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  • 88
    Gene Codes Inc sequencher dna sequencing software
    Sequencher Dna Sequencing Software, supplied by Gene Codes Inc, used in various techniques. Bioz Stars score: 88/100, based on 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    4Gene sequencher dna sequence analysis software
    Sequencher Dna Sequence Analysis Software, supplied by 4Gene, used in various techniques. Bioz Stars score: 92/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Genewiz dna sequence analysis
    The <t>bbb22</t> gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. <t>DNA</t> was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.
    Dna Sequence Analysis, supplied by Genewiz, used in various techniques. Bioz Stars score: 93/100, based on 311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Beckman Coulter dna sequencing analysis
    Mapping Transcription Start Sites (TSS) of E. chaffeensis genes by primer extension (PE) and 5’ <t>RACE.</t> A) The primer extended products resolved on sequencing gels with TSS identified for the genes dnaK, hup, DNAbp <t>(DNA</t> binding protein gene), clpA, clpB , operons of groE * ( groES and groEL genes), and hsIV* ( hslV and hslU genes). The location of the TSS for each gene was identified by comparing the Sanger’s DNA sequencing runs generated with the same primers used for the PE reactions, but using plasmid DNAs containing the respective gene segments. All genes had one TSS with the exception of hup, which contained two TSS. [The location of the TSS established from the PE results for all 7 genes (bold and underlined text) relative to the initiation codon of each gene were presented under gel data.] B) 5’RACE data identifying the TSS of the genes dksA and grpE , and the operon glyQ * ( glyQ, glyS and dnaJ genes). Sequences generated from the 5’RACE products are compared with the sequences generated with DNA templates and are shown for each gene relative to the initiation codon. TSS are identified with bold and underlined text. The underlined G rich tails are added upstream to TSS during the 5’RACE reaction.
    Dna Sequencing Analysis, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 92/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sequetech dna sequence analysis
    Mapping Transcription Start Sites (TSS) of E. chaffeensis genes by primer extension (PE) and 5’ <t>RACE.</t> A) The primer extended products resolved on sequencing gels with TSS identified for the genes dnaK, hup, DNAbp <t>(DNA</t> binding protein gene), clpA, clpB , operons of groE * ( groES and groEL genes), and hsIV* ( hslV and hslU genes). The location of the TSS for each gene was identified by comparing the Sanger’s DNA sequencing runs generated with the same primers used for the PE reactions, but using plasmid DNAs containing the respective gene segments. All genes had one TSS with the exception of hup, which contained two TSS. [The location of the TSS established from the PE results for all 7 genes (bold and underlined text) relative to the initiation codon of each gene were presented under gel data.] B) 5’RACE data identifying the TSS of the genes dksA and grpE , and the operon glyQ * ( glyQ, glyS and dnaJ genes). Sequences generated from the 5’RACE products are compared with the sequences generated with DNA templates and are shown for each gene relative to the initiation codon. TSS are identified with bold and underlined text. The underlined G rich tails are added upstream to TSS during the 5’RACE reaction.
    Dna Sequence Analysis, supplied by Sequetech, used in various techniques. Bioz Stars score: 92/100, based on 119 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Macrogen dna sequencing analysis
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing Analysis, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 44 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    GATC Biotech dna sequencing
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing, supplied by GATC Biotech, used in various techniques. Bioz Stars score: 93/100, based on 2099 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eton Bioscience dna sequencing
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing, supplied by Eton Bioscience, used in various techniques. Bioz Stars score: 93/100, based on 431 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    ACGT Inc dna sequencing analysis
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing Analysis, supplied by ACGT Inc, used in various techniques. Bioz Stars score: 92/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Geno Technology dna sequencing analysis
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing Analysis, supplied by Geno Technology, used in various techniques. Bioz Stars score: 90/100, based on 29 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins Genomics dna sequencing analysis
    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with <t>VacA</t> (150 µg mL −1 ) cells showing chromatin condensation and <t>DNA</t> fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.
    Dna Sequencing Analysis, supplied by Eurofins Genomics, used in various techniques. Bioz Stars score: 93/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins dna sequencing analysis
    Denaturing electrophoresis of RNA extracted from particles and particle migration under non-denaturing conditions . (A) Denaturing agarose electrophoresis of nucleic acid extracted from TCV and particles formed by the infiltration of leaves with <t>pEAQ-</t> HT -P38. Yellow numbering denotes the size of the RNA (kb) ladder in lane M, catalog number 15623-200 (Invitrogen). (B) Migration of denatured viral proteins in 12% NuPAGE gel. (C) Separation of particles in a non-denaturing 1.2% agarose gel. Upper panel stained with Coomassie Brilliant Blue, the lower panel stained with ethidium bromide. 1, TCV; 2, pEAQ- HT -P38; 3, pEAQ- HT -P29; 4, CPMV eVLPs; 5, CPMV. Right lane lower panel, a commercially available <t>DNA</t> ladder (Bioline) confirming that the gel was stained with ethidium bromide.
    Dna Sequencing Analysis, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 48 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher dna sequence analysis
    Detection of the mRNA expression levels of IgG and related enzymes, including RAG1, RAG2 and AID in podocytes. (A) Transcripts of Igγ, γ1, γ3, γ4, κ and λ C regions were detected by <t>RT-PCR.</t> CD19 was detected as a marker of B lymphocytes. The mRNA of Igγ and κ V regions was amplified by nested RT-PCR. (B) mRNA expression levels of AID, RAG1 and RAG2. PBMCs were used as a positive control; water instead of cDNA was used as a blank control; R (cDNA template replaced by DNase-treated RNA) was used as negative control. (C) <t>DNA</t> sequencing results. The sequences of the PCR products were aligned with the mRNA sequences available in the NCBI database. HPC, human podocytes; AID, activation-induced cytidine deaminase; C, constant; Ig, immunoglobulin; Igγ, IgG heavy chain; PBMCs, peripheral blood mononuclear cells; RAG. recombination activating gene; RT-PCR, reverse transcription-polymerase chain reaction; V, variable.
    Dna Sequence Analysis, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 1685 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MWG-Biotech dna sequencing analysis
    Detection of the mRNA expression levels of IgG and related enzymes, including RAG1, RAG2 and AID in podocytes. (A) Transcripts of Igγ, γ1, γ3, γ4, κ and λ C regions were detected by <t>RT-PCR.</t> CD19 was detected as a marker of B lymphocytes. The mRNA of Igγ and κ V regions was amplified by nested RT-PCR. (B) mRNA expression levels of AID, RAG1 and RAG2. PBMCs were used as a positive control; water instead of cDNA was used as a blank control; R (cDNA template replaced by DNase-treated RNA) was used as negative control. (C) <t>DNA</t> sequencing results. The sequences of the PCR products were aligned with the mRNA sequences available in the NCBI database. HPC, human podocytes; AID, activation-induced cytidine deaminase; C, constant; Ig, immunoglobulin; Igγ, IgG heavy chain; PBMCs, peripheral blood mononuclear cells; RAG. recombination activating gene; RT-PCR, reverse transcription-polymerase chain reaction; V, variable.
    Dna Sequencing Analysis, supplied by MWG-Biotech, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Elim Bio dna sequence analysis
    Determination of nucleotides at the 5′ and 3′ ends of <t>HCV</t> replicon RNAs recovered from G418-resistant Huh7 cells. (A) Schematic of the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) (Ambion). An adapter RNA (open bar) was ligated to the 5′ end of either the positive-strand (for determination of the 5′-end nucleotide) or the negative-strand (for determination of the 3′-end nucleotide) HCV replicon RNA isolated from different cell lines. The cDNAs of both positive and negative strands of HCV RNA were reverse transcribed by using HCV-specific primers and amplified by PCR with 5′RACE outer/inner primers (gray arrow) and HCV-specific primers (open arrow). The 5′-end nucleotides of both positive and negative strands of HCV RNA were determined by <t>DNA</t> sequence analysis. (B) 5′- and 3′-end nucleotides of HCV RNA determined by RLM-RACE. Letters indicate nucleotides at the 5′ and 3′ ends as shown on the top. The number indicates the number of cell clones from which HCV replicon RNAs were analyzed. Numbers in parentheses indicate the frequency of the nucleotides determined. The HCV replicon RNAs given on the left column were the ones transcribed in vitro by a T7 RNA polymerase.
    Dna Sequence Analysis, supplied by Elim Bio, used in various techniques. Bioz Stars score: 92/100, based on 63 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    MCLAB dna sequence analysis
    The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic <t>DNA</t> from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing <t>PCR</t> using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.
    Dna Sequence Analysis, supplied by MCLAB, used in various techniques. Bioz Stars score: 92/100, based on 19 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Gene Codes Inc sequencher dna sequence analysis software v2 1
    The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic <t>DNA</t> from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing <t>PCR</t> using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.
    Sequencher Dna Sequence Analysis Software V2 1, supplied by Gene Codes Inc, used in various techniques. Bioz Stars score: 86/100, based on 28 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Sangon Biotech dna sequencing analysis
    The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic <t>DNA</t> from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing <t>PCR</t> using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.
    Dna Sequencing Analysis, supplied by Sangon Biotech, used in various techniques. Bioz Stars score: 90/100, based on 20 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Cosmo Genetech Co dna sequencing analysis
    The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic <t>DNA</t> from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing <t>PCR</t> using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.
    Dna Sequencing Analysis, supplied by Cosmo Genetech Co, used in various techniques. Bioz Stars score: 92/100, based on 47 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    The bbb22 gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. DNA was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.

    Journal: Infection and Immunity

    Article Title: Molecular Dissection of a Borrelia burgdorferiIn Vivo Essential Purine Transport System

    doi: 10.1128/IAI.02859-14

    Figure Lengend Snippet: The bbb22 gene alone is not sufficient to maintain wild-type levels of spirochete loads in infected mouse tissues. DNA was isolated from ear, heart, and joint tissues of C3H/HeN mice inoculated with 1 × 10 4 bbb22-23 + or 22dist p -bbb22 + spirochetes.

    Article Snippet: Plasmids pBSV2G 22dist p - bbb22 + and pBSV2G 22prox p - bbb22 + were confirmed by restriction digestion and DNA sequence analysis (Genewiz).

    Techniques: Infection, Isolation, Mouse Assay

    Mapping Transcription Start Sites (TSS) of E. chaffeensis genes by primer extension (PE) and 5’ RACE. A) The primer extended products resolved on sequencing gels with TSS identified for the genes dnaK, hup, DNAbp (DNA binding protein gene), clpA, clpB , operons of groE * ( groES and groEL genes), and hsIV* ( hslV and hslU genes). The location of the TSS for each gene was identified by comparing the Sanger’s DNA sequencing runs generated with the same primers used for the PE reactions, but using plasmid DNAs containing the respective gene segments. All genes had one TSS with the exception of hup, which contained two TSS. [The location of the TSS established from the PE results for all 7 genes (bold and underlined text) relative to the initiation codon of each gene were presented under gel data.] B) 5’RACE data identifying the TSS of the genes dksA and grpE , and the operon glyQ * ( glyQ, glyS and dnaJ genes). Sequences generated from the 5’RACE products are compared with the sequences generated with DNA templates and are shown for each gene relative to the initiation codon. TSS are identified with bold and underlined text. The underlined G rich tails are added upstream to TSS during the 5’RACE reaction.

    Journal: PLoS ONE

    Article Title: Transcription of Ehrlichia chaffeensis Genes Is Accomplished by RNA Polymerase Holoenzyme Containing either Sigma 32 or Sigma 70

    doi: 10.1371/journal.pone.0081780

    Figure Lengend Snippet: Mapping Transcription Start Sites (TSS) of E. chaffeensis genes by primer extension (PE) and 5’ RACE. A) The primer extended products resolved on sequencing gels with TSS identified for the genes dnaK, hup, DNAbp (DNA binding protein gene), clpA, clpB , operons of groE * ( groES and groEL genes), and hsIV* ( hslV and hslU genes). The location of the TSS for each gene was identified by comparing the Sanger’s DNA sequencing runs generated with the same primers used for the PE reactions, but using plasmid DNAs containing the respective gene segments. All genes had one TSS with the exception of hup, which contained two TSS. [The location of the TSS established from the PE results for all 7 genes (bold and underlined text) relative to the initiation codon of each gene were presented under gel data.] B) 5’RACE data identifying the TSS of the genes dksA and grpE , and the operon glyQ * ( glyQ, glyS and dnaJ genes). Sequences generated from the 5’RACE products are compared with the sequences generated with DNA templates and are shown for each gene relative to the initiation codon. TSS are identified with bold and underlined text. The underlined G rich tails are added upstream to TSS during the 5’RACE reaction.

    Article Snippet: The final products were resolved in a 1% agarose gel; the major amplicons were gel isolated and used to perform DNA sequencing analysis. (5’RACE primers are listed in .)

    Techniques: Sequencing, Binding Assay, DNA Sequencing, Generated, Plasmid Preparation

    DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with VacA (150 µg mL −1 ) cells showing chromatin condensation and DNA fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.

    Journal: BioMed Research International

    Article Title: Sequence and Apoptotic Activity of VacA Cytotoxin Cloned from a Helicobacter pylori Thai Clinical Isolate

    doi: 10.1155/2014/398350

    Figure Lengend Snippet: DAPI staining of intestinal epithelial cells (T84) and Madin-Darby Canine Kidney (MDCK) cells. (a) Control of untreated T84 cells showing normal nuclei. (b) T84 cells treated with VacA (150 µg mL −1 ) cells showing chromatin condensation and DNA fragmentation. (c) Control of untreated MDCK cells. (d) MDCK cells treated with VacA (150 µg mL −1 ) showing chromatin condensation and DNA fragmentation. DAPI was used at 1000-fold dilution in buffer and the magnification of images is 1000×.

    Article Snippet: The correct sequence of the recombinant pTrcHisA/VacA construct was verified by restriction digestion and DNA sequencing analysis (Macrogen Inc., South Korea).

    Techniques: Staining

    Site directed mutagenesis of the KRRIH region of Msm UdgX and its activity assay. ( A ( i ) and B ( i )) Line diagrams of Msm UdgX and its mutants highlighting the regions targeted by site directed mutagenesis. Motif A, motif B and the KRRIH loop region are shown in red, blue and yellow boxes, respectively. ( A ( ii )) Uracil excision/binding assays with Msm UdgX (WT) and its R107S, H109S and SSAS mutant derivatives. ∼200 ng of each protein was reacted with 5′ 32 P-labeled SSU9, the reaction mix was resolved on 8 M urea-PAGE (15%) and analyzed by phosphor imaging. The presence of uracil excision activity is seen by the fast migrating product band, ‘P’ whereas the uracil-DNA binding activity by the slow migrating complex, ‘C’. ( B ( ii )) Uracil excision/binding assays with Msm UdgX (WT) and its F4, F1, LD and F4 LD mutant derivatives (∼200 ng each) using SSU9. ( C ) Uracil release assay of Msm UdgX H109S (∼200 ng) using 3 H-uracil containing DNA (21 000 c.p.m) as substrate. Released uracil was separated by TLC (CEL 300 PEI/UV254) and seen as a fast migrating species upon phosphor imaging (the radioactive spot comigrated with the cold uracil spot detected by UV shadowing). ( D ) Uracil excision/binding assay with Msm UdgX and its H109S, H109G, H109Q, H109A and C27S derivatives. ∼200 ng of each protein was used in a standard UDG assay with SSU9 substrate.

    Journal: Nucleic Acids Research

    Article Title: A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

    doi: 10.1093/nar/gkv854

    Figure Lengend Snippet: Site directed mutagenesis of the KRRIH region of Msm UdgX and its activity assay. ( A ( i ) and B ( i )) Line diagrams of Msm UdgX and its mutants highlighting the regions targeted by site directed mutagenesis. Motif A, motif B and the KRRIH loop region are shown in red, blue and yellow boxes, respectively. ( A ( ii )) Uracil excision/binding assays with Msm UdgX (WT) and its R107S, H109S and SSAS mutant derivatives. ∼200 ng of each protein was reacted with 5′ 32 P-labeled SSU9, the reaction mix was resolved on 8 M urea-PAGE (15%) and analyzed by phosphor imaging. The presence of uracil excision activity is seen by the fast migrating product band, ‘P’ whereas the uracil-DNA binding activity by the slow migrating complex, ‘C’. ( B ( ii )) Uracil excision/binding assays with Msm UdgX (WT) and its F4, F1, LD and F4 LD mutant derivatives (∼200 ng each) using SSU9. ( C ) Uracil release assay of Msm UdgX H109S (∼200 ng) using 3 H-uracil containing DNA (21 000 c.p.m) as substrate. Released uracil was separated by TLC (CEL 300 PEI/UV254) and seen as a fast migrating species upon phosphor imaging (the radioactive spot comigrated with the cold uracil spot detected by UV shadowing). ( D ) Uracil excision/binding assay with Msm UdgX and its H109S, H109G, H109Q, H109A and C27S derivatives. ∼200 ng of each protein was used in a standard UDG assay with SSU9 substrate.

    Article Snippet: The PCR product (662 bp) was blunt end ligated into pJET1.2 (MBI) vector to generate pJETMsm UdgX and confirmed by restriction digestion and DNA sequence analysis (Macrogen, S. Korea).

    Techniques: Mutagenesis, Activity Assay, Binding Assay, Labeling, Polyacrylamide Gel Electrophoresis, Imaging, Release Assay, Thin Layer Chromatography

    Activity assay of Msm UdgX. ( A ) Assay of Msm UdgX (100 ng) on 5′ 32 P-end labeled SSU9:G and SSU9 substrate, ‘S’ (∼10,000 c.p.m). Family 1 UDG (Ung) was used as control, Ugi was added as indicated. Reactions were resolved on 8 M Urea PAGE (15%) and analyzed by phosphor imaging. The presence of uracil excision activity can be seen as a fast migrating species forming a product ‘P’ whereas uracil binding activity form a slow migrating complex ‘C’ [panel (i) on left shows a diagrammatic sketch whereas the panel on right (ii) shows the results of the experiment]. ( B ) Proteinase K (1.2 U) digestion of the complex, ‘C’ observed in A, and re-resolved on 8 M Urea PAGE (15%) and analyzed by phosphor imaging. ( C ) Uracil release assay using 3 H-uracil containing DNA substrate. Msm UdgX (∼1 μg) or Ung (100 ng) was allowed to react with 3 H-uracil DNA substrate (∼21 000 c.p.m.), the samples were mixed with unlabeled uracil and spotted on the CEL 300 PEI/UV254, resolved using a mobile phase of 0.2% formic acid (v/v) containing 0.55 M LiCl at 4°C and analyzed by phosphor imaging. Released uracil is seen as a fast migrating species. ( D ) Binding of Msm UdgX (∼1 μg) with unlabeled uracil containing DNA oligomers (50 pmol) of different sizes resolved on 15% SDS PAGE and visualized by Coomassie blue staining. ( E ) Activity assay of Msm UdgX (4 pmol) in the presence of Msm Ung (4 pmol) using 5′ 32 P-labeled SSU9:G. Reactions were resolved on 8 M urea-PAGE (15%) and analyzed by phosphor imaging. The 1 st and 2 nd represents the order of addition, and ‘+’ in both Msm Ung and Msm UdgX indicates that both the protein are added simultaneously.

    Journal: Nucleic Acids Research

    Article Title: A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

    doi: 10.1093/nar/gkv854

    Figure Lengend Snippet: Activity assay of Msm UdgX. ( A ) Assay of Msm UdgX (100 ng) on 5′ 32 P-end labeled SSU9:G and SSU9 substrate, ‘S’ (∼10,000 c.p.m). Family 1 UDG (Ung) was used as control, Ugi was added as indicated. Reactions were resolved on 8 M Urea PAGE (15%) and analyzed by phosphor imaging. The presence of uracil excision activity can be seen as a fast migrating species forming a product ‘P’ whereas uracil binding activity form a slow migrating complex ‘C’ [panel (i) on left shows a diagrammatic sketch whereas the panel on right (ii) shows the results of the experiment]. ( B ) Proteinase K (1.2 U) digestion of the complex, ‘C’ observed in A, and re-resolved on 8 M Urea PAGE (15%) and analyzed by phosphor imaging. ( C ) Uracil release assay using 3 H-uracil containing DNA substrate. Msm UdgX (∼1 μg) or Ung (100 ng) was allowed to react with 3 H-uracil DNA substrate (∼21 000 c.p.m.), the samples were mixed with unlabeled uracil and spotted on the CEL 300 PEI/UV254, resolved using a mobile phase of 0.2% formic acid (v/v) containing 0.55 M LiCl at 4°C and analyzed by phosphor imaging. Released uracil is seen as a fast migrating species. ( D ) Binding of Msm UdgX (∼1 μg) with unlabeled uracil containing DNA oligomers (50 pmol) of different sizes resolved on 15% SDS PAGE and visualized by Coomassie blue staining. ( E ) Activity assay of Msm UdgX (4 pmol) in the presence of Msm Ung (4 pmol) using 5′ 32 P-labeled SSU9:G. Reactions were resolved on 8 M urea-PAGE (15%) and analyzed by phosphor imaging. The 1 st and 2 nd represents the order of addition, and ‘+’ in both Msm Ung and Msm UdgX indicates that both the protein are added simultaneously.

    Article Snippet: The PCR product (662 bp) was blunt end ligated into pJET1.2 (MBI) vector to generate pJETMsm UdgX and confirmed by restriction digestion and DNA sequence analysis (Macrogen, S. Korea).

    Techniques: Activity Assay, Labeling, Polyacrylamide Gel Electrophoresis, Imaging, Binding Assay, Release Assay, SDS Page, Staining

    Proposed pathway for repair of uracil- Msm UdgX complexes. Binding of Msm UdgX to uracil would stall replication which could in turn lead to generation of single stranded gaps or double stranded breaks. Activation of RecA protein upon binding to ss DNA would induce the SOS response through cleavage of LexA. RecBCD proteins would also repair the complex through homologous recombination repair pathway.

    Journal: Nucleic Acids Research

    Article Title: A unique uracil-DNA binding protein of the uracil DNA glycosylase superfamily

    doi: 10.1093/nar/gkv854

    Figure Lengend Snippet: Proposed pathway for repair of uracil- Msm UdgX complexes. Binding of Msm UdgX to uracil would stall replication which could in turn lead to generation of single stranded gaps or double stranded breaks. Activation of RecA protein upon binding to ss DNA would induce the SOS response through cleavage of LexA. RecBCD proteins would also repair the complex through homologous recombination repair pathway.

    Article Snippet: The PCR product (662 bp) was blunt end ligated into pJET1.2 (MBI) vector to generate pJETMsm UdgX and confirmed by restriction digestion and DNA sequence analysis (Macrogen, S. Korea).

    Techniques: Binding Assay, Activation Assay, Homologous Recombination

    Denaturing electrophoresis of RNA extracted from particles and particle migration under non-denaturing conditions . (A) Denaturing agarose electrophoresis of nucleic acid extracted from TCV and particles formed by the infiltration of leaves with pEAQ- HT -P38. Yellow numbering denotes the size of the RNA (kb) ladder in lane M, catalog number 15623-200 (Invitrogen). (B) Migration of denatured viral proteins in 12% NuPAGE gel. (C) Separation of particles in a non-denaturing 1.2% agarose gel. Upper panel stained with Coomassie Brilliant Blue, the lower panel stained with ethidium bromide. 1, TCV; 2, pEAQ- HT -P38; 3, pEAQ- HT -P29; 4, CPMV eVLPs; 5, CPMV. Right lane lower panel, a commercially available DNA ladder (Bioline) confirming that the gel was stained with ethidium bromide.

    Journal: Frontiers in Plant Science

    Article Title: The Generation of Turnip Crinkle Virus-Like Particles in Plants by the Transient Expression of Wild-Type and Modified Forms of Its Coat Protein

    doi: 10.3389/fpls.2015.01138

    Figure Lengend Snippet: Denaturing electrophoresis of RNA extracted from particles and particle migration under non-denaturing conditions . (A) Denaturing agarose electrophoresis of nucleic acid extracted from TCV and particles formed by the infiltration of leaves with pEAQ- HT -P38. Yellow numbering denotes the size of the RNA (kb) ladder in lane M, catalog number 15623-200 (Invitrogen). (B) Migration of denatured viral proteins in 12% NuPAGE gel. (C) Separation of particles in a non-denaturing 1.2% agarose gel. Upper panel stained with Coomassie Brilliant Blue, the lower panel stained with ethidium bromide. 1, TCV; 2, pEAQ- HT -P38; 3, pEAQ- HT -P29; 4, CPMV eVLPs; 5, CPMV. Right lane lower panel, a commercially available DNA ladder (Bioline) confirming that the gel was stained with ethidium bromide.

    Article Snippet: The sequence of all the resulting gene constructs, both in pDONR207 and in pEAQ-HT -DEST1 were subsequently confirmed by DNA sequencing analysis (Eurofins Genomics).

    Techniques: Electrophoresis, Migration, Agarose Gel Electrophoresis, Staining

    Detection of the mRNA expression levels of IgG and related enzymes, including RAG1, RAG2 and AID in podocytes. (A) Transcripts of Igγ, γ1, γ3, γ4, κ and λ C regions were detected by RT-PCR. CD19 was detected as a marker of B lymphocytes. The mRNA of Igγ and κ V regions was amplified by nested RT-PCR. (B) mRNA expression levels of AID, RAG1 and RAG2. PBMCs were used as a positive control; water instead of cDNA was used as a blank control; R (cDNA template replaced by DNase-treated RNA) was used as negative control. (C) DNA sequencing results. The sequences of the PCR products were aligned with the mRNA sequences available in the NCBI database. HPC, human podocytes; AID, activation-induced cytidine deaminase; C, constant; Ig, immunoglobulin; Igγ, IgG heavy chain; PBMCs, peripheral blood mononuclear cells; RAG. recombination activating gene; RT-PCR, reverse transcription-polymerase chain reaction; V, variable.

    Journal: International Journal of Molecular Medicine

    Article Title: Expression of immunoglobulin G in human podocytes, and its role in cell viability and adhesion

    doi: 10.3892/ijmm.2018.3525

    Figure Lengend Snippet: Detection of the mRNA expression levels of IgG and related enzymes, including RAG1, RAG2 and AID in podocytes. (A) Transcripts of Igγ, γ1, γ3, γ4, κ and λ C regions were detected by RT-PCR. CD19 was detected as a marker of B lymphocytes. The mRNA of Igγ and κ V regions was amplified by nested RT-PCR. (B) mRNA expression levels of AID, RAG1 and RAG2. PBMCs were used as a positive control; water instead of cDNA was used as a blank control; R (cDNA template replaced by DNase-treated RNA) was used as negative control. (C) DNA sequencing results. The sequences of the PCR products were aligned with the mRNA sequences available in the NCBI database. HPC, human podocytes; AID, activation-induced cytidine deaminase; C, constant; Ig, immunoglobulin; Igγ, IgG heavy chain; PBMCs, peripheral blood mononuclear cells; RAG. recombination activating gene; RT-PCR, reverse transcription-polymerase chain reaction; V, variable.

    Article Snippet: The reliability of PCR products was analyzed by DNA sequence analysis, which was performed by Invitrogen Trading (Shanghai) Co., Ltd. (Shanghai, China).

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Marker, Amplification, Positive Control, Negative Control, DNA Sequencing, Polymerase Chain Reaction, Activation Assay

    Determination of nucleotides at the 5′ and 3′ ends of HCV replicon RNAs recovered from G418-resistant Huh7 cells. (A) Schematic of the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) (Ambion). An adapter RNA (open bar) was ligated to the 5′ end of either the positive-strand (for determination of the 5′-end nucleotide) or the negative-strand (for determination of the 3′-end nucleotide) HCV replicon RNA isolated from different cell lines. The cDNAs of both positive and negative strands of HCV RNA were reverse transcribed by using HCV-specific primers and amplified by PCR with 5′RACE outer/inner primers (gray arrow) and HCV-specific primers (open arrow). The 5′-end nucleotides of both positive and negative strands of HCV RNA were determined by DNA sequence analysis. (B) 5′- and 3′-end nucleotides of HCV RNA determined by RLM-RACE. Letters indicate nucleotides at the 5′ and 3′ ends as shown on the top. The number indicates the number of cell clones from which HCV replicon RNAs were analyzed. Numbers in parentheses indicate the frequency of the nucleotides determined. The HCV replicon RNAs given on the left column were the ones transcribed in vitro by a T7 RNA polymerase.

    Journal: Journal of Virology

    Article Title: Effects of Mutations of the Initiation Nucleotides on Hepatitis C Virus RNA Replication in the Cell

    doi: 10.1128/JVI.78.7.3633-3643.2004

    Figure Lengend Snippet: Determination of nucleotides at the 5′ and 3′ ends of HCV replicon RNAs recovered from G418-resistant Huh7 cells. (A) Schematic of the RNA ligase-mediated rapid amplification of cDNA ends (RLM-RACE) (Ambion). An adapter RNA (open bar) was ligated to the 5′ end of either the positive-strand (for determination of the 5′-end nucleotide) or the negative-strand (for determination of the 3′-end nucleotide) HCV replicon RNA isolated from different cell lines. The cDNAs of both positive and negative strands of HCV RNA were reverse transcribed by using HCV-specific primers and amplified by PCR with 5′RACE outer/inner primers (gray arrow) and HCV-specific primers (open arrow). The 5′-end nucleotides of both positive and negative strands of HCV RNA were determined by DNA sequence analysis. (B) 5′- and 3′-end nucleotides of HCV RNA determined by RLM-RACE. Letters indicate nucleotides at the 5′ and 3′ ends as shown on the top. The number indicates the number of cell clones from which HCV replicon RNAs were analyzed. Numbers in parentheses indicate the frequency of the nucleotides determined. The HCV replicon RNAs given on the left column were the ones transcribed in vitro by a T7 RNA polymerase.

    Article Snippet: Further analyses of HCV RNA derived from this patient by cDNA cloning and DNA sequence analysis revealed that the mixture of HCV RNA consists of 60% of RNA with a 5′-end nucleotide G and 40% of RNA with a 5′-end nucleotide A.

    Techniques: Rapid Amplification of cDNA Ends, Isolation, Amplification, Polymerase Chain Reaction, Sequencing, Clone Assay, In Vitro

    The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic DNA from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing PCR using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.

    Journal: PLoS Genetics

    Article Title: The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

    doi: 10.1371/journal.pgen.1003386

    Figure Lengend Snippet: The effect of ATM deficiency on small deletions at interstitial and telomeric DSBs. The frequency of small deletions in genomic DNA from clones EDS-7F2 and EDS-6J8 that were infected with pQCXIH-ISceI and selected with hygromycin for 14 days was determined by first performing PCR using oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (see Figure 1 ). The fraction of cells in the population that contain small deletions at the I- Sce I site was then determined from the fraction of the PCR product that was not digested with I- Sce I (see Materials and Methods ). All samples were analyzed in triplicate. Error bars represent standard deviation of three separate experiments.

    Article Snippet: The conditions for PCR involved 94°C for 2 min, then 40 cycles of 94°C for 30 sec, 62°C for 30 sec, and 72°C for 45 sec. DNA sequence analysis was then performed directly on the PCR products using the EJ5-1 primer (MCLAB).

    Techniques: Clone Assay, Infection, Polymerase Chain Reaction, Plasmid Preparation, Standard Deviation

    DNA sequence analysis of recombination junctions involved in the formation of GCRs in DsRed + subclones. Genomic DNA was isolated from individual DsRed + subclones following expression of I- Sce I in clones EDS-6J7 and EDS-6J8 (with telomeric pEJ5-GFP and interstitial pDsRed-ISceI). The sites containing the recombination junctions were amplified by PCR using one primer in the pEJ5-GFP plasmid and one primer in the pDsRed-ISceI plasmid (see Figure 1 ). DNA sequence analysis was then performed on the PCR fragments to determine the structure of the recombination junction involved in the formation of the GCR. The location of the 4-nucleotide overhang generated by I- Sce I endonuclease (bold), deletions at the site of the DSB (dashes), and insertions (nucleotides between dashed lines) are shown.

    Journal: PLoS Genetics

    Article Title: The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

    doi: 10.1371/journal.pgen.1003386

    Figure Lengend Snippet: DNA sequence analysis of recombination junctions involved in the formation of GCRs in DsRed + subclones. Genomic DNA was isolated from individual DsRed + subclones following expression of I- Sce I in clones EDS-6J7 and EDS-6J8 (with telomeric pEJ5-GFP and interstitial pDsRed-ISceI). The sites containing the recombination junctions were amplified by PCR using one primer in the pEJ5-GFP plasmid and one primer in the pDsRed-ISceI plasmid (see Figure 1 ). DNA sequence analysis was then performed on the PCR fragments to determine the structure of the recombination junction involved in the formation of the GCR. The location of the 4-nucleotide overhang generated by I- Sce I endonuclease (bold), deletions at the site of the DSB (dashes), and insertions (nucleotides between dashed lines) are shown.

    Article Snippet: The conditions for PCR involved 94°C for 2 min, then 40 cycles of 94°C for 30 sec, 62°C for 30 sec, and 72°C for 45 sec. DNA sequence analysis was then performed directly on the PCR products using the EJ5-1 primer (MCLAB).

    Techniques: Sequencing, Isolation, Expressing, Clone Assay, Amplification, Polymerase Chain Reaction, Plasmid Preparation, Generated

    The structure of the plasmids used to monitor NHEJ, GCRs, and large deletions. (A) NHEJ was monitored using the pEJ5-GFP plasmid integrated at interstitial (not shown) or telomeric sites (shown). pEJ5-GFP contains a GFP gene that is initially inactive due to the presence of a puromycin-resistance (puro) gene located between the GFP gene and its promoter. NHEJ occurring between the distal ends of the I- Sce I-induced DSBs at either end of the puro gene results in the activation of the GFP gene. A PCR product generated with oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (red arrows) was digested with I- Sce I endonuclease to determine the frequency of small deletions at a single I- Sce I-induced DSB. Cell clones containing the pGFP-ISceI plasmid integrated at interstitial sites (not shown) or telomeric sites (shown) were used for analysis of large deletions that inactivate the GFP gene. (B) GCRs were monitored using cell clones that contain the pEJ5-GFP plasmid integrated at an interstitial (not shown) or telomeric (shown) site and the pDsRed-ISceI plasmid integrated at an interstitial site at a different location in the genome. The DsRed gene in the pDsRed-ISceI plasmid is initially inactive due to the lack of a promoter, but becomes activated following NHEJ between the I- Sce I-induced DSBs in the pEJ5-GFP and pDsRed-ISceI plasmids. The location of oligonucleotide primers used for PCR to analyze the junctions between the pEJ5-GFP and pDsRed-ISceI plasmids are shown (red arrows). The location of the ampicillin gene and plasmid origin of replication (Amp/ori), chicken β-actin promoter (promoter), puro gene (Puro), GFP coding sequence (GFP), and telomere are shown. Also show is the genomic DNA (solid line), directions of transcription (black arrows), and I- Sce I recognition sites (I- Sce I).

    Journal: PLoS Genetics

    Article Title: The Role of ATM in the Deficiency in Nonhomologous End-Joining near Telomeres in a Human Cancer Cell Line

    doi: 10.1371/journal.pgen.1003386

    Figure Lengend Snippet: The structure of the plasmids used to monitor NHEJ, GCRs, and large deletions. (A) NHEJ was monitored using the pEJ5-GFP plasmid integrated at interstitial (not shown) or telomeric sites (shown). pEJ5-GFP contains a GFP gene that is initially inactive due to the presence of a puromycin-resistance (puro) gene located between the GFP gene and its promoter. NHEJ occurring between the distal ends of the I- Sce I-induced DSBs at either end of the puro gene results in the activation of the GFP gene. A PCR product generated with oligonucleotide primers spanning one of the I- Sce I sites in the pEJ5-GFP plasmid (red arrows) was digested with I- Sce I endonuclease to determine the frequency of small deletions at a single I- Sce I-induced DSB. Cell clones containing the pGFP-ISceI plasmid integrated at interstitial sites (not shown) or telomeric sites (shown) were used for analysis of large deletions that inactivate the GFP gene. (B) GCRs were monitored using cell clones that contain the pEJ5-GFP plasmid integrated at an interstitial (not shown) or telomeric (shown) site and the pDsRed-ISceI plasmid integrated at an interstitial site at a different location in the genome. The DsRed gene in the pDsRed-ISceI plasmid is initially inactive due to the lack of a promoter, but becomes activated following NHEJ between the I- Sce I-induced DSBs in the pEJ5-GFP and pDsRed-ISceI plasmids. The location of oligonucleotide primers used for PCR to analyze the junctions between the pEJ5-GFP and pDsRed-ISceI plasmids are shown (red arrows). The location of the ampicillin gene and plasmid origin of replication (Amp/ori), chicken β-actin promoter (promoter), puro gene (Puro), GFP coding sequence (GFP), and telomere are shown. Also show is the genomic DNA (solid line), directions of transcription (black arrows), and I- Sce I recognition sites (I- Sce I).

    Article Snippet: The conditions for PCR involved 94°C for 2 min, then 40 cycles of 94°C for 30 sec, 62°C for 30 sec, and 72°C for 45 sec. DNA sequence analysis was then performed directly on the PCR products using the EJ5-1 primer (MCLAB).

    Techniques: Non-Homologous End Joining, Plasmid Preparation, Activation Assay, Polymerase Chain Reaction, Generated, Clone Assay, Sequencing