dna sequencing Search Results


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  • 99
    Thermo Fisher 3730xl dna analyzer
    3730xl Dna Analyzer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 46651 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Millipore dna sequence
    Generation of a <t>bb0028</t> mutant. A . An IPTG-regulatable bb0028 mutant was generated by inserting a streptomycin resistance cassette followed by the flacp promoter immediately upstream of bb0028 . Red arrows indicate primer-binding areas used to confirm insertion of the streptomycin cassette and flacp promoter into the borrelial genome. B . PCR amplification resulted in a 1.2 kb amplicon in the wildtype strain (WT), as expected, and a 3.0 kb amplicon in the BB0028 mutant (flacp::0028) indicating the streptomycin cassette and flacp promoter were inserted in the mutant. A PCR reaction with no <t>DNA</t> template (Neg) was included as a negative control. DNA sizes, in kilobase pairs, are indicated at left. C . Whole-cell lysate from the BB0028 mutant (flacp::0028) strain propagated with (+) or without (−) 1 mM IPTG, as well as from the parental, wildtype strain (WT), were subjected to immunoblot analysis using BB0028 specific antibodies (top panel) or FlaB antibodies (bottom panel). FlaB reactivity was used to ensure all lanes were loaded equally.
    Dna Sequence, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 1652 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher dna sequencer
    <t>RT-PCR</t> analysis of ovarian tissues and OVCAR-3 cells. ( A ) RT-PCR analysis of ovarian serous carcinoma. Primers specific for the four known human SAA genes were used, and PCR fragments were analyzed on a 2% agarose gel. Markers of a <t>DNA</t> ladder (100-bp
    Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 8489 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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    94
    Biotechnology Information dna sequences
    Agarose gels of <t>amplicons</t> generated by PCR amplification of enriched fecal specimens using the invE-A (457-bp amplicon) and hisJ (496-bp amplicon) primer sets. (A) Results using the invE-A primers. Lanes 1 and 6 contain molecular weight standards (100-bp <t>DNA</t> ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lane 3, DNA extracted from culture-negative clinical specimen; lane 4, negative control (distilled water); lane 5, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). (B) Results using hisJ primers. Lanes 1 and 9 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lanes 3 to 6, DNA extracted from culture-negative clinical specimens; lane 7, negative control (distilled water); lane 8, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). Arrows indicate amplicons produced in each reaction.
    Dna Sequences, supplied by Biotechnology Information, used in various techniques. Bioz Stars score: 94/100, based on 2081 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi prism 377 dna sequencer
    Agarose gels of <t>amplicons</t> generated by PCR amplification of enriched fecal specimens using the invE-A (457-bp amplicon) and hisJ (496-bp amplicon) primer sets. (A) Results using the invE-A primers. Lanes 1 and 6 contain molecular weight standards (100-bp <t>DNA</t> ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lane 3, DNA extracted from culture-negative clinical specimen; lane 4, negative control (distilled water); lane 5, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). (B) Results using hisJ primers. Lanes 1 and 9 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lanes 3 to 6, DNA extracted from culture-negative clinical specimens; lane 7, negative control (distilled water); lane 8, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). Arrows indicate amplicons produced in each reaction.
    Abi Prism 377 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 3170 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Bioedit Company dna sequence
    Construction and analysis of <t>sreA</t> strains. A : Construction of pTFCM-R-L and pTFCM- neo - sreA plasmids. B: PCR analysis of Δ sreA , and CO sreA transformants using primers sreA -F and sreA -R ( Table 1 ). C: Southern blot analysis of P . digitatum wild-type PdHS-F6 and Δ sreA , CO sreA strains using a probe specific to the 5’ region of Pdsre . 30μg genomic <t>DNA</t> was digested with Hind III and detected using a probe specific to the 5’ region of sreA gene.
    Dna Sequence, supplied by Bioedit Company, used in various techniques. Bioz Stars score: 93/100, based on 107 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 3730 dna sequencer
    Construction and analysis of <t>sreA</t> strains. A : Construction of pTFCM-R-L and pTFCM- neo - sreA plasmids. B: PCR analysis of Δ sreA , and CO sreA transformants using primers sreA -F and sreA -R ( Table 1 ). C: Southern blot analysis of P . digitatum wild-type PdHS-F6 and Δ sreA , CO sreA strains using a probe specific to the 5’ region of Pdsre . 30μg genomic <t>DNA</t> was digested with Hind III and detected using a probe specific to the 5’ region of sreA gene.
    Abi 3730 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 2098 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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    Illumina Inc illumina dna sequencing
    Relationship between the <t>Illumina</t> <t>DNA-sequencing</t> read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.
    Illumina Dna Sequencing, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 91/100, based on 950 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    GenScript dna sequences
    Large chromosomal deletions using two <t>TALENs.</t> (A) Schematic representation of the structure of partial BmBlos2 gene as described in Figure 3 . Red arrows at the top indicate primers used for PCR amplification. Blue lines at the bottom show the expected results of PCR amplification with (556 bp) or without (1351 bp) the large chromosomal deletion. (B) Gel analysis of PCR amplifications. WT represents the wild-type silkworm. Be-21 and Be-22 represent two G1 silkworm broods from G0 silkworms co-injected with B2 and B3, respectively. The numbers on the left represent the sizes of the <t>DNA</t> ladder (DL2000 plus). The blue arrows on the right indicate the two expected PCR products. (C) Sequences of the PCR products. The wild-type sequence is shown at the top with the full B2 site in red and the B3 site in blue. The TALEN recognition sites are underlined, and deletions are indicated by dashed lines.
    Dna Sequences, supplied by GenScript, used in various techniques. Bioz Stars score: 92/100, based on 590 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher abi prism 3100 dna sequencer
    The <t>DNA</t> sequencing of rs840088. Direct sequencing of a subgroup of samples with the same primers for PCR-RFLP was performed using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems), and analyzed on an <t>ABI</t> <t>PRISM</t> 3100 DNA sequencer (Applied Biosystems, Foster City, USA). (A) GG genotype. (B) GA genotype. (C) AA genotype. 82×48 mm (300×300 DPI).
    Abi Prism 3100 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1382 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Illumina Inc dna sequences
    Contents of biopolymers of the extracellular polymeric substances from <t>biofilm</t> samples harvested at T5. The <t>DNA</t> content was
    Dna Sequences, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 92/100, based on 483 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 377 dna sequencer
    Contents of biopolymers of the extracellular polymeric substances from <t>biofilm</t> samples harvested at T5. The <t>DNA</t> content was
    Abi 377 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 1253 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Eurofins dna sequencing
    Contents of biopolymers of the extracellular polymeric substances from <t>biofilm</t> samples harvested at T5. The <t>DNA</t> content was
    Dna Sequencing, supplied by Eurofins, used in various techniques. Bioz Stars score: 92/100, based on 4234 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 373a dna sequencer
    Fluorescent detection of RAF profiles. The oligonucleotide primer K-06 (5′-CACCTTTCCC-3′) was synthesized and tagged with FAM (6-carboxy-fluorescein), and then used in the RAF protocol with genomic <t>DNA</t> from sugarcane hybrid Q117. After the PCR, the RAF profiles were separated and detected on an Applied Biosystem <t>373A</t> DNA sequencer. The two panels represent RAF profiles amplified from replicate DNA extractions. The size of RAF markers (in bp) is shown on the X axis, and the relative intensity of the signals is shown on the Y axis. Larger RAF fragments were also amplified but these are not shown.
    373a Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 869 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Promega fmol dna cycle sequencing system
    Binding of Zur to the promoter regions of its potential target genes . The [γ- 32 P]-labeled upstream region of each genes (10 <t>fmol</t> of target <t>DNA</t> probes) were incubated with the purified Zur protein in the presence of 100 μM ZnCl 2 . 0, 1.25, 2.5, 5, 5, 5 and 0 pmol of Zur were used in lanes 1 to 4 and C1 to C3, respectively. The mixtures were directly subjected to 4% polyacrylamide gel electrophoresis. For lanes 1 to 4, the retarded DNA band with decreased mobility turned up, which presumably represented the Zur-DNA complex. To confirm the specificity of the binding complexes, either a 200-fold molar excess of nonspecific competitor (2 pmol of unlabeled znuA DNA without its predicted binding region in lane C1) or a 200-fold molar excess of specific competitor (2 pmol of unlabeled target DNA probe in lane C2) was added to the binding mixture. 2 pmol of an unrelated protein, i.e., purified rabbit anti-F1 antibody, were included in lane C3. Both znuA and znuC gave positive EMSA results. Since these two genes had overlapped upstream regions and shared a single predicted Zur site, the EMSA data of only znuA rather than znuC was presented herein.
    Fmol Dna Cycle Sequencing System, supplied by Promega, used in various techniques. Bioz Stars score: 91/100, based on 952 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher 377 dna sequencer
    Secondary structure data obtained for ES3 and ES6 in isolated yeast ( A ), wheat ( B ), and mouse ( C ) ribosomes. Native ribosomes isolated from yeast, wheat, and mouse were incubated in the presence of single-strand-specific reagents CMCT, final concentration 50 mM (blue), and DMS, final concentration 20 mM (red), or in the presence of the double-strand-specific ribonuclease V1, final concentration 1 unit/50 μL (green), as described in Materials and Methods. The generated modification patterns were analyzed using primer extension and gel electrophoresis in an ABI <t>377</t> automated <t>DNA</t> sequencer as described in Materials and Methods. Control samples (black) were incubated in the absence of modifying reagents but otherwise treated identically to the modified samples. The reagent-independent termination products seen in the control samples were used as internal standards for alignment and for adjusting the peak height for each of the individual overlaid lanes. Sequences are given in the 5′-to-3′ direction.
    377 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 749 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi 3100 dna sequencer
    Secondary structure data obtained for ES3 and ES6 in isolated yeast ( A ), wheat ( B ), and mouse ( C ) ribosomes. Native ribosomes isolated from yeast, wheat, and mouse were incubated in the presence of single-strand-specific reagents CMCT, final concentration 50 mM (blue), and DMS, final concentration 20 mM (red), or in the presence of the double-strand-specific ribonuclease V1, final concentration 1 unit/50 μL (green), as described in Materials and Methods. The generated modification patterns were analyzed using primer extension and gel electrophoresis in an ABI <t>377</t> automated <t>DNA</t> sequencer as described in Materials and Methods. Control samples (black) were incubated in the absence of modifying reagents but otherwise treated identically to the modified samples. The reagent-independent termination products seen in the control samples were used as internal standards for alignment and for adjusting the peak height for each of the individual overlaid lanes. Sequences are given in the 5′-to-3′ direction.
    Abi 3100 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1112 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Macrogen dna sequence analysis
    Site directed mutagenesis of the KRRIH region of Msm <t>UdgX</t> 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 <t>uracil-DNA</t> 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.
    Dna Sequence Analysis, supplied by Macrogen, used in various techniques. Bioz Stars score: 92/100, based on 193 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher 3730 dna sequencer
    Site directed mutagenesis of the KRRIH region of Msm <t>UdgX</t> 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 <t>uracil-DNA</t> 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.
    3730 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 612 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Thermo Fisher abi prism 3730 dna sequencer
    Site directed mutagenesis of the KRRIH region of Msm <t>UdgX</t> 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 <t>uracil-DNA</t> 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.
    Abi Prism 3730 Dna Sequencer, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 92/100, based on 793 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Generation of a bb0028 mutant. A . An IPTG-regulatable bb0028 mutant was generated by inserting a streptomycin resistance cassette followed by the flacp promoter immediately upstream of bb0028 . Red arrows indicate primer-binding areas used to confirm insertion of the streptomycin cassette and flacp promoter into the borrelial genome. B . PCR amplification resulted in a 1.2 kb amplicon in the wildtype strain (WT), as expected, and a 3.0 kb amplicon in the BB0028 mutant (flacp::0028) indicating the streptomycin cassette and flacp promoter were inserted in the mutant. A PCR reaction with no DNA template (Neg) was included as a negative control. DNA sizes, in kilobase pairs, are indicated at left. C . Whole-cell lysate from the BB0028 mutant (flacp::0028) strain propagated with (+) or without (−) 1 mM IPTG, as well as from the parental, wildtype strain (WT), were subjected to immunoblot analysis using BB0028 specific antibodies (top panel) or FlaB antibodies (bottom panel). FlaB reactivity was used to ensure all lanes were loaded equally.

    Journal: BMC Microbiology

    Article Title: Characterization of the β-barrel assembly machine accessory lipoproteins from Borrelia burgdorferi

    doi: 10.1186/s12866-015-0411-y

    Figure Lengend Snippet: Generation of a bb0028 mutant. A . An IPTG-regulatable bb0028 mutant was generated by inserting a streptomycin resistance cassette followed by the flacp promoter immediately upstream of bb0028 . Red arrows indicate primer-binding areas used to confirm insertion of the streptomycin cassette and flacp promoter into the borrelial genome. B . PCR amplification resulted in a 1.2 kb amplicon in the wildtype strain (WT), as expected, and a 3.0 kb amplicon in the BB0028 mutant (flacp::0028) indicating the streptomycin cassette and flacp promoter were inserted in the mutant. A PCR reaction with no DNA template (Neg) was included as a negative control. DNA sizes, in kilobase pairs, are indicated at left. C . Whole-cell lysate from the BB0028 mutant (flacp::0028) strain propagated with (+) or without (−) 1 mM IPTG, as well as from the parental, wildtype strain (WT), were subjected to immunoblot analysis using BB0028 specific antibodies (top panel) or FlaB antibodies (bottom panel). FlaB reactivity was used to ensure all lanes were loaded equally.

    Article Snippet: The DNA sequence encoding mature BB0028, lacking the N-terminal leader peptide, was amplified from B. burgdorferi B31 genomic DNA using primers bb0028 (NheI) F and bb0028 (XhoI) R. The bamA P1-4 + 14 and bb0028 amplicons were then cloned into NheI and XhoI restriction sites of the the pET23a cloning vector (EMD Millipore, Darmstadt, Germany), and the resulting constructs were electroporated into E. coli Overexpress™ C41(DE3) (Lucigen Corp, Middleton, WI).

    Techniques: Mutagenesis, Generated, Binding Assay, Polymerase Chain Reaction, Amplification, Negative Control

    RT-PCR analysis of ovarian tissues and OVCAR-3 cells. ( A ) RT-PCR analysis of ovarian serous carcinoma. Primers specific for the four known human SAA genes were used, and PCR fragments were analyzed on a 2% agarose gel. Markers of a DNA ladder (100-bp

    Journal: Journal of Histochemistry and Cytochemistry

    Article Title: Expression of Serum Amyloid A in Human Ovarian Epithelial Tumors: Implication for a Role in Ovarian Tumorigenesis

    doi: 10.1369/jhc.2010.956821

    Figure Lengend Snippet: RT-PCR analysis of ovarian tissues and OVCAR-3 cells. ( A ) RT-PCR analysis of ovarian serous carcinoma. Primers specific for the four known human SAA genes were used, and PCR fragments were analyzed on a 2% agarose gel. Markers of a DNA ladder (100-bp

    Article Snippet: The identity of the PCR products was confirmed by their sequencing in an automated DNA sequencer (Applied Biosystems 3730 DNA Analyzer; Hy Laboratories; Rehovot, Israel).

    Techniques: Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Agarose Gel Electrophoresis

    Sequence of the FGF-2 mRNA leader. (A) The sequence of the FGF-2 cDNA 5′ region was obtained by using a DNA analyzer (see Materials and Methods) and compared to the two published sequences. Under the schema of the FGF-2 cDNA indicating the two Hga ). Line 3 is the sequence obtained in this study for both DNAs with the DNA analyzer. The new Hga I site is underlined (position 153). (B) The pFC1 plasmid with the 5′ region of the FGF-2 cDNA, its homolog with the corresponding genomic sequence (kindly provided by R. Florkiewicz), and a pFC1-derived plasmid lacking the Hga ]) were Bss HII digested. The 291-nt-long resulting fragments were dephosphorylated, kinase treated in the presence of [ 32 P]ATP, and then digested with enzyme Hga I. Each step was followed by a G50 column purification. The restriction fragments were fractionated on a 15% polyacrylamide–Tris-borate-EDTA gel, which was dried and autoradiographed. Sizes of the expected fragments are (i) 121, 85, 51, and 34 nt in the presence of the new Hga I site and (ii) 121 nt plus a doublet at 85 nt in its absence. The DNA origin (cDNA or genomic) is indicated at the top; control corresponds to the plasmid lacking the Hga I site at position 153. (C) Representation of the new reading frame given by the DNA sequence shown in A (line 3). The sequence between nt 80 and 163 is shown. The two potential initiation codons are shown in boldface; the Hga I site (positions 153 to 157) is underlined.

    Journal: Molecular and Cellular Biology

    Article Title: A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor

    doi:

    Figure Lengend Snippet: Sequence of the FGF-2 mRNA leader. (A) The sequence of the FGF-2 cDNA 5′ region was obtained by using a DNA analyzer (see Materials and Methods) and compared to the two published sequences. Under the schema of the FGF-2 cDNA indicating the two Hga ). Line 3 is the sequence obtained in this study for both DNAs with the DNA analyzer. The new Hga I site is underlined (position 153). (B) The pFC1 plasmid with the 5′ region of the FGF-2 cDNA, its homolog with the corresponding genomic sequence (kindly provided by R. Florkiewicz), and a pFC1-derived plasmid lacking the Hga ]) were Bss HII digested. The 291-nt-long resulting fragments were dephosphorylated, kinase treated in the presence of [ 32 P]ATP, and then digested with enzyme Hga I. Each step was followed by a G50 column purification. The restriction fragments were fractionated on a 15% polyacrylamide–Tris-borate-EDTA gel, which was dried and autoradiographed. Sizes of the expected fragments are (i) 121, 85, 51, and 34 nt in the presence of the new Hga I site and (ii) 121 nt plus a doublet at 85 nt in its absence. The DNA origin (cDNA or genomic) is indicated at the top; control corresponds to the plasmid lacking the Hga I site at position 153. (C) Representation of the new reading frame given by the DNA sequence shown in A (line 3). The sequence between nt 80 and 163 is shown. The two potential initiation codons are shown in boldface; the Hga I site (positions 153 to 157) is underlined.

    Article Snippet: By sequencing this region of the FGF-2 cDNA with an automatic DNA sequencer (Applied Biosystems), we obtained the DNA sequence 153-GACGCGGT downstream from position 153, which still differed from the published sequences of cDNA and genomic DNA, i.e., 153-GACGGCT and 153-GACGGT, respectively (Fig. A, line 3).

    Techniques: Sequencing, Plasmid Preparation, Derivative Assay, Purification

    Analysis of the ability of the FGF-2 mRNA 5′ UTR to promote translation initiation of the 34-kDa FGF-2. Expression of the 34-kDa FGF-2 was analyzed for three DNA constructs either by in vitro transcription and translation in RRL or by COS-7 cell transfection followed by Western immunoblotting (see Materials and Methods). (A) Schema of the mRNAs expressed from the different constructs (see Materials and Methods). WT5′-FGF corresponds to the FGF-2 mRNA with a complete 5′ leader; Δ1-257 corresponds to a deletion of nt 1 to 257 in the FGF-2 mRNA 5′ end; WT5′CAT corresponds to a fusion of FGF-2 cDNA nt 1 to 312 with the CAT sequence (devoid of an AUG start codon). (B) Analysis of mRNA expression either by in vitro translation (lanes 1 and 2) or by Western immunoblotting (lanes 3 to 6). Names of the antibodies (αFGF-2, αCAT, and αN23) are indicated at the bottom. Migration of size standards is indicated on the right; migration of FGF-2 isoforms is indicated on the left.

    Journal: Molecular and Cellular Biology

    Article Title: A New 34-Kilodalton Isoform of Human Fibroblast Growth Factor 2 Is Cap Dependently Synthesized by Using a Non-AUG Start Codon and Behaves as a Survival Factor

    doi:

    Figure Lengend Snippet: Analysis of the ability of the FGF-2 mRNA 5′ UTR to promote translation initiation of the 34-kDa FGF-2. Expression of the 34-kDa FGF-2 was analyzed for three DNA constructs either by in vitro transcription and translation in RRL or by COS-7 cell transfection followed by Western immunoblotting (see Materials and Methods). (A) Schema of the mRNAs expressed from the different constructs (see Materials and Methods). WT5′-FGF corresponds to the FGF-2 mRNA with a complete 5′ leader; Δ1-257 corresponds to a deletion of nt 1 to 257 in the FGF-2 mRNA 5′ end; WT5′CAT corresponds to a fusion of FGF-2 cDNA nt 1 to 312 with the CAT sequence (devoid of an AUG start codon). (B) Analysis of mRNA expression either by in vitro translation (lanes 1 and 2) or by Western immunoblotting (lanes 3 to 6). Names of the antibodies (αFGF-2, αCAT, and αN23) are indicated at the bottom. Migration of size standards is indicated on the right; migration of FGF-2 isoforms is indicated on the left.

    Article Snippet: By sequencing this region of the FGF-2 cDNA with an automatic DNA sequencer (Applied Biosystems), we obtained the DNA sequence 153-GACGCGGT downstream from position 153, which still differed from the published sequences of cDNA and genomic DNA, i.e., 153-GACGGCT and 153-GACGGT, respectively (Fig. A, line 3).

    Techniques: Expressing, Construct, In Vitro, Transfection, Western Blot, Sequencing, Migration

    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

    Agarose gels of amplicons generated by PCR amplification of enriched fecal specimens using the invE-A (457-bp amplicon) and hisJ (496-bp amplicon) primer sets. (A) Results using the invE-A primers. Lanes 1 and 6 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lane 3, DNA extracted from culture-negative clinical specimen; lane 4, negative control (distilled water); lane 5, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). (B) Results using hisJ primers. Lanes 1 and 9 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lanes 3 to 6, DNA extracted from culture-negative clinical specimens; lane 7, negative control (distilled water); lane 8, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). Arrows indicate amplicons produced in each reaction.

    Journal: Journal of Clinical Microbiology

    Article Title: Identification of Two Phylogenetically Related Organisms from Feces by PCR for Detection of Salmonella spp.

    doi: 10.1128/JCM.40.4.1487-1492.2002

    Figure Lengend Snippet: Agarose gels of amplicons generated by PCR amplification of enriched fecal specimens using the invE-A (457-bp amplicon) and hisJ (496-bp amplicon) primer sets. (A) Results using the invE-A primers. Lanes 1 and 6 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lane 3, DNA extracted from culture-negative clinical specimen; lane 4, negative control (distilled water); lane 5, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). (B) Results using hisJ primers. Lanes 1 and 9 contain molecular weight standards (100-bp DNA ladder). DNA used for amplification was as follows: lane 2, DNA extracted from culture-positive clinical specimen; lanes 3 to 6, DNA extracted from culture-negative clinical specimens; lane 7, negative control (distilled water); lane 8, positive control (purified DNA from S. enterica serovar Infantis isolate 978-05817). Arrows indicate amplicons produced in each reaction.

    Article Snippet: To do this, the DNA sequences of the culture-negative and -positive amplicons and the negative and positive consensus sequences were submitted to the National Center for Biotechnology Information (NCBI) (National Library of Medicine, National Institutes of Health, Bethesda, Md.) for a nucleotide BLAST ( , ) search of the GenBank (NCBI), EMBL, DNA Databank of Japan (DDBJ), and Protein Data Bank (PDB) genetic sequence databases.

    Techniques: Generated, Polymerase Chain Reaction, Amplification, Molecular Weight, Negative Control, Positive Control, Purification, Produced

    Construction and analysis of sreA strains. A : Construction of pTFCM-R-L and pTFCM- neo - sreA plasmids. B: PCR analysis of Δ sreA , and CO sreA transformants using primers sreA -F and sreA -R ( Table 1 ). C: Southern blot analysis of P . digitatum wild-type PdHS-F6 and Δ sreA , CO sreA strains using a probe specific to the 5’ region of Pdsre . 30μg genomic DNA was digested with Hind III and detected using a probe specific to the 5’ region of sreA gene.

    Journal: PLoS ONE

    Article Title: A Novel Sterol Regulatory Element-Binding Protein Gene (sreA) Identified in Penicilliumdigitatum Is Required for Prochloraz Resistance, Full Virulence and erg11 (cyp51) Regulation

    doi: 10.1371/journal.pone.0117115

    Figure Lengend Snippet: Construction and analysis of sreA strains. A : Construction of pTFCM-R-L and pTFCM- neo - sreA plasmids. B: PCR analysis of Δ sreA , and CO sreA transformants using primers sreA -F and sreA -R ( Table 1 ). C: Southern blot analysis of P . digitatum wild-type PdHS-F6 and Δ sreA , CO sreA strains using a probe specific to the 5’ region of Pdsre . 30μg genomic DNA was digested with Hind III and detected using a probe specific to the 5’ region of sreA gene.

    Article Snippet: DNA and protein analysis The DNA sequence of sreA was analyzed using NCBI BLAST and BioEdit software.

    Techniques: Polymerase Chain Reaction, Southern Blot

    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

    Relationship between the Illumina DNA-sequencing read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

    doi: 10.1093/dnares/dsy005

    Figure Lengend Snippet: Relationship between the Illumina DNA-sequencing read depth and the copy number inferred by ddPCR. (A) The Y-axis represents copy numbers per μl inferred by ddPCR. Black circles (gray background) and open circles (black background) indicate three-copy genes and single-copy genes, respectively. All points and error bars represent averages of four replicates and 95% CIs. (B) Each dot represents an A. halleri gene. The X-axis represents the Illumina DNA-sequencing read depth, which is the number of reads per 1 Kbp per 1 million reads. The Y-axis represents copy numbers per μl, which were inferred by ddPCR. The regression line was calculated with the simple formula Y = αX; α was inferred by the least squares method.

    Article Snippet: We then inferred SSDs in a species after the divergence of closed species throughout the reading depth of Illumina DNA sequencing.

    Techniques: DNA Sequencing

    Three sets of OGGs among non- Arabidopsis species, A. thaliana , A. lyrata and A. halleri. OGGs between A. lyrata and A. halleri were defined as AL–AH OGGs. There were 25,833, 26,428 and 26,007 AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among A. thaliana , A. lyrata and A. halleri were defined as AT–AL–AH OGGs. There were 22,105, 22,684 and 21,537 AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among non- Arabidopsis species ( B. rapa , B. stricta , C. grandiflora , C. rubella , E. salsugineum ), A. thaliana , A. lyrata and A. halleri were defined as nonA-AT–AL–AH OGGs. There were 17,669 and 17,925 non-A-AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes and the available A. halleri genome, respectively.

    Journal: DNA Research: An International Journal for Rapid Publication of Reports on Genes and Genomes

    Article Title: Functional divergence of duplicate genes several million years after gene duplication in Arabidopsis

    doi: 10.1093/dnares/dsy005

    Figure Lengend Snippet: Three sets of OGGs among non- Arabidopsis species, A. thaliana , A. lyrata and A. halleri. OGGs between A. lyrata and A. halleri were defined as AL–AH OGGs. There were 25,833, 26,428 and 26,007 AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among A. thaliana , A. lyrata and A. halleri were defined as AT–AL–AH OGGs. There were 22,105, 22,684 and 21,537 AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes, Illumina paired-end DNA-sequencing reads including pseudogene-like genes and the available A. halleri genome, respectively. OGGs among non- Arabidopsis species ( B. rapa , B. stricta , C. grandiflora , C. rubella , E. salsugineum ), A. thaliana , A. lyrata and A. halleri were defined as nonA-AT–AL–AH OGGs. There were 17,669 and 17,925 non-A-AT–AL–AH OGGs based on Illumina paired-end DNA-sequencing reads without any pseudogene-like genes and the available A. halleri genome, respectively.

    Article Snippet: We then inferred SSDs in a species after the divergence of closed species throughout the reading depth of Illumina DNA sequencing.

    Techniques: DNA Sequencing

    Identification of minicircles in experimentally infected Beta vulgaris plants by the NGS approach. a Diagram of the bioinformatic pipeline used for the detection of minicircles. The tools used in each step are indicated in parentheses (refer to the Methods section for details). b Schematic representation of the eight BCTIV/ Beta vulgaris hybrid scaffolds assembled after the filtering step described in a . The junction coordinates referring to the BCTIV genome are indicated above each recombination point. The sizes (nt) of the B. vulgaris DNA fragments (blue portions) are reported on the right. c In vivo validation of the circular nature of the scaffolds reported in b , performed by inverse PCR (see also Supplementary Figure 3). The approximate positions of the primers specific for the non-viral portions of each minicircle (in blue) are indicated by arrows. DNA from mock-inoculated B. vulgaris . d Distribution of minicircle junctions between viral and host DNA. The proximal and distal junctions are marked with red and black arrows, respectively. BCTIV ORFs are represented by yellow boxes. e Relative contributions of B. vulgaris and BCTIV DNA to minicircles identified by NGS. f

    Journal: Nature Communications

    Article Title: Virus-mediated export of chromosomal DNA in plants

    doi: 10.1038/s41467-018-07775-w

    Figure Lengend Snippet: Identification of minicircles in experimentally infected Beta vulgaris plants by the NGS approach. a Diagram of the bioinformatic pipeline used for the detection of minicircles. The tools used in each step are indicated in parentheses (refer to the Methods section for details). b Schematic representation of the eight BCTIV/ Beta vulgaris hybrid scaffolds assembled after the filtering step described in a . The junction coordinates referring to the BCTIV genome are indicated above each recombination point. The sizes (nt) of the B. vulgaris DNA fragments (blue portions) are reported on the right. c In vivo validation of the circular nature of the scaffolds reported in b , performed by inverse PCR (see also Supplementary Figure 3). The approximate positions of the primers specific for the non-viral portions of each minicircle (in blue) are indicated by arrows. DNA from mock-inoculated B. vulgaris . d Distribution of minicircle junctions between viral and host DNA. The proximal and distal junctions are marked with red and black arrows, respectively. BCTIV ORFs are represented by yellow boxes. e Relative contributions of B. vulgaris and BCTIV DNA to minicircles identified by NGS. f

    Article Snippet: The genomic DNA sequencing libraries were prepared using the TruSeq DNA PCR-Free LT Library Prep Kit following the manufacturer’s instructions (Illumina, San Diego, CA), starting from 1.1 µg of sonicated DNA.

    Techniques: Infection, Next-Generation Sequencing, In Vivo, Inverse PCR

    Large chromosomal deletions using two TALENs. (A) Schematic representation of the structure of partial BmBlos2 gene as described in Figure 3 . Red arrows at the top indicate primers used for PCR amplification. Blue lines at the bottom show the expected results of PCR amplification with (556 bp) or without (1351 bp) the large chromosomal deletion. (B) Gel analysis of PCR amplifications. WT represents the wild-type silkworm. Be-21 and Be-22 represent two G1 silkworm broods from G0 silkworms co-injected with B2 and B3, respectively. The numbers on the left represent the sizes of the DNA ladder (DL2000 plus). The blue arrows on the right indicate the two expected PCR products. (C) Sequences of the PCR products. The wild-type sequence is shown at the top with the full B2 site in red and the B3 site in blue. The TALEN recognition sites are underlined, and deletions are indicated by dashed lines.

    Journal: PLoS ONE

    Article Title: Highly Efficient and Specific Genome Editing in Silkworm Using Custom TALENs

    doi: 10.1371/journal.pone.0045035

    Figure Lengend Snippet: Large chromosomal deletions using two TALENs. (A) Schematic representation of the structure of partial BmBlos2 gene as described in Figure 3 . Red arrows at the top indicate primers used for PCR amplification. Blue lines at the bottom show the expected results of PCR amplification with (556 bp) or without (1351 bp) the large chromosomal deletion. (B) Gel analysis of PCR amplifications. WT represents the wild-type silkworm. Be-21 and Be-22 represent two G1 silkworm broods from G0 silkworms co-injected with B2 and B3, respectively. The numbers on the left represent the sizes of the DNA ladder (DL2000 plus). The blue arrows on the right indicate the two expected PCR products. (C) Sequences of the PCR products. The wild-type sequence is shown at the top with the full B2 site in red and the B3 site in blue. The TALEN recognition sites are underlined, and deletions are indicated by dashed lines.

    Article Snippet: The designed amino acid sequences of TALENs (designated as B2L, B2R, B3L, and B3R) were converted to DNA sequences that correspond with the codon usage in silkworm and then synthesized using a commercial service (GenScript).

    Techniques: TALENs, Polymerase Chain Reaction, Amplification, Injection, Sequencing

    Design and construction of TALENs that target the BmBlos2 gene. (A) Schematic representation of the BmBlos2 gene structure depicting the introns (broken lines), exons (colored boxes) and TALEN target sequences (sequences at the bottom). The numbers indicate the exact lengths of the introns and exons. ATG and TAA indicate the translation start and stop sites, respectively. The underlined sequence represents the recognition site of the corresponding TALEN monomer. (B) Schematic representation of TALEN binding to its target DNA. A triple flag tag and a nuclear localization signal (NLS) (grey boxes) are fused to the N-terminal of each TALEN monomer. The Fok I domain (red boxes) is linked to the C-terminal of each TALEN monomer through a flexible linker. The TAL effector domain is composed of an N-terminal (blue box labelled NT), a C-terminal (blue box labelled CT), and a tandem array of repeats (an array of colored boxes between NT and CT). The TALEN binding site is composed of a left binding site (underlined), right binding site (underlined) and a spacer (NNN…). (C) TALEN target sequences and the corresponding RVDs within each repeat. The number at the top of the left panel indicates the position of the repeat. The letters at the top of each TALEN monomer represent the target sequence and the letters below represent the RVDs of the corresponding repeat. The right panel represents the RVD usage in the design. The yellow coloured RVDs are different from the commonly used RVDs.

    Journal: PLoS ONE

    Article Title: Highly Efficient and Specific Genome Editing in Silkworm Using Custom TALENs

    doi: 10.1371/journal.pone.0045035

    Figure Lengend Snippet: Design and construction of TALENs that target the BmBlos2 gene. (A) Schematic representation of the BmBlos2 gene structure depicting the introns (broken lines), exons (colored boxes) and TALEN target sequences (sequences at the bottom). The numbers indicate the exact lengths of the introns and exons. ATG and TAA indicate the translation start and stop sites, respectively. The underlined sequence represents the recognition site of the corresponding TALEN monomer. (B) Schematic representation of TALEN binding to its target DNA. A triple flag tag and a nuclear localization signal (NLS) (grey boxes) are fused to the N-terminal of each TALEN monomer. The Fok I domain (red boxes) is linked to the C-terminal of each TALEN monomer through a flexible linker. The TAL effector domain is composed of an N-terminal (blue box labelled NT), a C-terminal (blue box labelled CT), and a tandem array of repeats (an array of colored boxes between NT and CT). The TALEN binding site is composed of a left binding site (underlined), right binding site (underlined) and a spacer (NNN…). (C) TALEN target sequences and the corresponding RVDs within each repeat. The number at the top of the left panel indicates the position of the repeat. The letters at the top of each TALEN monomer represent the target sequence and the letters below represent the RVDs of the corresponding repeat. The right panel represents the RVD usage in the design. The yellow coloured RVDs are different from the commonly used RVDs.

    Article Snippet: The designed amino acid sequences of TALENs (designated as B2L, B2R, B3L, and B3R) were converted to DNA sequences that correspond with the codon usage in silkworm and then synthesized using a commercial service (GenScript).

    Techniques: TALENs, Sequencing, Binding Assay, FLAG-tag

    The DNA sequencing of rs840088. Direct sequencing of a subgroup of samples with the same primers for PCR-RFLP was performed using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems), and analyzed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, USA). (A) GG genotype. (B) GA genotype. (C) AA genotype. 82×48 mm (300×300 DPI).

    Journal: Yonsei Medical Journal

    Article Title: The Association of SERPINE2 Gene with COPD in a Chinese Han Population

    doi: 10.3349/ymj.2011.52.6.953

    Figure Lengend Snippet: The DNA sequencing of rs840088. Direct sequencing of a subgroup of samples with the same primers for PCR-RFLP was performed using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems), and analyzed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, USA). (A) GG genotype. (B) GA genotype. (C) AA genotype. 82×48 mm (300×300 DPI).

    Article Snippet: Sequencing was performed using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems), and analyzed on an ABI PRISM 3100 DNA sequencer (Applied Biosystems, Foster City, CA, USA).

    Techniques: DNA Sequencing, Sequencing, Polymerase Chain Reaction

    Contents of biopolymers of the extracellular polymeric substances from biofilm samples harvested at T5. The DNA content was

    Journal: Applied and Environmental Microbiology

    Article Title: Metagenome Survey of a Multispecies and Alga-Associated Biofilm Revealed Key Elements of Bacterial-Algal Interactions in Photobioreactors

    doi: 10.1128/AEM.01641-13

    Figure Lengend Snippet: Contents of biopolymers of the extracellular polymeric substances from biofilm samples harvested at T5. The DNA content was

    Article Snippet: To further verify these data, we analyzed the DNA sequences of the mature PBR biofilm obtained by pyrosequencing and Illumina-based sequencing for the presence of hypervariable regions of rRNA gene fragments.

    Techniques:

    Fluorescent detection of RAF profiles. The oligonucleotide primer K-06 (5′-CACCTTTCCC-3′) was synthesized and tagged with FAM (6-carboxy-fluorescein), and then used in the RAF protocol with genomic DNA from sugarcane hybrid Q117. After the PCR, the RAF profiles were separated and detected on an Applied Biosystem 373A DNA sequencer. The two panels represent RAF profiles amplified from replicate DNA extractions. The size of RAF markers (in bp) is shown on the X axis, and the relative intensity of the signals is shown on the Y axis. Larger RAF fragments were also amplified but these are not shown.

    Journal: Journal of Biomedicine and Biotechnology

    Article Title: Randomly Amplified DNA Fingerprinting: A Culmination of DNA Marker Technologies Based on Arbitrarily-Primed PCR Amplification

    doi: 10.1155/S1110724302206026

    Figure Lengend Snippet: Fluorescent detection of RAF profiles. The oligonucleotide primer K-06 (5′-CACCTTTCCC-3′) was synthesized and tagged with FAM (6-carboxy-fluorescein), and then used in the RAF protocol with genomic DNA from sugarcane hybrid Q117. After the PCR, the RAF profiles were separated and detected on an Applied Biosystem 373A DNA sequencer. The two panels represent RAF profiles amplified from replicate DNA extractions. The size of RAF markers (in bp) is shown on the X axis, and the relative intensity of the signals is shown on the Y axis. Larger RAF fragments were also amplified but these are not shown.

    Article Snippet: In these cases, the fluorescent RAF profiles were detected on an Applied Biosystem 373A DNA sequencer.

    Techniques: Synthesized, Polymerase Chain Reaction, Amplification

    Binding of Zur to the promoter regions of its potential target genes . The [γ- 32 P]-labeled upstream region of each genes (10 fmol of target DNA probes) were incubated with the purified Zur protein in the presence of 100 μM ZnCl 2 . 0, 1.25, 2.5, 5, 5, 5 and 0 pmol of Zur were used in lanes 1 to 4 and C1 to C3, respectively. The mixtures were directly subjected to 4% polyacrylamide gel electrophoresis. For lanes 1 to 4, the retarded DNA band with decreased mobility turned up, which presumably represented the Zur-DNA complex. To confirm the specificity of the binding complexes, either a 200-fold molar excess of nonspecific competitor (2 pmol of unlabeled znuA DNA without its predicted binding region in lane C1) or a 200-fold molar excess of specific competitor (2 pmol of unlabeled target DNA probe in lane C2) was added to the binding mixture. 2 pmol of an unrelated protein, i.e., purified rabbit anti-F1 antibody, were included in lane C3. Both znuA and znuC gave positive EMSA results. Since these two genes had overlapped upstream regions and shared a single predicted Zur site, the EMSA data of only znuA rather than znuC was presented herein.

    Journal: BMC Microbiology

    Article Title: Characterization of Zur-dependent genes and direct Zur targets in Yersinia pestis

    doi: 10.1186/1471-2180-9-128

    Figure Lengend Snippet: Binding of Zur to the promoter regions of its potential target genes . The [γ- 32 P]-labeled upstream region of each genes (10 fmol of target DNA probes) were incubated with the purified Zur protein in the presence of 100 μM ZnCl 2 . 0, 1.25, 2.5, 5, 5, 5 and 0 pmol of Zur were used in lanes 1 to 4 and C1 to C3, respectively. The mixtures were directly subjected to 4% polyacrylamide gel electrophoresis. For lanes 1 to 4, the retarded DNA band with decreased mobility turned up, which presumably represented the Zur-DNA complex. To confirm the specificity of the binding complexes, either a 200-fold molar excess of nonspecific competitor (2 pmol of unlabeled znuA DNA without its predicted binding region in lane C1) or a 200-fold molar excess of specific competitor (2 pmol of unlabeled target DNA probe in lane C2) was added to the binding mixture. 2 pmol of an unrelated protein, i.e., purified rabbit anti-F1 antibody, were included in lane C3. Both znuA and znuC gave positive EMSA results. Since these two genes had overlapped upstream regions and shared a single predicted Zur site, the EMSA data of only znuA rather than znuC was presented herein.

    Article Snippet: For sequencing, the fmol® DNA Cycle Sequencing System (Promega) was used.

    Techniques: Binding Assay, Labeling, Incubation, Purification, Polyacrylamide Gel Electrophoresis

    Secondary structure data obtained for ES3 and ES6 in isolated yeast ( A ), wheat ( B ), and mouse ( C ) ribosomes. Native ribosomes isolated from yeast, wheat, and mouse were incubated in the presence of single-strand-specific reagents CMCT, final concentration 50 mM (blue), and DMS, final concentration 20 mM (red), or in the presence of the double-strand-specific ribonuclease V1, final concentration 1 unit/50 μL (green), as described in Materials and Methods. The generated modification patterns were analyzed using primer extension and gel electrophoresis in an ABI 377 automated DNA sequencer as described in Materials and Methods. Control samples (black) were incubated in the absence of modifying reagents but otherwise treated identically to the modified samples. The reagent-independent termination products seen in the control samples were used as internal standards for alignment and for adjusting the peak height for each of the individual overlaid lanes. Sequences are given in the 5′-to-3′ direction.

    Journal: RNA

    Article Title: Secondary structure of two regions in expansion segments ES3 and ES6 with the potential of forming a tertiary interaction in eukaryotic 40S ribosomal subunits

    doi: 10.1261/rna.5135204

    Figure Lengend Snippet: Secondary structure data obtained for ES3 and ES6 in isolated yeast ( A ), wheat ( B ), and mouse ( C ) ribosomes. Native ribosomes isolated from yeast, wheat, and mouse were incubated in the presence of single-strand-specific reagents CMCT, final concentration 50 mM (blue), and DMS, final concentration 20 mM (red), or in the presence of the double-strand-specific ribonuclease V1, final concentration 1 unit/50 μL (green), as described in Materials and Methods. The generated modification patterns were analyzed using primer extension and gel electrophoresis in an ABI 377 automated DNA sequencer as described in Materials and Methods. Control samples (black) were incubated in the absence of modifying reagents but otherwise treated identically to the modified samples. The reagent-independent termination products seen in the control samples were used as internal standards for alignment and for adjusting the peak height for each of the individual overlaid lanes. Sequences are given in the 5′-to-3′ direction.

    Article Snippet: The primer extension products were analyzed on 4.75% (w/v) acrylamide sequencing gels in an Applied Biosystems 377 DNA Sequencer as described ( ).

    Techniques: Isolation, Incubation, Concentration Assay, Generated, Modification, Nucleic Acid Electrophoresis

    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