hfx volcanii dna  (Roche)


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    Structured Review

    Roche hfx volcanii dna
    Phylogenetic analysis of archaeal ATP-dependent <t>DNA</t> ligases . Phylogenetic tree showing the evolutionary relationship of 41 archaeal ATP-dependent DNA ligase proteins together with the closely-related enzyme encoded by the eubacterium Aquifex aeolicus . Crenarchaeal species are shown in red, whereas halophilic euryarchaeal species (such as <t>Hfx.volcanii</t> ) are shown in blue. The Aquifex enzyme ( Aae , indicated in green) can be seen clustering with the hyperthermophilic (non-marine) crenarchaeal species shown at the top of the figure. Archaeal species abbreviations: Hvo ( Haloferax volcanii ), Aam ( Acidianus ambivalens ), Afu (Archaeoglobus fulgidus), Ape ( Aeropyrum pernix ), Csy ( Cenarcheon symbiosum ), Hma ( Haloarcula marismortui ), Fac (Ferroplasma acidarmanus), Nph ( Natronomonas pharaosis ), Hqw ( Haloquadratum walsbyi ), Has ( Halobacterium salinarum ), Mac ( Methanosarcina acetivorans ), Mba ( Methanosarcina barkeri ), Mbu ( Methanococcoides burtonii ), Mhu ( Methanospirillum hungatei ), Mja ( Methanocaldococcus jannaschii ), Mka ( Methanopyrus kandleri ), Mma ( Methanosarcina mazei ), Mmr ( Methanococcus maripaludis ), Mpt ( Methanosaeta thermophila ), Mst ( Methanosphaera stadtmanae ), Mth ( Methanothermobacter thermautotrophicus ), Neq ( Nanoarchaeum equitans ), Pab ( Pyrococcus abyssi ), Pae ( Pyrobaculum aerophilum ), Pfu ( Pyrococcus furiosus ), Pho ( Pyrococcus horikoshii ), Pto ( Picrophilus torridus ), Sac ( Sulfolobus acidocaldarius ), Ssh ( Sulfolobus shibatae ), Sso (Sulfolobus solfataricus), Sto ( Sulfolobus tokodaii ), Tac ( Thermoplasma acidophilum ), Tfu ( Thermococcus fumicolans ), Tko ( Thermococcus kodakarensis ), Tsp ( Thermococcus sp . NA1), Tvo ( Thermoplasma volcanium ), 74A4 ( uncultured crenarchaeote 74A4 ). The tree was generated using TreeView X [39] from sequence alignment data generated using Clustal X [40]. A full listing of the database accession numbers of the proteins used to construct the tree can be obtained from the authors on request.
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    Images

    1) Product Images from "Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations"

    Article Title: Biochemical characterisation of LigN, an NAD+-dependent DNA ligase from the halophilic euryarchaeon Haloferax volcanii that displays maximal in vitro activity at high salt concentrations

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-7-44

    Phylogenetic analysis of archaeal ATP-dependent DNA ligases . Phylogenetic tree showing the evolutionary relationship of 41 archaeal ATP-dependent DNA ligase proteins together with the closely-related enzyme encoded by the eubacterium Aquifex aeolicus . Crenarchaeal species are shown in red, whereas halophilic euryarchaeal species (such as Hfx.volcanii ) are shown in blue. The Aquifex enzyme ( Aae , indicated in green) can be seen clustering with the hyperthermophilic (non-marine) crenarchaeal species shown at the top of the figure. Archaeal species abbreviations: Hvo ( Haloferax volcanii ), Aam ( Acidianus ambivalens ), Afu (Archaeoglobus fulgidus), Ape ( Aeropyrum pernix ), Csy ( Cenarcheon symbiosum ), Hma ( Haloarcula marismortui ), Fac (Ferroplasma acidarmanus), Nph ( Natronomonas pharaosis ), Hqw ( Haloquadratum walsbyi ), Has ( Halobacterium salinarum ), Mac ( Methanosarcina acetivorans ), Mba ( Methanosarcina barkeri ), Mbu ( Methanococcoides burtonii ), Mhu ( Methanospirillum hungatei ), Mja ( Methanocaldococcus jannaschii ), Mka ( Methanopyrus kandleri ), Mma ( Methanosarcina mazei ), Mmr ( Methanococcus maripaludis ), Mpt ( Methanosaeta thermophila ), Mst ( Methanosphaera stadtmanae ), Mth ( Methanothermobacter thermautotrophicus ), Neq ( Nanoarchaeum equitans ), Pab ( Pyrococcus abyssi ), Pae ( Pyrobaculum aerophilum ), Pfu ( Pyrococcus furiosus ), Pho ( Pyrococcus horikoshii ), Pto ( Picrophilus torridus ), Sac ( Sulfolobus acidocaldarius ), Ssh ( Sulfolobus shibatae ), Sso (Sulfolobus solfataricus), Sto ( Sulfolobus tokodaii ), Tac ( Thermoplasma acidophilum ), Tfu ( Thermococcus fumicolans ), Tko ( Thermococcus kodakarensis ), Tsp ( Thermococcus sp . NA1), Tvo ( Thermoplasma volcanium ), 74A4 ( uncultured crenarchaeote 74A4 ). The tree was generated using TreeView X [39] from sequence alignment data generated using Clustal X [40]. A full listing of the database accession numbers of the proteins used to construct the tree can be obtained from the authors on request.
    Figure Legend Snippet: Phylogenetic analysis of archaeal ATP-dependent DNA ligases . Phylogenetic tree showing the evolutionary relationship of 41 archaeal ATP-dependent DNA ligase proteins together with the closely-related enzyme encoded by the eubacterium Aquifex aeolicus . Crenarchaeal species are shown in red, whereas halophilic euryarchaeal species (such as Hfx.volcanii ) are shown in blue. The Aquifex enzyme ( Aae , indicated in green) can be seen clustering with the hyperthermophilic (non-marine) crenarchaeal species shown at the top of the figure. Archaeal species abbreviations: Hvo ( Haloferax volcanii ), Aam ( Acidianus ambivalens ), Afu (Archaeoglobus fulgidus), Ape ( Aeropyrum pernix ), Csy ( Cenarcheon symbiosum ), Hma ( Haloarcula marismortui ), Fac (Ferroplasma acidarmanus), Nph ( Natronomonas pharaosis ), Hqw ( Haloquadratum walsbyi ), Has ( Halobacterium salinarum ), Mac ( Methanosarcina acetivorans ), Mba ( Methanosarcina barkeri ), Mbu ( Methanococcoides burtonii ), Mhu ( Methanospirillum hungatei ), Mja ( Methanocaldococcus jannaschii ), Mka ( Methanopyrus kandleri ), Mma ( Methanosarcina mazei ), Mmr ( Methanococcus maripaludis ), Mpt ( Methanosaeta thermophila ), Mst ( Methanosphaera stadtmanae ), Mth ( Methanothermobacter thermautotrophicus ), Neq ( Nanoarchaeum equitans ), Pab ( Pyrococcus abyssi ), Pae ( Pyrobaculum aerophilum ), Pfu ( Pyrococcus furiosus ), Pho ( Pyrococcus horikoshii ), Pto ( Picrophilus torridus ), Sac ( Sulfolobus acidocaldarius ), Ssh ( Sulfolobus shibatae ), Sso (Sulfolobus solfataricus), Sto ( Sulfolobus tokodaii ), Tac ( Thermoplasma acidophilum ), Tfu ( Thermococcus fumicolans ), Tko ( Thermococcus kodakarensis ), Tsp ( Thermococcus sp . NA1), Tvo ( Thermoplasma volcanium ), 74A4 ( uncultured crenarchaeote 74A4 ). The tree was generated using TreeView X [39] from sequence alignment data generated using Clustal X [40]. A full listing of the database accession numbers of the proteins used to construct the tree can be obtained from the authors on request.

    Techniques Used: Microscale Thermophoresis, Generated, Sequencing, Construct

    Recombinant LigN is active as a DNA ligase . A. Schematic representation of Hfx.volcanii LigN protein structure showing the six structural domains that characterise this family of enzymes. B. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. C. Adenylation assay. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) were incubated with [ 32 P]NAD + , as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. D. Ligase activity assays carried out using λ Bst EII-digested DNA, as described in the Methods. Reactions were performed in 2.5 M KCl at 45°C with no added enzyme (lane 1, 10 minute incubation), LigN (lanes 2 – 5, incubation for 2.5, 5, 7.5 and 10 minutes), LigN-K139A (lane 6, 10 minute incubation) and LigN-K141A (lane 7, 10 minute incubation). E. Purified LigN and LigN-ΔC visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. F. Adenylation assay. Purified LigN (lane 1) and LigN-ΔC (lane 2) were incubated with [ 32 P]NAD + , as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. G. DNA ligase activity assays performed as described in Methods without added enzyme (lane 1) or using LigN (lane 2) or LigN-ΔC (lane 3). LigN-ΔC was inactive in the assay.
    Figure Legend Snippet: Recombinant LigN is active as a DNA ligase . A. Schematic representation of Hfx.volcanii LigN protein structure showing the six structural domains that characterise this family of enzymes. B. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. C. Adenylation assay. Purified LigN, LigN-K139A and LigN-D141A proteins (lanes 1, 2 and 3 respectively) were incubated with [ 32 P]NAD + , as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. D. Ligase activity assays carried out using λ Bst EII-digested DNA, as described in the Methods. Reactions were performed in 2.5 M KCl at 45°C with no added enzyme (lane 1, 10 minute incubation), LigN (lanes 2 – 5, incubation for 2.5, 5, 7.5 and 10 minutes), LigN-K139A (lane 6, 10 minute incubation) and LigN-K141A (lane 7, 10 minute incubation). E. Purified LigN and LigN-ΔC visualised by PAGE-Blue G90 staining following 10% SDS-PAGE. 5 μg of each protein was loaded per lane. The lane marked M contains molecular weight markers with molecular weights (in kDa) shown to the left. F. Adenylation assay. Purified LigN (lane 1) and LigN-ΔC (lane 2) were incubated with [ 32 P]NAD + , as described in the Methods, before being subjected to SDS-PAGE. Adenylated proteins were detected by autoradiography of the dried gel. G. DNA ligase activity assays performed as described in Methods without added enzyme (lane 1) or using LigN (lane 2) or LigN-ΔC (lane 3). LigN-ΔC was inactive in the assay.

    Techniques Used: Recombinant, Purification, Polyacrylamide Gel Electrophoresis, Staining, SDS Page, Molecular Weight, Incubation, Autoradiography, Activity Assay

    Phylogenetic analysis of archaeal and eubacterial NAD + -dependent DNA ligases . Phylogenetic tree showing the evolutionary relationship of 106 eubacterial and archaeal NAD + -dependent DNA ligase proteins. The tree was generated using TreeView X [39] from sequence alignment data generated using Clustal X [40]. For clarity, only the five proteins of archaeal origin are indicated by name, as follows: Hvo ( Haloferax volcanii ), Hma ( Haloarcula marismortui ), Nph ( Natronomonas pharaosis ), Hqw ( Haloquadratum walsbyi HQ2659A protein) and HF70 ( uncultured marine archaeon HF70_B12 ). The colour key is shown bottom right. The category labelled Other eubacteria includes representatives of the Chloroflexi , Bacteroidetes and Deinococci groups, amongst others. A full listing of the identities and database accession numbers of the proteins used to construct the tree can be obtained from the authors on request.
    Figure Legend Snippet: Phylogenetic analysis of archaeal and eubacterial NAD + -dependent DNA ligases . Phylogenetic tree showing the evolutionary relationship of 106 eubacterial and archaeal NAD + -dependent DNA ligase proteins. The tree was generated using TreeView X [39] from sequence alignment data generated using Clustal X [40]. For clarity, only the five proteins of archaeal origin are indicated by name, as follows: Hvo ( Haloferax volcanii ), Hma ( Haloarcula marismortui ), Nph ( Natronomonas pharaosis ), Hqw ( Haloquadratum walsbyi HQ2659A protein) and HF70 ( uncultured marine archaeon HF70_B12 ). The colour key is shown bottom right. The category labelled Other eubacteria includes representatives of the Chloroflexi , Bacteroidetes and Deinococci groups, amongst others. A full listing of the identities and database accession numbers of the proteins used to construct the tree can be obtained from the authors on request.

    Techniques Used: Generated, Sequencing, Construct

    2) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Techniques Used: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
    Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Techniques Used: Synthesized, Mutagenesis

    3) Product Images from "Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region"

    Article Title: Evidence for a rapid rate of molecular evolution at the hypervariable and immunogenic Mycobacterium tuberculosis PPE38 gene region

    Journal: BMC Evolutionary Biology

    doi: 10.1186/1471-2148-9-237

    Schematic representations of the PPE38 gene region in the H37 reference strain published sequences . The PPE38 region from the published H37Rv (2a) and H37Ra (2b) sequences are shown. Colour coding as follows: PPE38 pale blue, PPE71 dark blue, MRA_ 2374 pale green, MRA_ 2375 dark green. Locations of the PPE38F/R and PPE38 IntF/R primers are shown. 2a. H37Rv ATCC reference strain (published whole genome sequence) The published H37Rv sequence [ 1 ] represents the RvD7 genotype. Recombination between PPE38 and PPE71 results in a single PPE38/71 gene (Rv 2352c ) and loss of the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . The PPE38F/R primers (black arrows) are predicted to produce an amplicon of 1335 bp from the RvD7 genotype. It is impossible to determine which PPE38/71 gene has been deleted hence the mixture of colours used. The published H37Rv sequence is not representative of the H37Rv ATCC reference strain, most clinical isolates, or the H37Ra whole genome sequence [ 19 ]. This genotype is also seen in strains SAWC 2240 (CAS, F20), SAWC 1748 (Pre-Haarlem, F24), SAWC 1595 (Quebec/S), SAWC 1841 (Haarlem, F4), CPHL_A (WA-1, M. africanum ), T17 (PGG1, EAI), EAS054 (PGG1, EAI), strain C (LCC,
    Figure Legend Snippet: Schematic representations of the PPE38 gene region in the H37 reference strain published sequences . The PPE38 region from the published H37Rv (2a) and H37Ra (2b) sequences are shown. Colour coding as follows: PPE38 pale blue, PPE71 dark blue, MRA_ 2374 pale green, MRA_ 2375 dark green. Locations of the PPE38F/R and PPE38 IntF/R primers are shown. 2a. H37Rv ATCC reference strain (published whole genome sequence) The published H37Rv sequence [ 1 ] represents the RvD7 genotype. Recombination between PPE38 and PPE71 results in a single PPE38/71 gene (Rv 2352c ) and loss of the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . The PPE38F/R primers (black arrows) are predicted to produce an amplicon of 1335 bp from the RvD7 genotype. It is impossible to determine which PPE38/71 gene has been deleted hence the mixture of colours used. The published H37Rv sequence is not representative of the H37Rv ATCC reference strain, most clinical isolates, or the H37Ra whole genome sequence [ 19 ]. This genotype is also seen in strains SAWC 2240 (CAS, F20), SAWC 1748 (Pre-Haarlem, F24), SAWC 1595 (Quebec/S), SAWC 1841 (Haarlem, F4), CPHL_A (WA-1, M. africanum ), T17 (PGG1, EAI), EAS054 (PGG1, EAI), strain C (LCC, "3 bander") and Haarlem (PGG2, F4) [see additional file 1 ]. 2b. H37Rv ATCC reference strain (actual) and H37Ra (published whole genome sequence) This represents the ancestral MTBC genotype that is also seen in M. canettii . It contains the 2 identical PPE38 (MRA_ 2373) and PPE71 (MRA_ 2376 ) genes separated by the 2 esx -like genes MRA_ 2374 and MRA_ 2375 . Gene annotations are as reported for the H37Ra published sequence [ 19 ]. Locations of primers used for PCR and sequence analysis are indicated (black arrows). This is also the true genotype of the ATTC reference strain H37Rv.

    Techniques Used: Sequencing, Amplification, Polymerase Chain Reaction

    4) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    5) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
    Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    6) Product Images from "Mechanical properties of DNA-like polymers"

    Article Title: Mechanical properties of DNA-like polymers

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt808

    Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).
    Figure Legend Snippet: Characterization of DNA analogs. ( A ) PCR assays analyzed by 5% native polyacrylamide gel electrophoresis. Total PCR volume 100 µl: 20 ng 418-bp DNA template (pJ1506), 0.4 mM each LJM-3222 (5'-G TA CGC AG T ) and LJM-3223 (5'-TGTGAGT AGCTCACTCAT AG ), 0.2 mM each dNTP with indicated analog triphosphate ( 1–9 ) completely replacing appropriate dNTP, and 5 U DNA polymerase (indicated with plus symbol) with associated buffer and cycle conditions. Taq DNA polymerase ( Taq ) conditions: Taq DNA polymerase buffer with 100 mg/ml BSA and 2 mM MgCl ; 98°C (3 min), 30 cycles of [94°C (30 s), 60°C (30 s), and 72°C (45 s)], 72°C (5 min). PrimeSTAR HS DNA polymerase (PS) conditions: PrimeSTAR GC buffer with 2 M betaine; 98°C (3 min), 30 cycles of [98°C (15 s), 60°C (5 s), and 72°C (45 s)], 72°C (5 min). Pwo SuperYield DNA Polymerase ( Pwo ) conditions: Pwo PCR buffer with GC-rich solution and 2 M betaine; 98°C (3 min), 30 cycles of [98°C (1 min), 60°C (2 min), and 72°C (8 min)], 72°C (5 min). Lane 1 is marker (M) DNA (100 bp DNA ladder, Invitrogen) with 400 - and 500-bp bands indicated. ( B ) Anion exchange chromatography of 98-bp DNA-like polymers (pJ1923). Following equilibration in 20 mM Tris–HCl, pH 8 (buffer A), samples were eluted over 25 min at a 1 ml/min flow rate in a linear gradient from 50 to 100% buffer B (buffer A plus 1 M NaCl). Eluent absorbance at 260 nm (milli-absorbance units) was monitored with elution time (min).

    Techniques Used: Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Marker, Chromatography, Flow Cytometry

    7) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    8) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    9) Product Images from "Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma"

    Article Title: Aberrant Methylation Inactivates Transforming Growth Factor β Receptor I in Head and Neck Squamous Cell Carcinoma

    Journal: International Journal of Otolaryngology

    doi: 10.1155/2009/848695

    Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.
    Figure Legend Snippet: Analysis of T β R - I promoter status and gene function in HNSCCs. (a) Representative examples of restriction enzyme-mediated PCR (MSRE) experiments. Analyses were performed for each tumor in the presence (+) and in the absence (−) of Bst UI as described in Materials and Methods. Presence of PCR products in (+) lanes indicates methylated DNA. Methylation of T β R - I was detected for carcinomas 6, 8, 30, 37, and 46. A positive control of peripheral blood lymphocytes DNA (H) shows unmethylated DNA. A negative (N) control without DNA was used in each assay. M: molecular size marker 100 bp. (b) Methylation-specific PCR for bisulfite-modified DNA that was amplified with primers specific for methylated alleles, as described in Materials and Methods. The presence of PCR products (Lanes 1 to 9 and 11 to 12) is indicative of a methylated T β R - I gene promoter. Lane 10 (HNSCC no. 39) shows an unmethylated DNA. (c) Semiquantitative RT-PCR analysis of T β R - I gene expression in representative samples of HNSCCs. Expression of ACTB gene was used as a control for RNA integrity. Relative mRNA level was normalized based on that of β -actin (153 bp). The length of the T β R - I PCR product is 186 bp. The agarose gel image was taken from a 30-cycle PCR. T β R - I (a) and ACTB (b) PCR products were visualized after electrophoresis through 2.5% agarose. HNSCC samples 28, 16, 38, 19, 23, 32 have lost or show reduced mRNA expression. HNSCC sample 39 had preserved mRNA expression. M: molecular size marker 50 bp.

    Techniques Used: Polymerase Chain Reaction, Methylation, DNA Methylation Assay, Positive Control, Marker, Modification, Amplification, Reverse Transcription Polymerase Chain Reaction, Expressing, Agarose Gel Electrophoresis, Electrophoresis

    10) Product Images from "PCR-based rapid genotyping of Stenotrophomonas maltophilia isolates"

    Article Title: PCR-based rapid genotyping of Stenotrophomonas maltophilia isolates

    Journal: BMC Microbiology

    doi: 10.1186/1471-2180-8-202

    A) Organization of the repetitive SMAG-positive loci in the genome of the S. maltophilia K279a strain . The structure of loci I to XII is diagramatically shown. SMAG sequences are represented as boxes. Repeat units at each locus are highlighted and their size in bp is indicated. B) Allelic variations at SMAG + loci . Amplimers spanning the loci IX and X derived from the DNA of the indicated S. maltophilia strains were electrophoresed on a 2% agarose gel. Numbers below bands mark the number of repeat units within each amplimer. The 100 bp ladder was used as molecular weight (MW) DNA marker.
    Figure Legend Snippet: A) Organization of the repetitive SMAG-positive loci in the genome of the S. maltophilia K279a strain . The structure of loci I to XII is diagramatically shown. SMAG sequences are represented as boxes. Repeat units at each locus are highlighted and their size in bp is indicated. B) Allelic variations at SMAG + loci . Amplimers spanning the loci IX and X derived from the DNA of the indicated S. maltophilia strains were electrophoresed on a 2% agarose gel. Numbers below bands mark the number of repeat units within each amplimer. The 100 bp ladder was used as molecular weight (MW) DNA marker.

    Techniques Used: Derivative Assay, Agarose Gel Electrophoresis, Molecular Weight, Marker

    PFGE profiles of S. maltophilia strains . The DNA of the indicated isolates was restricted with Xba I. The digestion products were resolved on 1.2% agarose gels (see Methods). The percentage genetic similarity is shown above the dendrogram. Strain numbers and PT types are shown on the right of each PFGE profile. Numbers at the bottom refer to the size in kb of the MW DNA.
    Figure Legend Snippet: PFGE profiles of S. maltophilia strains . The DNA of the indicated isolates was restricted with Xba I. The digestion products were resolved on 1.2% agarose gels (see Methods). The percentage genetic similarity is shown above the dendrogram. Strain numbers and PT types are shown on the right of each PFGE profile. Numbers at the bottom refer to the size in kb of the MW DNA.

    Techniques Used:

    11) Product Images from "Current Situation of Antimicrobial Resistance and Genetic Differences in Stenotrophomonas maltophilia Complex Isolates by Multilocus Variable Number of Tandem Repeat Analysis"

    Article Title: Current Situation of Antimicrobial Resistance and Genetic Differences in Stenotrophomonas maltophilia Complex Isolates by Multilocus Variable Number of Tandem Repeat Analysis

    Journal: Infection & Chemotherapy

    doi: 10.3947/ic.2016.48.4.285

    Dendrogram demonstrating genetic disimilarity of 118 Stenotrophomonas maltophilia complex isolates based on MLVA genotyping method in amplifications of all eight SMAGs loci.
    Figure Legend Snippet: Dendrogram demonstrating genetic disimilarity of 118 Stenotrophomonas maltophilia complex isolates based on MLVA genotyping method in amplifications of all eight SMAGs loci.

    Techniques Used:

    12) Product Images from "A Single Nucleotide Variant in the FMR1 CGG Repeat Results in a "Pseudodeletion" and Is Not Associated with the Fragile X Syndrome Phenotype"

    Article Title: A Single Nucleotide Variant in the FMR1 CGG Repeat Results in a "Pseudodeletion" and Is Not Associated with the Fragile X Syndrome Phenotype

    Journal:

    doi: 10.2353/jmoldx.2008.070163

    FMR1 Sequencing
    Figure Legend Snippet: FMR1 Sequencing

    Techniques Used: Sequencing

    13) Product Images from "Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines"

    Article Title: Estrogen Receptor β Agonists Differentially Affect the Growth of Human Melanoma Cell Lines

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0134396

    ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells. (A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E 2 (10 −8 M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E 2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.
    Figure Legend Snippet: ERβ activation induces global DNA methylation reprogramming in BLM melanoma cells. (A) Preliminary experiments were carried out to analyze the global DNA methylation status of BLM cells when compared to that of human normal melanocytes (hMel). To this purpose, a restriction enzymatic assay was employed. For each DNA sample, two restriction digests were performed: one with RsaI and MspI, and one with RsaI and HpaII. RsaI is methylation insensitive, while MspI and HpaII are sensitive to DNA methylation and are able to cut only unmethylated restriction sites. The digests were then amplified by PCR. Data are expressed as the MspI/RsaI or HpaII/RsaI ratios relative to the intensity of the bands. BLM melanoma cells were found to be globally hypomethylated when compared to normal melanocytes, when both MspI and HpaII restriction enzymes were utilized. One representative of three different experiments, which gave similar results, is reported. (B) Experiments were performed to evaluate whether activation of ERβ might affect the global DNA hypomethylation status observed in melanoma cells. BLM cells were treated with either DPN or E 2 (10 −8 M) for 24 or 48 h; the DNA methylation status was then evaluated as described above. Both DPN (at 24 and 48 h) and E 2 (at 24 h) increased the DNA methylation profile of BLM cells, indicating that ERβ activation reverts the DNA hypomethylation status in melanoma cells. One representative of three different experiments, which gave similar results, is reported.

    Techniques Used: Activation Assay, DNA Methylation Assay, Enzymatic Assay, Methylation, Amplification, Polymerase Chain Reaction

    14) Product Images from "A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands"

    Article Title: A role for heparan sulfate proteoglycans in Plasmodium falciparum sporozoite invasion of anopheline mosquito salivary glands

    Journal: Biochemical Journal

    doi: 10.1042/BJ20110694

    Enzymatic characterization of AgOXT1 RP-HPLC-based assays were used to determine the percentage conversion over 2 h into product under various conditions to assess ( A ) optimum pH, ( B ) cation dependence, ( C ) optimum temperature and ( D ) K m value for the donor substrate UDP-Xyl (Hanes–Woolf plot); the inset in ( D ) shows the kinetic curve plotting UDP-Xyl concentration against percentage conversion.
    Figure Legend Snippet: Enzymatic characterization of AgOXT1 RP-HPLC-based assays were used to determine the percentage conversion over 2 h into product under various conditions to assess ( A ) optimum pH, ( B ) cation dependence, ( C ) optimum temperature and ( D ) K m value for the donor substrate UDP-Xyl (Hanes–Woolf plot); the inset in ( D ) shows the kinetic curve plotting UDP-Xyl concentration against percentage conversion.

    Techniques Used: High Performance Liquid Chromatography, Concentration Assay

    MALDI–TOF-MS and HPLC-based assays for xylosyltransferase activity ( A – H ) Incubation of selected peptides with the recombinant enzyme preparation was performed overnight in the presence ( A , C , E , G ) or absence ( B , D , F , H ) of UDP-Xyl as enzyme donor. In all cases, the xylosyltransferase transferred Xyl (indicated by Δ m/z =132; one in the case of the Bik, Syn and Syn2 peptides, and two in the case of the Perl peptide); asterisks in ( G and H ) indicate contaminating peptides originating from the culture medium. ( I ) A typical RP-HPLC assay is depicted showing the UDP-Xyl-dependent formation over 2 h by AgOXT1 of a product with a slightly earlier elution time; product (Xyl-Syn) and substrate (Syn) peaks were collected and subject to MALDI–TOF-MS ( J , K ) to show the respective presence of species of m/z 1239 and 1107. The comparative MALDI–TOF-MS/MS data for the two peaks ( L , M ) verify the transfer of Xyl to the second serine residue of the DDDSIEGSGGR sequence of the Syn peptide (fragments of m/z 564 in D and 432 in both D and E); loss of Xyl directly from the parent ion in D is also apparent. The Δ m/z values between peaks correspond to either single amino acids (D, E, G, R), dipeptides (G+G, I+S, S+G) or xylosylserine (S+Xyl).
    Figure Legend Snippet: MALDI–TOF-MS and HPLC-based assays for xylosyltransferase activity ( A – H ) Incubation of selected peptides with the recombinant enzyme preparation was performed overnight in the presence ( A , C , E , G ) or absence ( B , D , F , H ) of UDP-Xyl as enzyme donor. In all cases, the xylosyltransferase transferred Xyl (indicated by Δ m/z =132; one in the case of the Bik, Syn and Syn2 peptides, and two in the case of the Perl peptide); asterisks in ( G and H ) indicate contaminating peptides originating from the culture medium. ( I ) A typical RP-HPLC assay is depicted showing the UDP-Xyl-dependent formation over 2 h by AgOXT1 of a product with a slightly earlier elution time; product (Xyl-Syn) and substrate (Syn) peaks were collected and subject to MALDI–TOF-MS ( J , K ) to show the respective presence of species of m/z 1239 and 1107. The comparative MALDI–TOF-MS/MS data for the two peaks ( L , M ) verify the transfer of Xyl to the second serine residue of the DDDSIEGSGGR sequence of the Syn peptide (fragments of m/z 564 in D and 432 in both D and E); loss of Xyl directly from the parent ion in D is also apparent. The Δ m/z values between peaks correspond to either single amino acids (D, E, G, R), dipeptides (G+G, I+S, S+G) or xylosylserine (S+Xyl).

    Techniques Used: Mass Spectrometry, High Performance Liquid Chromatography, Activity Assay, Incubation, Recombinant, Sequencing

    RNAi-mediated knockdown of HS biosynthesis in salivary glands affects P. falciparum sporozoite invasion ( A ) Experimental time line for RNAi experiments shown in Figure 3 ( C )–3( D ) and the salivary gland invasion inhibition assay in Table 1 . Each time point represents steps in the experimental process and indicates the age of the mosquito at the time of manipulation, the point of delivery of the P. falciparum infected bloodmeal, and the point of salivary gland harvest, which is 4 days post-dsRNA injection (day 12 post-infectious blood feeding). The gold arrow highlights the oocyst development period on the mosquito midgut. The red arrow highlights the period when sporozoites are escaping the ruptured oocyst and begin invasion of the salivary glands. Note that some oocysts will rupture earlier given the asynchronous nature of oocyst development in the mosquito. The green arrow highlights the 72 h window wherein AgOXT1 mRNA and HS biosynthesis is knocked down (kd). ( B ) Immunofluorescence staining of the basal lamina of a fixed non-permeabilized A. gambiae salivary gland. DAPI staining identifies nuclei in the tissue and appears blue (panel a). The arrow highlights the surface staining of the medial (ML) and distal lateral (DL) lobes of salivary glands using the RB4Ea12 scFv antibody, which recognizes 6-O-sulfated GlcNS/NAc-containing disaccharides, as detected by mouse anti-VSV secondary antibody and goat-anti mouse Alexa Fluor® 594-conjugated tertiary antibody and appears red (panel b). Close-up images using ×400 magnification. ( C ) RNAi-mediated knockdown of AgOXT1 transcript in salivary glands measured by semi-quantitative RT–PCR (upper panel) as well as a representative Western blot using RB4Ea12 scFv antibody of HSPGs that remain present on Anopheles gambiae salivary glands at 72 h post-dsRNA delivery. The wild-type salivary gland HS banding profile is represented by the ds GFP control group. Following ds AgOXT1 injection, the HS staining is reduced (lower panel). Antibodies against AgLDH (molecular mass ~35 kDa) were used as a protein loading control. Images were acquired using the Odyssey® near-infrared system. Arrowheads indicate two faint bands that are barely detectable in salivary glands from ds AgOXT1 -treated mosquitoes (molecular mass ~70 kDa). ( D ) Bright-field images and immunofluorescence staining of salivary glands, confirming that repression of HS biosynthesis is lost at 96 h, which correlated with the mRNA rebound observed at 96 h post-ds AgOXT1 inoculation (panels a–c) as compared with ds GFP controls (panels d–f). RB4Ea12 scFv was detected by Alexa Fluor® 488-conjugated secondary antibody, and appears green. The images for bright-field and fluorescence were acquired at ×200 and ×400 magnification respectively, and are representative images of salivary gland matched pairs for mosquitoes from two biological cohorts. Image acquisition and analysis was performed using exactly the same parameters for treatment and control glands. DAPI staining identifies nuclei in the tissue and appears blue. It is clear from the results that the window of opportunity to assess the role of HS in sporozoite invasion of the salivary glands is within the first 72 h following dsRNA delivery (indicated as a green asterisk on the timeline shown in ( A ). Salivary gland harvest at this time point should capture only the first 2–3 days of sporozoite tissue invasion events.
    Figure Legend Snippet: RNAi-mediated knockdown of HS biosynthesis in salivary glands affects P. falciparum sporozoite invasion ( A ) Experimental time line for RNAi experiments shown in Figure 3 ( C )–3( D ) and the salivary gland invasion inhibition assay in Table 1 . Each time point represents steps in the experimental process and indicates the age of the mosquito at the time of manipulation, the point of delivery of the P. falciparum infected bloodmeal, and the point of salivary gland harvest, which is 4 days post-dsRNA injection (day 12 post-infectious blood feeding). The gold arrow highlights the oocyst development period on the mosquito midgut. The red arrow highlights the period when sporozoites are escaping the ruptured oocyst and begin invasion of the salivary glands. Note that some oocysts will rupture earlier given the asynchronous nature of oocyst development in the mosquito. The green arrow highlights the 72 h window wherein AgOXT1 mRNA and HS biosynthesis is knocked down (kd). ( B ) Immunofluorescence staining of the basal lamina of a fixed non-permeabilized A. gambiae salivary gland. DAPI staining identifies nuclei in the tissue and appears blue (panel a). The arrow highlights the surface staining of the medial (ML) and distal lateral (DL) lobes of salivary glands using the RB4Ea12 scFv antibody, which recognizes 6-O-sulfated GlcNS/NAc-containing disaccharides, as detected by mouse anti-VSV secondary antibody and goat-anti mouse Alexa Fluor® 594-conjugated tertiary antibody and appears red (panel b). Close-up images using ×400 magnification. ( C ) RNAi-mediated knockdown of AgOXT1 transcript in salivary glands measured by semi-quantitative RT–PCR (upper panel) as well as a representative Western blot using RB4Ea12 scFv antibody of HSPGs that remain present on Anopheles gambiae salivary glands at 72 h post-dsRNA delivery. The wild-type salivary gland HS banding profile is represented by the ds GFP control group. Following ds AgOXT1 injection, the HS staining is reduced (lower panel). Antibodies against AgLDH (molecular mass ~35 kDa) were used as a protein loading control. Images were acquired using the Odyssey® near-infrared system. Arrowheads indicate two faint bands that are barely detectable in salivary glands from ds AgOXT1 -treated mosquitoes (molecular mass ~70 kDa). ( D ) Bright-field images and immunofluorescence staining of salivary glands, confirming that repression of HS biosynthesis is lost at 96 h, which correlated with the mRNA rebound observed at 96 h post-ds AgOXT1 inoculation (panels a–c) as compared with ds GFP controls (panels d–f). RB4Ea12 scFv was detected by Alexa Fluor® 488-conjugated secondary antibody, and appears green. The images for bright-field and fluorescence were acquired at ×200 and ×400 magnification respectively, and are representative images of salivary gland matched pairs for mosquitoes from two biological cohorts. Image acquisition and analysis was performed using exactly the same parameters for treatment and control glands. DAPI staining identifies nuclei in the tissue and appears blue. It is clear from the results that the window of opportunity to assess the role of HS in sporozoite invasion of the salivary glands is within the first 72 h following dsRNA delivery (indicated as a green asterisk on the timeline shown in ( A ). Salivary gland harvest at this time point should capture only the first 2–3 days of sporozoite tissue invasion events.

    Techniques Used: Inhibition, Infection, Injection, Immunofluorescence, Staining, Quantitative RT-PCR, Western Blot, Fluorescence

    15) Product Images from "The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA"

    Article Title: The G-rich Repeats in FMR1 and C9orf72 Loci Are Hotspots for Local Unpairing of DNA

    Journal: Genetics

    doi: 10.1534/genetics.118.301672

    Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.
    Figure Legend Snippet: Bisulfite footprinting by deep-sequencing across the FMR1 repeats in wild-type (WT) hESCs. (A) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in WT XY hESC (WT-ES-4). This was followed by a bioinformatic analysis which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns. Next, the reads were clustered into heatmaps. For simplicity, the template strand is presented in an opposite orientation (from 3′ to 5′ similar to the nontemplate strand orientation). The total read count appears on the y -axis. The length of the analyzed region is 250 bp with 80 C sites for the nontemplate and 100 C sites for the template strand. Dark gray and blue represent double-strand DNA (dsDNA) at the nontemplate and template strands, respectively, red represents single-strand DNA (ssDNA), and black represents sequencing errors. The TSS site and the repeats are designated with yellow lines. (B) DNA bisulfite footprinting by deep-sequencing was carried out using unconverted primers in a FXS XX hESC lines with an unmethylated full expansion (uFM) with skewed X-inactivation of the WT allele (uFM-ES-2), which allowed the selective amplification of a methylated WT allele. This was followed by a bioinformatic analysis, which separated the reads into nontemplate (G-rich; left panel) and template (C-rich; right panel) strands, according to conversion patterns.

    Techniques Used: Footprinting, Sequencing, Amplification, Methylation

    Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.
    Figure Legend Snippet: Proposed model for the formation of noncanonical structures by the G/C-rich repeats at the FMR1 and C9orf72 loci. Four potential configurations can be formed at the repeats in FMR1 and C9orf72 ). Such G-rich hybrid structures are expected to be particularly stable and difficult for the cell to resolve, thus providing a potent source of repeat instability. Blue, red, and green lines designate the DNA (paired and unpaired), the CGG/GGGGCC repeats on the nontemplate/template strand, and the newly synthesized RNA molecules, respectively. FM, full mutation; WT, wild type.

    Techniques Used: Synthesized, Mutagenesis

    16) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Pedigrees of the LQTS families and sequence electropherograms. (a) Family 1 segregates the c.781_782delinsTC mutation in the KCNQ1 gene. The proband (II-1) is indicated by the black arrow. (b) Family 2 segregates the c.2437-5C > A mutation in the SCN5A gene. The proband (II-4) is indicated by the black arrow. The location of the mutation in the sequence electropherograms is indicated by a red box.
    Figure Legend Snippet: Pedigrees of the LQTS families and sequence electropherograms. (a) Family 1 segregates the c.781_782delinsTC mutation in the KCNQ1 gene. The proband (II-1) is indicated by the black arrow. (b) Family 2 segregates the c.2437-5C > A mutation in the SCN5A gene. The proband (II-4) is indicated by the black arrow. The location of the mutation in the sequence electropherograms is indicated by a red box.

    Techniques Used: Sequencing, Mutagenesis

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    Diagrammatic representation of the KCNQ1 and SCN5A proteins. (a) The KCNQ1 protein is composed of six transmembrane domains (S1-S6) with a pore region between S5 and S6 (shown in orange). The section encompassed by the two red scissors and colored in light blue is encoded by exon 6 of the KCNQ1 gene. (b) The SCN5A protein is composed of four homologous transmembrane domains (DI-DIV) with six transmembrane segments (S1-S6) in each section. The four sections’ S1-S4 domains form the channel’s voltage-sensing region, and the four S5-S6 domains with the intervening loop region form the central pore region and selective filter. The section encompassed by the two red scissors and colored in light green is encoded by exon 16 of the SCN5A gene.
    Figure Legend Snippet: Diagrammatic representation of the KCNQ1 and SCN5A proteins. (a) The KCNQ1 protein is composed of six transmembrane domains (S1-S6) with a pore region between S5 and S6 (shown in orange). The section encompassed by the two red scissors and colored in light blue is encoded by exon 6 of the KCNQ1 gene. (b) The SCN5A protein is composed of four homologous transmembrane domains (DI-DIV) with six transmembrane segments (S1-S6) in each section. The four sections’ S1-S4 domains form the channel’s voltage-sensing region, and the four S5-S6 domains with the intervening loop region form the central pore region and selective filter. The section encompassed by the two red scissors and colored in light green is encoded by exon 16 of the SCN5A gene.

    Techniques Used:

    Summary of splice variants reported in HGMD-Pro for the KCNQ1 , KCNH2 and SCN5A genes. Blue boxes represent splice variants that have been characterized by in silico prediction studies and transcript studies; green boxes represent splice variants that have been characterized by transcript studies; orange boxes represent variants that have been predicted to have an effect on splicing using in silico prediction programmes (most using one programme described by Xiong et al [ 11 ]); red boxes represent splice variants that have not been characterized at all by transcript studies or in silico prediction programmes. Dashed purple boxes represent the last exonic nucleotide.
    Figure Legend Snippet: Summary of splice variants reported in HGMD-Pro for the KCNQ1 , KCNH2 and SCN5A genes. Blue boxes represent splice variants that have been characterized by in silico prediction studies and transcript studies; green boxes represent splice variants that have been characterized by transcript studies; orange boxes represent variants that have been predicted to have an effect on splicing using in silico prediction programmes (most using one programme described by Xiong et al [ 11 ]); red boxes represent splice variants that have not been characterized at all by transcript studies or in silico prediction programmes. Dashed purple boxes represent the last exonic nucleotide.

    Techniques Used: In Silico

    17) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    18) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

    19) Product Images from "Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity"

    Article Title: Splice Site Variants in the KCNQ1 and SCN5A Genes: Transcript Analysis as a Tool in Supporting Pathogenicity

    Journal: Journal of Clinical Medicine Research

    doi: 10.14740/jocmr2894w

    Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.2437-5C > A mutation in the SCN5A gene, together with transcript analysis. (a) Diagrammatic representation of the genomic location of the c.2437-5C > A mutation in the SCN5A gene in relation to the other exons (indicated by the red arrow; top), and the amplicon sizes of the expected PCR products for an unaffected individual (bottom left), and the shortened amplicon sizes for the carriers of the mutation if exon 16 were spliced out (bottom right). The diagram only shows a partial representation of the SCN5A genomic/mRNA sequence instead of all 16 exons. (b) The 2% agarose gel showing the results of PCR amplification of cDNA from the proband’s sister (II-3) and an unrelated control. The sequence electropherogram of the higher molecular weight amplicon (green box) shows the junction between exons 15 and 16 (gray dashed line). The sequence electropherogram of the lower molecular weight amplicon (blue box) shows the in-frame fusion of exons 15 and 17 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Polymerase Chain Reaction, Sequencing, Agarose Gel Electrophoresis, Molecular Weight

    Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).
    Figure Legend Snippet: Predicted outcomes for carriers of the c.781_782delinsTC mutation in the KCNQ1 gene, together with transcript analysis. (a) The wild-type (WT) KCNQ1 gene transcript would be unaffected and produce normal KCNQ1, but the mutation could cause an amino acid change at residue 261 of glutamic acid to alanine (p.E261A) or cause exon 6 to be spliced out. Primers for the amplification of cDNA are shown as blue arrows with the lengths of anticipated amplicons shown above the relevant primer pairs. The diagram only shows a partial representation of the KCNQ1 mRNA sequence instead of all 16 exons. The thick red line shows the location of the c.781_782delinsTC mutation in relation to the rest of the exons. (b) The 2% agarose gel showing the results of the PCR amplification of cDNA from the proband’s mother (I-1) and an unrelated control. The highest molecular weight product is a heteroduplex of the two smaller amplicons (representing non-excised and excised exon 6 transcripts). The sequence electropherogram of the about 470 bp amplicon (green box) shows the sequence of the junction between exons 5 and 6 (gray dashed line) and heterozygosity for the c.781_782delinsTC mutation (red box). The approximate 330 bp amplicon (blue box) shows the in-frame fusion of exon 5 and exon 7 (gray dashed line).

    Techniques Used: Mutagenesis, Amplification, Sequencing, Agarose Gel Electrophoresis, Polymerase Chain Reaction, Molecular Weight

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    Article Snippet: .. All PCR assays were carried out in 25-µl volumes containing 200 ng of template DNA, 0.1 units of Taq DNA polymerase (Roche Diagnostics), 25 pmol of each primer, and 10 nmol of dNTPs. ..

    Article Title: Expression and Antigenic Evaluation of VacA Antigenic Fragment of Helicobacter Pylori
    Article Snippet: .. PCR amplification was performed in a 50 μl total volume containing 3 μl of template DNA, 2 μl of each primers (10 picomole), 8 μl MgCl₂ (25 mM), 1.5 μl dNTP mixture, 5 μl PCR buffer (10X) and 1 μl of expand DNA polymerase (Roche, Germany). .. The following conditions were used for amplification: initial denaturation at 94°C for 5 min followed by 30 cycles of denaturation at 94°C for 1 min, annealing at 53°C for 1 min and extension at 72°C for 1 min and further extension for 5 min at 72°C ( ).

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

    Article Title: Preferential MGMT methylation could predispose a subset of KIT/PDGFRA-WT GISTs, including SDH-deficient ones, to respond to alkylating agents
    Article Snippet: .. PCR was carried out in a total volume of 25 μl consisting in 20 ng of DNA, 10 × PCR buffer, MgCl2, dNTP, primers (10 pM each), and 1 U FastStart DNA Taq polymerase (Roche). .. PCR conditions were an initial denaturation of 95 °C for 5 min, followed by 40 cycles of 95 °C for 30 s, 52–64 °C for 30 s, and 72 °C for 30 s. PCR products were purified with the Qiaquick PCR purification kit (Qiagen) and sequenced on both strands using the Big Dye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems).

    Article Title: Full-length 16S rRNA gene amplicon analysis of human gut microbiota using MinION™ nanopore sequencing confers species-level resolution
    Article Snippet: .. PCR amplification of 16S rRNA genes was conducted using the KAPA2G™ Robust HotStart ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA) in a total volume of 25 µl containing inner primer pairs (50 nM each) and the barcoded outer primer mixture (3%) from the PCR Barcoding Kit (SQK-PBK004; Oxford Nanopore Technologies, Oxford, UK). .. Amplification was performed with the following PCR conditions: initial denaturation at 95 °C for 3 min, 5 cycles of 95 °C for 15 sec, 55 °C for 15 sec, and 72 °C for 30 sec, 30 cycles of 95 °C for 15 sec, 62 °C for 15 sec, and 72 °C for 30 sec, followed by a final extension at 72 °C for 1 min. Amplified DNA was purified using AMPure® XP (Beckman Coulter) and quantified by a NanoDrop® 1000 (Thermo Fischer Scientific, Waltham, MA, USA).

    Modification:

    Article Title: Detection of methylation in promoter sequences by melting curve analysis-based semiquantitative real time PCR
    Article Snippet: .. MSP The methylation specific PCRs were carried out with 60 ng of bisulfite modified DNA in a total volume of 25 μl, which contained 2.5 μl 10× reaction buffer, 2–2.5 mM MgCl2 , 0.2 mM of each dNTP, 10 pmol forward and reverse primers, 5% DMSO and one unit of AmpliTaq Gold™ polymerase (Roche), in a T3 thermocycler of Biometra® . .. Oligonucleotides for RASSF1A and MGMT (Table ) were designed using the MethPrimer software [ ].

    Sonication:

    Article Title: Identification and characterisation of a regulatory region in the Toxoplasma gondii hsp70genomic locus✩
    Article Snippet: .. Parasites were sonicated in buffer B [20 mM Hepes (pH 8.0), 0.1 KCL, 0.5 mM DTT, 0.2 mM EDTA, 20% glycerol and one protease inhibitor cocktail tablet (Roche, Germany) per 10 ml of solution]. ..

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Enrichment Followed by Quantitative PCR both for Rapid Detection and as a Tool for Quantitative Risk Assessment of Food-Borne Thermotolerant Campylobacters
    Article Snippet: .. The 25-μl real-time PCR mixture contained 1× PCR buffer for Tth DNA polymerase (Roche A/S, Hvidovre, Denmark), 1 U of Tth DNA polymerase (Roche A/S), 0.4 mM deoxynucleoside triphosphate mixture (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom), 0.44 μM forward primer 5′ CTG CTT AAC ACA AGT TGA GTA GG 3′, 0.48 μM reverse primer 5′ TTC CTT AGG TAC CGT CAG AA 3′ (DNA Technology, Århus, Denmark; C. jejuni 16S rRNA; GenBank accession no. ), 2.5 mM MgCl2 (Applied Biosystems), 30 μg of bovine serum albumin (BSA) for chicken samples and 5 μg of BSA for pure DNA (Roche A/S), 20 nM target Campylobacter probe labeled with 6-carboxyfluorescein (FAM; reporter dye) and 6-carboxytetramethylrhodamine (TAMRA; quencher dye) (5′ FAM-TGT CAT CCT CCA CGC GGC GTT GCT GC-TAMRA 3′; DNA Technology), 50 nM IAC probe (5′ VIC-TTC ATG AGG ACA CCT GAG TTG A-TAMRA 3′; Applied Biosystems), 5 × 103 copies of IAC (124 bp), and 5 μl of DNA sample. .. The cycle profile was as follows: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 15 s and 58°C for 60 s. Fluorescence measurements were obtained online and analyzed on the ABI-PRISM with the SDS software (version 1.7a; Applied Biosystems) and on the RotorGene with the version 4.6 software (Corbett Research).

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