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    BsaAI
    Description:
    BsaAI 2 500 units
    Catalog Number:
    R0531L
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    Category:
    Restriction Enzymes
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    2 500 units
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    New England Biolabs bsaai
    BsaAI
    BsaAI 2 500 units
    https://www.bioz.com/result/bsaai/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaai - by Bioz Stars, 2021-05
    92/100 stars

    Images

    1) Product Images from "Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping"

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkv506

    TALEN-mediated destruction of the −82 kb PAG1 HRE. ( A ) PCR analysis of representative HeLa and MCF-7 clones following co-transfection of two independent TALEN pairs (TPI and TPII) targeting the HBS within the −82 kb PAG1 HRE. A 317 bp fragment encompassing the putative HBS was amplified by PCR and restriction digested with (lower panel) or without BsaAI (upper panel). ( B ) DNA sequence analysis of the cell clones shown in (A). Dashes indicate deleted bases. Wild-type (wt) and targeted HeLa ( C ) and MCF-7 ( D ) clones were exposed to 20% O 2 or 0.2% O 2 for 24 h, followed by quantification of PAG1 and CAIX mRNA levels. Results of three independent experiments are shown relative to the β-actin control mRNA levels. Statistical analyses were performed with unpaired Student's t-tests (* P
    Figure Legend Snippet: TALEN-mediated destruction of the −82 kb PAG1 HRE. ( A ) PCR analysis of representative HeLa and MCF-7 clones following co-transfection of two independent TALEN pairs (TPI and TPII) targeting the HBS within the −82 kb PAG1 HRE. A 317 bp fragment encompassing the putative HBS was amplified by PCR and restriction digested with (lower panel) or without BsaAI (upper panel). ( B ) DNA sequence analysis of the cell clones shown in (A). Dashes indicate deleted bases. Wild-type (wt) and targeted HeLa ( C ) and MCF-7 ( D ) clones were exposed to 20% O 2 or 0.2% O 2 for 24 h, followed by quantification of PAG1 and CAIX mRNA levels. Results of three independent experiments are shown relative to the β-actin control mRNA levels. Statistical analyses were performed with unpaired Student's t-tests (* P

    Techniques Used: Polymerase Chain Reaction, Clone Assay, Cotransfection, Amplification, Sequencing

    2) Product Images from "Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells"

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-24

    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.
    Figure Legend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Techniques Used: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Autoradiography

    3) Product Images from "Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences"

    Article Title: Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0118813

    Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5. (A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).
    Figure Legend Snippet: Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5. (A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).

    Techniques Used: Plasmid Preparation, Mutagenesis, Sequencing, Nucleic Acid Electrophoresis, Electrophoresis, Produced

    Related Articles

    Methylation:

    Article Title: (CME)
    Article Snippet: .. D4Z4 methylation levels were determined in the proximal D4Z4 repeat units of chromosomes 4q and 10q using the (methylation-sensitive) restriction enzymes Bln I (Takara), Bgl II, Cpo I, Eco 91I, Eco RI, Xap I (MBI Fermentas), Bsa AI, and Fse I (New England BioLabs). .. Briefly, 4 μg of genomic DNA was digested overnight with a combination of (methylation-sensitive) restriction enzymes followed by linear gel electrophoresis, Southern blotting, and hybridization with the 32 P-labeled probe p13E-11.

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Polymerase Chain Reaction:

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping
    Article Snippet: Following expansion of the cloned cells, genomic DNA was isolated and the −82 kb PAG1 region amplified by PCR as described above. .. PCR products were either digested with BsaAI (New England Biolabs, Ipswich, MA, USA) and analyzed by 1% NuSieve agarose gel electrophoresis (Lonza, Basel, Switzerland) or cloned into a plasmid vector and sequenced (Microsynth). .. Chromatin immunoprecipitation and chromosome conformation capture (3C) Chromatin immunoprecipitation (ChIP) experiments using HIF-1α, HIF-2α, HIFβ, histone modifications or p300 occupancy were performed as described previously ( , ).

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

    Agarose Gel Electrophoresis:

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping
    Article Snippet: Following expansion of the cloned cells, genomic DNA was isolated and the −82 kb PAG1 region amplified by PCR as described above. .. PCR products were either digested with BsaAI (New England Biolabs, Ipswich, MA, USA) and analyzed by 1% NuSieve agarose gel electrophoresis (Lonza, Basel, Switzerland) or cloned into a plasmid vector and sequenced (Microsynth). .. Chromatin immunoprecipitation and chromosome conformation capture (3C) Chromatin immunoprecipitation (ChIP) experiments using HIF-1α, HIF-2α, HIFβ, histone modifications or p300 occupancy were performed as described previously ( , ).

    Clone Assay:

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping
    Article Snippet: Following expansion of the cloned cells, genomic DNA was isolated and the −82 kb PAG1 region amplified by PCR as described above. .. PCR products were either digested with BsaAI (New England Biolabs, Ipswich, MA, USA) and analyzed by 1% NuSieve agarose gel electrophoresis (Lonza, Basel, Switzerland) or cloned into a plasmid vector and sequenced (Microsynth). .. Chromatin immunoprecipitation and chromosome conformation capture (3C) Chromatin immunoprecipitation (ChIP) experiments using HIF-1α, HIF-2α, HIFβ, histone modifications or p300 occupancy were performed as described previously ( , ).

    Plasmid Preparation:

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping
    Article Snippet: Following expansion of the cloned cells, genomic DNA was isolated and the −82 kb PAG1 region amplified by PCR as described above. .. PCR products were either digested with BsaAI (New England Biolabs, Ipswich, MA, USA) and analyzed by 1% NuSieve agarose gel electrophoresis (Lonza, Basel, Switzerland) or cloned into a plasmid vector and sequenced (Microsynth). .. Chromatin immunoprecipitation and chromosome conformation capture (3C) Chromatin immunoprecipitation (ChIP) experiments using HIF-1α, HIF-2α, HIFβ, histone modifications or p300 occupancy were performed as described previously ( , ).

    Article Title: Neisseria meningitidis RTX Protein FrpC Induces High Levels of Serum Antibodies during Invasive Disease: Polymorphism of frpC Alleles and Purification of Recombinant FrpC
    Article Snippet: Plasmid pTZ19R Mlu I is a construct prepared by ligation of Pst I-digested pTZ19R (Pharmacia) with a double-stranded adapter, 5′-TACGCGTATGCA, introducing a new Mlu I site. .. Plasmid pT7-7Δ Bsa AI was constructed by digestion of pT7-7 ( ) with Bsa AI and religation of the vector with a double-stranded synthetic hexanucleotide 5′-GGATCC. pTYB2 (the intein-mediated purification with an affinity chitin-binding tag protocol [IMPACT] T7 system) was from New England Biolabs. ..

    Injection:

    Article Title: Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences
    Article Snippet: Products were cloned and sequenced to confirm DUX4 mRNA amplification. .. For the restriction digest assay, 3’ RACE products (n = 4 injected legs per assay) were digested with BsaAI (New England Biolabs) overnight at 37°C. .. Samples were electrophoresed on a 1.5% agarose gel for 3 hrs and 100V and stained with ethidium bromide for UV visualization.

    Construct:

    Article Title: Neisseria meningitidis RTX Protein FrpC Induces High Levels of Serum Antibodies during Invasive Disease: Polymorphism of frpC Alleles and Purification of Recombinant FrpC
    Article Snippet: Plasmid pTZ19R Mlu I is a construct prepared by ligation of Pst I-digested pTZ19R (Pharmacia) with a double-stranded adapter, 5′-TACGCGTATGCA, introducing a new Mlu I site. .. Plasmid pT7-7Δ Bsa AI was constructed by digestion of pT7-7 ( ) with Bsa AI and religation of the vector with a double-stranded synthetic hexanucleotide 5′-GGATCC. pTYB2 (the intein-mediated purification with an affinity chitin-binding tag protocol [IMPACT] T7 system) was from New England Biolabs. ..

    Purification:

    Article Title: Neisseria meningitidis RTX Protein FrpC Induces High Levels of Serum Antibodies during Invasive Disease: Polymorphism of frpC Alleles and Purification of Recombinant FrpC
    Article Snippet: Plasmid pTZ19R Mlu I is a construct prepared by ligation of Pst I-digested pTZ19R (Pharmacia) with a double-stranded adapter, 5′-TACGCGTATGCA, introducing a new Mlu I site. .. Plasmid pT7-7Δ Bsa AI was constructed by digestion of pT7-7 ( ) with Bsa AI and religation of the vector with a double-stranded synthetic hexanucleotide 5′-GGATCC. pTYB2 (the intein-mediated purification with an affinity chitin-binding tag protocol [IMPACT] T7 system) was from New England Biolabs. ..

    Article Title: Evidence for high bi-allelic expression of activating Ly49 receptors
    Article Snippet: Only fragments that were originally methylated in the gDNA and therefore not converted by sodium bisulfite treatment are cut. .. Gel purified Ly49h fragments were digested with Taqα I (NEB) and BsaAI (NEB) restriction enzymes. .. RNA extraction, RT–PCR and 5′ amplification of cDNA ends cDNA was generated from total B6 and NOD/ShiLtJ spleens and FACS sorted B6 T-cell RNA per SuperScript III protocol.

    Incubation:

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells
    Article Snippet: .. Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia). .. Genomic DNA isolated from human blood serum served as an unmethylated COBRA control.

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    New England Biolabs bsaai
    TALEN-mediated destruction of the −82 kb PAG1 HRE. ( A ) <t>PCR</t> analysis of representative HeLa and MCF-7 clones following co-transfection of two independent TALEN pairs (TPI and TPII) targeting the HBS within the −82 kb PAG1 HRE. A 317 bp fragment encompassing the putative HBS was amplified by PCR and restriction digested with (lower panel) or without <t>BsaAI</t> (upper panel). ( B ) DNA sequence analysis of the cell clones shown in (A). Dashes indicate deleted bases. Wild-type (wt) and targeted HeLa ( C ) and MCF-7 ( D ) clones were exposed to 20% O 2 or 0.2% O 2 for 24 h, followed by quantification of PAG1 and CAIX mRNA levels. Results of three independent experiments are shown relative to the β-actin control mRNA levels. Statistical analyses were performed with unpaired Student's t-tests (* P
    Bsaai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/bsaai/product/New England Biolabs
    Average 92 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    bsaai - by Bioz Stars, 2021-05
    92/100 stars
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    TALEN-mediated destruction of the −82 kb PAG1 HRE. ( A ) PCR analysis of representative HeLa and MCF-7 clones following co-transfection of two independent TALEN pairs (TPI and TPII) targeting the HBS within the −82 kb PAG1 HRE. A 317 bp fragment encompassing the putative HBS was amplified by PCR and restriction digested with (lower panel) or without BsaAI (upper panel). ( B ) DNA sequence analysis of the cell clones shown in (A). Dashes indicate deleted bases. Wild-type (wt) and targeted HeLa ( C ) and MCF-7 ( D ) clones were exposed to 20% O 2 or 0.2% O 2 for 24 h, followed by quantification of PAG1 and CAIX mRNA levels. Results of three independent experiments are shown relative to the β-actin control mRNA levels. Statistical analyses were performed with unpaired Student's t-tests (* P

    Journal: Nucleic Acids Research

    Article Title: Destruction of a distal hypoxia response element abolishes trans-activation of the PAG1 gene mediated by HIF-independent chromatin looping

    doi: 10.1093/nar/gkv506

    Figure Lengend Snippet: TALEN-mediated destruction of the −82 kb PAG1 HRE. ( A ) PCR analysis of representative HeLa and MCF-7 clones following co-transfection of two independent TALEN pairs (TPI and TPII) targeting the HBS within the −82 kb PAG1 HRE. A 317 bp fragment encompassing the putative HBS was amplified by PCR and restriction digested with (lower panel) or without BsaAI (upper panel). ( B ) DNA sequence analysis of the cell clones shown in (A). Dashes indicate deleted bases. Wild-type (wt) and targeted HeLa ( C ) and MCF-7 ( D ) clones were exposed to 20% O 2 or 0.2% O 2 for 24 h, followed by quantification of PAG1 and CAIX mRNA levels. Results of three independent experiments are shown relative to the β-actin control mRNA levels. Statistical analyses were performed with unpaired Student's t-tests (* P

    Article Snippet: PCR products were either digested with BsaAI (New England Biolabs, Ipswich, MA, USA) and analyzed by 1% NuSieve agarose gel electrophoresis (Lonza, Basel, Switzerland) or cloned into a plasmid vector and sequenced (Microsynth).

    Techniques: Polymerase Chain Reaction, Clone Assay, Cotransfection, Amplification, Sequencing

    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Journal: BMC Molecular Biology

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

    doi: 10.1186/1471-2199-15-24

    Figure Lengend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Article Snippet: Restriction fragment length analysis was performed on PCR products incubated with the restriction endonucleases AciI (methylated 50 bp + 167 bp, unmethylated 217 bp), PvuI-HF (methylated 37 bp + 180 bp, unmethylated 217 bp) and BsaAI (methylated 192 bp + 25 bp, unmethylated 217 bp) (New England BioLabs Inc., Genesearch Pty Ltd., Arundal, Queensland, Australia).

    Techniques: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Autoradiography

    Fig. 2. The cyclin B1 promoter is accessible to restriction endonucleases in vivo . Nuclei isolated from asynchronous (Async.) (20 µg of DNA) and permeabilized MSO cells were partially digested with 100 U of Bsa AI ( A ), 100 U of Sau 96I ( B ), 200 U of Xba I ( C ) or 100 U of Sal I ( D ). Purified DNA was fully digested with Nco I and Xba I when Bsa AI was used for the partial digestion, and with Nco I when Xba I was used for the partial digestion. The probe used for the hybridization is shown at the bottom of each panel. Arrows mark the digested and undigested bands. The schematic diagram at the bottom of each panel indicates the position of the digested bands in the cyclin B1 promoter. The percent digestion is shown below each lane.

    Journal: EMBO Reports

    Article Title: The cyclin B1 gene is actively transcribed during mitosis in HeLa cells

    doi: 10.1093/embo-reports/kve223

    Figure Lengend Snippet: Fig. 2. The cyclin B1 promoter is accessible to restriction endonucleases in vivo . Nuclei isolated from asynchronous (Async.) (20 µg of DNA) and permeabilized MSO cells were partially digested with 100 U of Bsa AI ( A ), 100 U of Sau 96I ( B ), 200 U of Xba I ( C ) or 100 U of Sal I ( D ). Purified DNA was fully digested with Nco I and Xba I when Bsa AI was used for the partial digestion, and with Nco I when Xba I was used for the partial digestion. The probe used for the hybridization is shown at the bottom of each panel. Arrows mark the digested and undigested bands. The schematic diagram at the bottom of each panel indicates the position of the digested bands in the cyclin B1 promoter. The percent digestion is shown below each lane.

    Article Snippet: The following enzymes were used: Bsa AI, Sau 96I and Xba I from New England Biolabs, and Sal I from Boehringer Mannheim.

    Techniques: In Vivo, Isolation, Purification, Hybridization

    Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5. (A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).

    Journal: PLoS ONE

    Article Title: Aberrant Splicing in Transgenes Containing Introns, Exons, and V5 Epitopes: Lessons from Developing an FSHD Mouse Model Expressing a D4Z4 Repeat with Flanking Genomic Sequences

    doi: 10.1371/journal.pone.0118813

    Figure Lengend Snippet: Repaired AAV.D4Z4.V5.pLAM-2.0 vector produces full-length DUX4.V5. (A) Mutation of the DUX4-V5 splice donor from GGT to GGG (lowercase red letters) maintained the V5 glycine residue but destroyed the invariant T required for splicing. Note the yellow boxed area containing linker sequences joining the V5 epitope to the natural DUX4 exon 1 untranslated region. The red uppercase G (indicated by arrow) at the beginning of the linker indicates a second aberrant splice site utilized instead of the natural DUX4 exon 1 splice donor, only in the repaired sequence. BsaAI restriction sites are indicated. (B) Schematic of 3’ RACE/BsaAI restriction digestion assay to identify mis-spliced DUX4-V5 products. The BsaAI site is located in the V5 tag. Removal of this sequence by mis-splicing creates a BsaAI-resistant 3’ RACE product of 383 bp, evident by gel electrophoresis (C, original V5). The repaired vector incorporated the V5 tag and its resident BsaAI, making this product susceptible to digestion by the enzyme. The expected full-length and BsaAI-digested products were empirically smaller following electrophoresis (C, mutated V5). (D) Sequence chromatogram of the mutated DUX4.V5 transcript revealed that full-length DUX4.V5 was produced, but a second splice donor sequence was encountered and preferentially utilized instead of the natural DUX4 exon 1 donor. Arrow points to the G residue indicated in the linker sequence of (A).

    Article Snippet: For the restriction digest assay, 3’ RACE products (n = 4 injected legs per assay) were digested with BsaAI (New England Biolabs) overnight at 37°C.

    Techniques: Plasmid Preparation, Mutagenesis, Sequencing, Nucleic Acid Electrophoresis, Electrophoresis, Produced