article Title: The triple combination of tenofovir, emtricitabine and efavirenz shows synergistic anti-HIV-1 activity in vitro: a mechanism of action study
    Article Snippet: DNA primers D19, D25, and D26 were 5'-end labeled with T4 polynucleotide kinase (New England Biolabs, Ipswich, MA) and [γ-32 P]ATP as previously described [ ]. .. The annealed primer/templates were analyzed by non-denaturing polyacrylamide gel (4–20% TBE) electrophoresis to ensure that the proper annealing had taken place.

    Concentration Assay:

    Article Title: The yeast high mobility group protein HMO2, a subunit of the chromatin-remodeling complex INO80, binds DNA ends
    Article Snippet: The top strand was 5′-end labeled using T4 polynucleotide kinase (New England Biolabs) and [γ32 P] ATP. .. Similarly, a 50 bp DNA duplex and duplex with either a single or tandem loops was generated by extending the 37 bp construct at both ends.

    Lysis:

    Article Title: Dissection of a complex transcriptional response using genome-wide transcriptional modelling
    Article Snippet: Nuclei were prepared from the irradiated cells by washing the collected cells twice in 500 μl hypotonic buffer, followed by lysis in hypotonic buffer (10 mM Tris–HCl (pH 7.8), 5 mM KCl, 2 mM MgCl2 , 1 mM DTT, complete protease inhibitors (Roche Diagnostics)), supplemented with 0.25% IGEPAL CA-630 (Sigma), for 5 min on ice. .. DNA probes were prepared by end-labelling bo th strands of the oligonucleotide using [γ-32 P]ATP (Amersham Biosciences) and T4 polynucleotide kinase (New England Biolabs).

    Variant Assay:

    Article Title: Human telomeres that contain (CTAGGG)n repeats show replication dependent instability in somatic cells and the male germline
    Article Snippet: An electrophoretic mobility shift assay using hPOT1 was performed on the consensus TTA-22mer telomeric sequence (5′-AGGGTTAGGGTTAGGGTTAGGG-3′), on the CTA-22mer variant-repeat sequence (5′-AGGGCTAGGGCTAGGGCTAGGG-3′) or on the TCA-22mer variant-repeat sequence (5′-AGGGTCAGGGTCAGGGTCAGGG-3′). .. Oligonucleotides were labelled at the 5′ end with [γ-32 P]-ATP using T4 Polynucleotide Kinase (New England BioLabs®).

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  • 99
    New England Biolabs 5 phosphorylated
    Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with <t>5′-phosphorylated</t> pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.
    5 Phosphorylated, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 9 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    78
    New England Biolabs 5 ends
    Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between  E.coli  0157 and  E.coli  K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.
    5 Ends, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 78/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with 5′-phosphorylated pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Structure of a ssDNA molecule bound within the active site of LHK-Exo. ( A ) The structure of LHK-Exo complexed with 5′-phosphorylated pentamer of oligothymidine [5′-PO 4 -(dT) 5 ]. The oligonucleotide is shown in yellow-colored stick form and is covered by a 2 F o – F c electron density map contoured at 1σ (calculated at 2.80 Å). Bound Mg 2+ ions are shown as gray spheres, and the residues that interact with the ssDNA molecule are labeled and shown in stick form. dAMP ligand (green) is superposed on the ssDNA molecule and the 5′-phosphate groups of dAMP and ssDNA bind in identical positions. ( B ) After mapping the electrostatic potential onto the protein surface, four residues of the ssDNA ligand (green) can be seen to fit comfortably into a binding channel within one of the LHK-Exo monomers. The 5′- and 3′-termini of the complexed ssDNA molecule are indicated. The trimer is shown with the wide-end of the tapered central channel facing the front, i.e. DNA enters from the front face. ( C and D ) Surface representations of the native structures of λ-exonuclease (PDB code: 1AVQ) and the HS_1420 exonuclease from Haemophilus somnus 129PT (PDB code: 3K93), respectively. Both exonucleases contain similar channels (one is indicated with a yellow arrow) that may accommodate the 5′-end of the DNA strand to be hydrolyzed; with a more ‘closed’ channel observable for λ-exonuclease (C). Both protein trimers are shown orientated in a manner analogous to that of LHK-Exo in B, revealing an equivalent positioning of the ssDNA-binding channels adjacent to the central tapered channel.

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Labeling, Binding Assay

    Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Qualitative analysis of ssDNA and dsDNA hydrolysis activities of LHK-Exo. ( A ) dsDNA exonuclease activities. Agarose gel showing aliquots taken (0–15 min) from an incubation of LHK-Exo (30 µg, 0.41 nmol of trimers) and BamHI-linearized pET28a (1.8 µg, 0.54 pmol) in Tris–HCl (pH 8.0, 50 mM), 50 mM NaCl, 7.5 mM MgCl 2 at 37°C. ( B ) Polarity of dsDNA digestion. A total of 6 µg of LHK-Exo (82 pmol of trimers, lanes 2–5) or λ-exonuclease (74 pmol of trimers, lanes 6–9) protein was incubated with 0.1 µg (0.23 pmol) of a 712-bp linear 5′-phosphorylated dsDNA substrate (‘unmodified’; lanes 2, 3, 6 and 7), or an analogous 5′-phosphorylated linear dsDNA substrate containing three consecutive ‘nuclease-resistant’ phosphorothioate linkages at its 5′-termini (‘PT-modified’; lanes 4, 5, 8, 9). Assays were quenched immediately (0 min) or incubated at 37°C for 20 min, before analysis of digestion products on 1% agarose gels. ( C ) Digestion of 5′-phosphorylated ssDNA. Reaction mixtures (80 µl) containing LHK-Exo (4.5 µg, 61.4 pmol of trimers) and 5′-PO 4 -(dT) 50 (0.4 nmol) in 25 mM Tris–HCl (pH 8.0), 7.5 mM MgCl 2 , 1 mM DTT were incubated at 37°C. Aliquots (20 µl) were withdrawn after 0, 0.5, 5 and 20 min, and immediately quenched. Reaction products were analyzed by denaturing gel electrophoresis. ( D ) Digestion of non-phosphorylated ssDNA. Analogous sets of assays were performed using non-phosphorylated 50-mers of oligothymidine [5′-OH-(dT) 50 ]. Fluorescent gel images were scanned after SYBR Gold staining. A ssDNA ladder [Oligo Length Standards 20/100 Ladder (IDT)] is included in lane 1.

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Agarose Gel Electrophoresis, Incubation, Modification, Nucleic Acid Electrophoresis, Staining

    Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Journal: Nucleic Acids Research

    Article Title: Structural and functional insight into the mechanism of an alkaline exonuclease from Laribacter hongkongensis

    doi: 10.1093/nar/gkr660

    Figure Lengend Snippet: Processivity of double strand DNA digestion by wild-type LHK-Exo and Arg12Ala mutant. Time course analysis of the digestion of 5′-phosphorylated double strand DNA (5′-PO 4 -dsDNA: EcoRV-linearized pMal-c2) and 5′-dephosphorylated double strand DNA (5′-OH-dsDNA: 5′dephosphorylated EcoRV-linearized pMal-c2) substrates by wild-type LHK-Exo and the Arg12Ala mutant form using a ‘heparin trap’ approach. A total of 6 µg (82 pmol of trimers) of LHK-Exo or Arg12Ala mutant protein was incubated at 25°C with 60 ng (0.015 pmol) of 5′-PO 4 -dsDNA or 5′-OH-dsDNA in Tris–HCl (25 mM, pH 8.0), 1 mM DTT, 7.5 mM MgCl 2 . After 15 s, excess heparin was added to sequester all unbound protein, and to prevent disassociated protein from re-binding. Aliquots were removed at various time points (0–20 min), and dsDNA levels were determined using fluorescent PicoGreen assays, to enable the extent of DNA digestion to be calculated. In one set of assays, heparin was added to LHK-Exo prior to the addition of dsDNA substrate, to confirm the efficacy of the heparin trap method (filled black squares, green line). Graphs show the mean number of nucleotides digested from each terminus (±SD; y -axis) plotted against the time of analysis (in minutes; x -axis).

    Article Snippet: Both PCR-amplified substrates were 5′-phosphorylated (T4 polynucleotide kinase, NEB) and purified (QIAquick PCR purification kit, QIAgen) prior to use.

    Techniques: Mutagenesis, Incubation, Binding Assay

    The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in  a - d  were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Journal: Nature structural & molecular biology

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    doi: 10.1038/nsmb.1440

    Figure Lengend Snippet: The antigenomic HDV small RNA is 2′-3′ hydroxylated and has an mRNA-like 5′ cap (Northern Blot, 293 cells, RNA induction). ( a ) 3′ end by β-elimination. The mobility of the HDV small RNA is increased following β-elimination. miR-15a: 2′-3′ hydroxylated positive control; +β: +β-elimination; -β: untreated RNA. ( b ) 5′ end by enzymatic analysis. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Decapping enzyme (TAP; +HDV); 5: T4 RNA Ligase (+HDV); 6: Terminator Exonuclease (+HDV). The size of the HDV small RNA was estimated to be ∼24nt based on the largely 22nt, 5′ phosphorylated miR15-a shown in the inset (IS). ( c ) Confirmation that the 5′ end of the HDV small RNA is capped, not triphosphorylated (enlarged image to emphasize changes in gel mobility for miR-15a, but not HDV small RNA). 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: Antarctic Phosphatase (+HDV); 4: Antarctic Phosphatase followed by T4 PNK (+HDV). ( d ) RNA immunoprecipitation with anti-2,2,7-trimethylguanosine antibody K121. The immunoprecipitation efficiency of the HDV small RNA, U5 snRNA (positive control) and microRNAs miR-15a and let-7a (negative controls) was analysed by Northern blot. ‘S’: supernatant; ‘I’: IP fraction. ( e ) Predicted structure of the HDV small RNA. The various RNAs in a - d were detected after stripping and rehybridisation to the same blot. M: RNA marker.

    Article Snippet: The primer extension oligo was end-labeled with T4 PNK (NEB) and γ-32 P ATP (PerkinElmer).

    Techniques: Northern Blot, Positive Control, Immunoprecipitation, Stripping Membranes, Marker

    Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in  Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9  particles). MR: RNA Marker. The various RNAs in  c-f  were detected after stripping and re-hybridization to the same blot.

    Journal: Nature structural & molecular biology

    Article Title: Capped small RNAs and MOV10 in Human Hepatitis Delta Virus replication

    doi: 10.1038/nsmb.1440

    Figure Lengend Snippet: Cloning and characterization of an HDV small RNA of genomic polarity. ( a ) Relative location and cloning frequency of sequenced HDV small RNAs derived from the genomic and antigenomic pode hairpins (main species highlighted in red). ( b ) Detection of genomic HDV small RNA by Northern Blot (293 cells, day 5). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. ( c ) Enzymatic analysis of genomic small RNA 5′ end. 1: mock-treated (+HDV); 2: mock-treated (no HDV); 3: PNK (+HDV); 4: Antarctic Phosphatase (+HDV); 5: Antarctic Phosphatase followed by T4 PNK (+HDV); 6: Decapping enzyme (TAP; +HDV); 7: T4 RNA Ligase (+HDV); 8: Terminator Exonuclease (+HDV). Note that unlike the antigenomic small RNA, a minor fraction of the genomic small RNA does not appear to be shifted following TAP treatment. ( d - f ) Localization of the HDV small RNAs. ( d ) Nuclear-cytoplasmic fractionation of antigenomic HDV small RNA (polyacrylamide gel) and full-length antigenomic and genomic HDV RNA (denaturing agarose gel). The main species in the full-length genomic/antigenomic RNA blot corresponds to the monomer, the higher molecular weight species to dimer, trimer etc. 1: DNA induction, mutant HDAg; 2: DNA induction, wt HDAg; 3: untransfected. ( e ) Genomic small RNA is restricted to the nucleus (nuclear-cytoplasmic fractionation). 1: DNA induction, wt HDAg; 2: DNA induction, mutant HDAg. miR-15a and U6 snRNA chosen as largely cytoplasmic and nuclear RNA controls, respectively. ( f ) The HDV small RNA can be found in the HDV virion. 1: RNA induction (same RNA as in Fig. 2c ); 2: virion RNA isolated from tissue culture media (∼1.25×10 9 particles). MR: RNA Marker. The various RNAs in c-f were detected after stripping and re-hybridization to the same blot.

    Article Snippet: The primer extension oligo was end-labeled with T4 PNK (NEB) and γ-32 P ATP (PerkinElmer).

    Techniques: Clone Assay, Derivative Assay, Northern Blot, Mutagenesis, Fractionation, Agarose Gel Electrophoresis, Northern blot, Molecular Weight, Isolation, Marker, Stripping Membranes, Hybridization

    Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between  E.coli  0157 and  E.coli  K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.

    Journal: Nucleic Acids Research

    Article Title: Detection of 5?- and 3?-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

    doi: 10.1093/nar/gki256

    Figure Lengend Snippet: Northern analysis of the ( A ) RyfB, RyfC and ( B ) RyfD RNAs derived from intergenic sequences. The samples were the same as in Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Arrows indicate predominant bands. The arrangement of adjacent genes is shown below each panel as in Figure 2 . Sequences capable of base pairing and diagrams of possible pairing arrangements are given below the gene arrangements. Bases that differ between E.coli 0157 and E.coli K12 are indicated in red. The 5′ ends of the RyfB, RyfC and RyfD RNAs (indicated by balls) all correspond to the actual transcription start.

    Article Snippet: The ligation products were phosphorylated at their 5′ ends (30 μl reaction, 37°C, 1 h, 2 mM ATP and 5 U T4 polynucleotide kinase; New England Biolabs Inc., Beverly MA).

    Techniques: Northern Blot, Derivative Assay, Marker

    Northern analysis of antisense RNAs expressed near the ( A ) 5′ end or ( B ) 3′ end of the mRNA encoded on the opposing strand. The samples were the same as in   Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of adjacent genes is shown below each panel as in   Figure 2 . The 5′ ends of the RyjB and RyjC (indicated by balls) correspond to the actual transcription start.

    Journal: Nucleic Acids Research

    Article Title: Detection of 5?- and 3?-UTR-derived small RNAs and cis-encoded antisense RNAs in Escherichia coli

    doi: 10.1093/nar/gki256

    Figure Lengend Snippet: Northern analysis of antisense RNAs expressed near the ( A ) 5′ end or ( B ) 3′ end of the mRNA encoded on the opposing strand. The samples were the same as in Figure 1 , and the RNA marker lane similarly corresponds to one of the panels. Small arrows indicate the full-length mRNA and large arrows indicate processed transcripts. The arrangement of adjacent genes is shown below each panel as in Figure 2 . The 5′ ends of the RyjB and RyjC (indicated by balls) correspond to the actual transcription start.

    Article Snippet: The ligation products were phosphorylated at their 5′ ends (30 μl reaction, 37°C, 1 h, 2 mM ATP and 5 U T4 polynucleotide kinase; New England Biolabs Inc., Beverly MA).

    Techniques: Northern Blot, Marker