bisulfite-treated dna Search Results


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  • 99
    Millipore bisulfite treated dna
    The Recruitment of CHD4 to Oxidative <t>DNA</t> Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time <t>RT-PCR.</t> Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .
    Bisulfite Treated Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 261 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    TaKaRa bisulfite treated dna
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Dna, supplied by TaKaRa, used in various techniques. Bioz Stars score: 94/100, based on 366 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    ATUM bisulfite treated dna
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 92/100, based on 79 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    ATUM bisulfite treated template dna
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Template Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 91/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen bisulfite treated dna
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Dna, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 1037 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore bisulfite treated cpgenome universalunmethylated dna
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Cpgenome Universalunmethylated Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    BIOTAGE bisulfite treated dna pyrosequencing
    TCF- and LEF-binding motifs contained in the Klotho promoter . The <t>DNA</t> sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The <t>PCR</t> products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].
    Bisulfite Treated Dna Pyrosequencing, supplied by BIOTAGE, used in various techniques. Bioz Stars score: 85/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Illumina Inc bisulfite treated dna
    Effect of FoxA-deletion on <t>HBV</t> <t>DNA</t> methylation distribution in adult mouse liver. The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.
    Bisulfite Treated Dna, supplied by Illumina Inc, used in various techniques. Bioz Stars score: 94/100, based on 371 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    ATUM sequencing amplified bisulfite treated dna
    Effect of FoxA-deletion on <t>HBV</t> <t>DNA</t> methylation distribution in adult mouse liver. The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.
    Sequencing Amplified Bisulfite Treated Dna, supplied by ATUM, used in various techniques. Bioz Stars score: 88/100, based on 22 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Roche bisulfite treated dna
    <t>DNA</t> methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time <t>PCR</t> amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.
    Bisulfite Treated Dna, supplied by Roche, used in various techniques. Bioz Stars score: 93/100, based on 162 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Thermo Fisher bisulfite treated dna
    Genomic methylation assay for <t>IAP</t> LTR and α-actin sequences. (A) Southern blots of total <t>DNA</t> extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.
    Bisulfite Treated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 93/100, based on 1052 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    EpigenDx bisulfite treated dna
    Genomic methylation assay for <t>IAP</t> LTR and α-actin sequences. (A) Southern blots of total <t>DNA</t> extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.
    Bisulfite Treated Dna, supplied by EpigenDx, used in various techniques. Bioz Stars score: 92/100, based on 24 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Jena Bioscience bisulfite treated dna
    Genomic methylation assay for <t>IAP</t> LTR and α-actin sequences. (A) Southern blots of total <t>DNA</t> extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.
    Bisulfite Treated Dna, supplied by Jena Bioscience, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Epigenomics bisulfite treated dna
    Amplification of 24 individual human genomic loci. Comparison of <t>PCR</t> performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic <t>DNA.</t> Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).
    Bisulfite Treated Dna, supplied by Epigenomics, used in various techniques. Bioz Stars score: 92/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Millipore bisulfite treated cpgenome universal methylated dna
    Amplification of 24 individual human genomic loci. Comparison of <t>PCR</t> performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic <t>DNA.</t> Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).
    Bisulfite Treated Cpgenome Universal Methylated Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 91 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore bisulfite treated cpgenometm universal methylated dna
    Amplification of 24 individual human genomic loci. Comparison of <t>PCR</t> performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic <t>DNA.</t> Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).
    Bisulfite Treated Cpgenometm Universal Methylated Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 85/100, based on 6 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    EpigenDx bisulfite treated buccal dna
    Amplification of 24 individual human genomic loci. Comparison of <t>PCR</t> performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic <t>DNA.</t> Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).
    Bisulfite Treated Buccal Dna, supplied by EpigenDx, used in various techniques. Bioz Stars score: 85/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    97
    Millipore sodium bisulfite treated dna
    Amplification of 24 individual human genomic loci. Comparison of <t>PCR</t> performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic <t>DNA.</t> Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).
    Sodium Bisulfite Treated Dna, supplied by Millipore, used in various techniques. Bioz Stars score: 97/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    TIB MOLBIOL unmethylated bisulfite treated dna
    HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated <t>DNA</t> indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and <t>LCRed640-labeled</t> detection probes specific for methylated bisulfite-treated DNA.
    Unmethylated Bisulfite Treated Dna, supplied by TIB MOLBIOL, used in various techniques. Bioz Stars score: 85/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Qiagen bisulfite treated control dna samples
    HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated <t>DNA</t> indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and <t>LCRed640-labeled</t> detection probes specific for methylated bisulfite-treated DNA.
    Bisulfite Treated Control Dna Samples, supplied by Qiagen, used in various techniques. Bioz Stars score: 99/100, based on 10 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    91
    Thermo Fisher sodium bisulfite treated dna
    HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated <t>DNA</t> indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and <t>LCRed640-labeled</t> detection probes specific for methylated bisulfite-treated DNA.
    Sodium Bisulfite Treated Dna, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    85
    Millipore plates bisulfite treated cpgenome universal methylated dna
    HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated <t>DNA</t> indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and <t>LCRed640-labeled</t> detection probes specific for methylated bisulfite-treated DNA.
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    The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .

    Journal: Cancer cell

    Article Title: CHD4 Has Oncogenic Functions in Initiating and Maintaining Epigenetic Suppression of Multiple Tumor Suppressor Genes

    doi: 10.1016/j.ccell.2017.04.005

    Figure Lengend Snippet: The Recruitment of CHD4 to Oxidative DNA Damage Sites Depends on OGG1 (A) CoIPs of lysates from SW480 cells untreated or treated with 2 mM H 2 O 2 for 30 min were performed with the indicated antibodies. (B) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer. The immunoprecipitated samples were detected by western blot analyses using the antibodies indicated. (C) After SW480 OGG1 KO cells were transfected with pCMV-Taq or pCMV-OGG1 for 48 hr, the cells were untreated or treated with 2mMH 2 O 2 for 30 min. Whole-cell extracts and the tight chromatin fractions were analyzed by immunoblotting with the indicated antibodies. (D) Whole-cell extracts and the tight chromatin fractions from SW480 CHD4 KD cells untreated (Un) or treated with 2 mM H 2 O 2 for 30 min were analyzed by immunoblotting as in (C). (E) Purified OGG1 and Flag-CHD4 were incubated with antibodies against Flag or OGG1 in IP buffer with or without 8-OHdG oligonucleotide. The immuno-precipitated samples were detected by western blot analyses using the antibodies indicated. (F) Biotin labeled 8-OHdG oligonucleotide incubated with OGG1 and Flag-CHD4 was pulled down using streptavidin beads. Bound proteins were eluted and analyzed by immunoblotting with the indicated antibodies. (G) SW480 OGG1 KO cells were untreated or treated with 2mMH 2 O 2 for 30 min followed by ChIP for control IgG, 8-OHdG, and CHD4 at the promoter CpG islands of eight representative genes and analyzed by real-time RT-PCR. Data are represented as mean ± SEM for triplicate experiments. (H) Cells were untreated or treated with 2 mM H 2 O 2 for 30 min. Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG at the promoter CpG islands of eight TSGs. Data are represented as mean ± SEM for triplicate experiments. (I) Cells were untreated or treated with 2mMH 2 O 2 for 30 min, and nascent RNA was labeled concurrently. Real-time RT-PCR data are presented as mean ± SEM of the treated over untreated values for triplicate experiments. (J) Sequential ChIP analyses were performed to test the co-occupancy of CHD4 and 8-OHdG or epigenetic silencing proteins at the promoter CpG islands of eight representative TSGs in fresh frozen human CRC tissues (n = 20) and normal colon epithelial tissues (n = 6). Data are represented as mean ± SEM. .

    Article Snippet: Each methylation-specific PCR reaction incorporated 100 ng of bisulfite-treated DNA as template, 10 pmol/L of each primer, 100 pmol/L deoxynucleoside triphosphate, 10 PCR buffer, and 1 unit of JumpStart Red Taq Polymerase (Sigma-Aldrich, St. Louis, MO) in a final reaction volume of 25 μl.

    Techniques: Purification, Incubation, Immunoprecipitation, Western Blot, Transfection, Labeling, Chromatin Immunoprecipitation, Quantitative RT-PCR

    TCF- and LEF-binding motifs contained in the Klotho promoter . The DNA sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The PCR products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].

    Journal: Arthritis Research & Therapy

    Article Title: The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells

    doi: 10.1186/ar3830

    Figure Lengend Snippet: TCF- and LEF-binding motifs contained in the Klotho promoter . The DNA sequence of the promoter region of the Klotho gene. TCF/LEF (CTTTT, CTTTG, or CAAAG) consensus sequences are marked in bold type and underlined. The arrows indicate the starting location of the primers used to generate the promoter constructs. The transcription start site is marked as +1; CGC marks the translation start site. The PCR products were digested with Kpn I and Xho I, and ligated into the Kpn I and Xho I sites of the pGL3-basic vector [ 29 ].

    Article Snippet: The bisulfite-treated DNA was amplified by PCR with Takara Taq Hot Start Version (Takara Bio) and T-Vector pMD20 (Takara Bio) for cytosine-phosphate-guanosine (CpG)-rich regions around the rat Klotho gene.

    Techniques: Binding Assay, Sequencing, Construct, Polymerase Chain Reaction, Plasmid Preparation

    Effect of FoxA-deletion on HBV DNA methylation distribution in adult mouse liver. The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: Effect of FoxA-deletion on HBV DNA methylation distribution in adult mouse liver. The CpG DNA methylation frequency distribution across the 11, 38 and 14 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2131–2441, respectively, from male (A, C and E) and female (B, D and F) FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-); M A1A2A3Cre(-) and F A1A2A3Cre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+); M A1A2A3Cre(+) and F A1A2A3Cre(+)) HBV transgenic mice is shown.

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: DNA Methylation Assay, Expressing, Transgenic Assay, Mouse Assay

    RNA (Northern) filter hybridization and RT-qPCR analysis of HBV transcripts in the livers of 1, 2, 3, 4 and 7 week old HBV transgenic mice. (A) RNA (Northern) filter hybridization analysis of representative mice of each genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probes used were HBV ayw genomic DNA plus GAPDH cDNA. FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A1A2A3). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5kb RNA. (B) Quantitative analysis by RNA (Northern) filter hybridization of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: RNA (Northern) filter hybridization and RT-qPCR analysis of HBV transcripts in the livers of 1, 2, 3, 4 and 7 week old HBV transgenic mice. (A) RNA (Northern) filter hybridization analysis of representative mice of each genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probes used were HBV ayw genomic DNA plus GAPDH cDNA. FoxA-expressing (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A1A2A3). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5kb RNA. (B) Quantitative analysis by RNA (Northern) filter hybridization of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. The levels of the transcripts which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: Northern Blot, Hybridization, Quantitative RT-PCR, Transgenic Assay, Mouse Assay, Expressing, Quantitation Assay

    RNA (Northern) filter hybridization and RT-qPCR analysis of HBV transcripts in the livers of adult HBV transgenic mice. (A) RNA (Northern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probes used were HBV ayw genomic DNA plus GAPDH cDNA. FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5kb RNA. (B) Quantitative analysis by RNA (Northern) filter hybridization of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. Average number of mice per group was 6.6±1.9 (Range: 4–9). The levels of the HBV 3.5kb transcript which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: RNA (Northern) filter hybridization and RT-qPCR analysis of HBV transcripts in the livers of adult HBV transgenic mice. (A) RNA (Northern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probes used were HBV ayw genomic DNA plus GAPDH cDNA. FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The glyceraldehyde 3-phosphate dehydrogenase (GAPDH) transcript was used as an internal control for the quantitation of the HBV 3.5kb RNA. (B) Quantitative analysis by RNA (Northern) filter hybridization of the HBV 3.5kb transcript in the HBV transgenic mice. The mean HBV 3.5kb transcript levels plus standard deviations are indicated. Average number of mice per group was 6.6±1.9 (Range: 4–9). The levels of the HBV 3.5kb transcript which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: Northern Blot, Hybridization, Quantitative RT-PCR, Transgenic Assay, Mouse Assay, Expressing, Quantitation Assay

    Tissue-specific and developmental regulation of HBV DNA methylation. (A) The percentage of CpG DNA methylation at each position within the HBV genome from a male (A) and female (B) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The percentage of CpG DNA methylation at each position within the HBV genome from three individual 0.5 day old neonatal wild-type HBV transgenic mouse livers is shown.

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: Tissue-specific and developmental regulation of HBV DNA methylation. (A) The percentage of CpG DNA methylation at each position within the HBV genome from a male (A) and female (B) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The percentage of CpG DNA methylation at each position within the HBV genome from three individual 0.5 day old neonatal wild-type HBV transgenic mouse livers is shown.

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: DNA Methylation Assay, Transgenic Assay, Binding Assay

    DNA (Southern) filter hybridization analysis of HBV DNA replication intermediates in the livers of adult HBV transgenic mice. (A) DNA (Southern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probe used was HBVayw genomic DNA. FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The HBV transgene (Tg) was used as an internal control for the quantitation of the HBV replication intermediates. Tg = HBV transgene; RC = HBV relaxed circular replication intermediates; SS = HBV single stranded replication intermediates. (B) Quantitative analysis of the HBV DNA replication intermediate (RI) levels in HBV transgenic mice. The mean DNA replication intermediate levels plus standard deviations are indicated. Average number of mice per group was 6.7±1.8 (Range: 4–9). The levels of replication intermediates which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: DNA (Southern) filter hybridization analysis of HBV DNA replication intermediates in the livers of adult HBV transgenic mice. (A) DNA (Southern) filter hybridization analysis of representative mice of each sex and genotype are shown. Noncontiguous lanes from multiple analysis are presented. The probe used was HBVayw genomic DNA. FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice are indicated (Genotype A2, A1A2 and A1A2A3, respectively). The HBV transgene (Tg) was used as an internal control for the quantitation of the HBV replication intermediates. Tg = HBV transgene; RC = HBV relaxed circular replication intermediates; SS = HBV single stranded replication intermediates. (B) Quantitative analysis of the HBV DNA replication intermediate (RI) levels in HBV transgenic mice. The mean DNA replication intermediate levels plus standard deviations are indicated. Average number of mice per group was 6.7±1.8 (Range: 4–9). The levels of replication intermediates which are statistically significantly different between Cre(-) and Cre(+) HBV transgenic mice by a Student’s t-test (p

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: Hybridization, Transgenic Assay, Mouse Assay, Expressing, Quantitation Assay

    Tissue-specific and developmental regulation of HBV DNA methylation distribution. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from male (A-C) and female (D-F) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain DNA is shown. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from three independent 0.5 day old neonatal (G-I) wild-type HBV transgenic mouse liver DNA is shown.

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: Tissue-specific and developmental regulation of HBV DNA methylation distribution. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from male (A-C) and female (D-F) wild-type HBV transgenic mouse liver, kidney, muscle, spleen, lung and brain DNA is shown. The CpG DNA methylation frequency distribution across the 11, 38 and 13 sites within HBV nucleotide coordinates 341–711, 1215–1629 and 2264–2474, respectively, from three independent 0.5 day old neonatal (G-I) wild-type HBV transgenic mouse liver DNA is shown.

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: DNA Methylation Assay, Transgenic Assay

    Effect of FoxA-deletion on HBV DNA methylation in adult mouse liver. The percentage of CpG DNA methylation at each position within the HBV genome from male (A) and female (B) FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The average percent methylation of the CpG sites spanning nucleotide coordinate 1–706 is correlated with the level of serum HBeAg.

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: Effect of FoxA-deletion on HBV DNA methylation in adult mouse liver. The percentage of CpG DNA methylation at each position within the HBV genome from male (A) and female (B) FoxA-expressing (HBVFoxA2 fl/fl AlbCre(-), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(-) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(-)) and FoxA-deleted (HBVFoxA2 fl/fl AlbCre(+), HBVFoxA1 fl/fl FoxA2 fl/fl AlbCre(+) and HBVFoxA1 fl/fl FoxA2 fl/fl FoxA3 +/- AlbCre(+)) HBV transgenic mice is shown. The positions of the viral transcription initiation sites for the X-gene (X RNA), the precore/pregenomic transcripts (C RNA), the large surface antigen transcript (PS RNA) and the middle/major surface antigen transcript (S RNA) are shown. The locations of the FoxA binding sites and CpG island within the HBV genome are also indicated. (C) The average percent methylation of the CpG sites spanning nucleotide coordinate 1–706 is correlated with the level of serum HBeAg.

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: DNA Methylation Assay, Expressing, Transgenic Assay, Mouse Assay, Binding Assay, Methylation

    Model for the tissue-specific and developmental regulation of HBV DNA methylation and transcription by the pioneer transcription factor, FoxA. (A) In the neonatal (P0.5) wild-type HBV transgenic mice, the HBV transgene DNA is extensively methylated but FoxA is expressed and marks the HBV genome for later developmental expression upon recruitment of additional transcription factors to the viral promoters. In the adult wild-type HBV transgenic mice, the HBV transgene DNA is unmethylated, FoxA plus additional transcription factors are recruited to the viral promoters and HBV RNA and DNA synthesis is observed. (B) In the neonatal (P0.5) FoxA-deficient HBV transgenic mice, the HBV transgene DNA is extensively methylated while limiting levels of FoxA fail to mark the HBV genome for later developmental DNA demethylation (due to the failure to recruit TET for active DNA demethylation and/or inhibition of DNA methylation during replication), and hence subsequent recruitment of additional transcription factors to the viral promoters with concomitant viral gene expression. In the adult FoxA-deficient HBV transgenic mice, the HBV genome remains extensively methylated because the limiting abundance of FoxA throughout development fails to mark the viral transgene DNA for demethylation which is essential for HBV RNA and DNA synthesis. (C) In the neonatal (P0.5) wild-type HBV transgenic mice, the essential liver-specific genes which are dependent on FoxA for their expression at this stage of development have presumably been marked by this pioneer transcription factor leading to the recruitment of additional transcription factors necessary for the demethylation and subsequent expression of these genes. Increasing levels of liver-specific transcription factors associated with liver maturation may be associated with increased levels of gene expression in the adult mice. (D) In contrast to the effect of limiting FoxA abundance on HBV DNA methylation and transcription, limiting postnatal FoxA abundance in the hepatocytes of these mice must be sufficient to support gene expression levels consistent with host viability presumably by marking these genes for DNA demethylation and transcription at the neonatal stage of development. The size of the transcription factors (FoxA and TFs) reflects the number of hepatocytes expressing HBV or essential liver-specific genes and/or the overall level of gene expression. The size of the arrow reflects the level of gene transcription. 5MeC, 5-methylcytosine; C, cytosine.

    Journal: PLoS Pathogens

    Article Title: Hepatic deficiency of the pioneer transcription factor FoxA restricts hepatitis B virus biosynthesis by the developmental regulation of viral DNA methylation

    doi: 10.1371/journal.ppat.1006239

    Figure Lengend Snippet: Model for the tissue-specific and developmental regulation of HBV DNA methylation and transcription by the pioneer transcription factor, FoxA. (A) In the neonatal (P0.5) wild-type HBV transgenic mice, the HBV transgene DNA is extensively methylated but FoxA is expressed and marks the HBV genome for later developmental expression upon recruitment of additional transcription factors to the viral promoters. In the adult wild-type HBV transgenic mice, the HBV transgene DNA is unmethylated, FoxA plus additional transcription factors are recruited to the viral promoters and HBV RNA and DNA synthesis is observed. (B) In the neonatal (P0.5) FoxA-deficient HBV transgenic mice, the HBV transgene DNA is extensively methylated while limiting levels of FoxA fail to mark the HBV genome for later developmental DNA demethylation (due to the failure to recruit TET for active DNA demethylation and/or inhibition of DNA methylation during replication), and hence subsequent recruitment of additional transcription factors to the viral promoters with concomitant viral gene expression. In the adult FoxA-deficient HBV transgenic mice, the HBV genome remains extensively methylated because the limiting abundance of FoxA throughout development fails to mark the viral transgene DNA for demethylation which is essential for HBV RNA and DNA synthesis. (C) In the neonatal (P0.5) wild-type HBV transgenic mice, the essential liver-specific genes which are dependent on FoxA for their expression at this stage of development have presumably been marked by this pioneer transcription factor leading to the recruitment of additional transcription factors necessary for the demethylation and subsequent expression of these genes. Increasing levels of liver-specific transcription factors associated with liver maturation may be associated with increased levels of gene expression in the adult mice. (D) In contrast to the effect of limiting FoxA abundance on HBV DNA methylation and transcription, limiting postnatal FoxA abundance in the hepatocytes of these mice must be sufficient to support gene expression levels consistent with host viability presumably by marking these genes for DNA demethylation and transcription at the neonatal stage of development. The size of the transcription factors (FoxA and TFs) reflects the number of hepatocytes expressing HBV or essential liver-specific genes and/or the overall level of gene expression. The size of the arrow reflects the level of gene transcription. 5MeC, 5-methylcytosine; C, cytosine.

    Article Snippet: The 99 CpG sequences in the HBV DNA genome were targeted using PCR amplification of the bisulfite-treated DNA, followed by sequencing of the amplicons on an Illumina MiSeq instrument.

    Techniques: DNA Methylation Assay, Transgenic Assay, Mouse Assay, Methylation, Expressing, DNA Synthesis, Inhibition

    DNA methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.

    Journal: BMC Cancer

    Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

    doi: 10.1186/1471-2407-13-526

    Figure Lengend Snippet: DNA methylation and expression level of the PHD3 gene in HCT116 and DLD-1 CRC cells. A. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles. B. Cells were cultured in DMEM either in hypoxic (1%O 2 ) or normoxic conditions for 48 hrs. After incubation, the cells were used for total RNA isolation and reverse transcription. The PHD3 cDNA levels were determined by RQ-PCR relative quantification analysis. RQ-PCR results were standardized by the geometric mean of PBGD and hMRPL19 cDNA levels. PHD3 cDNA levels are expressed as a multiplicity of these cDNA copies in the cell line’s calibrator. C. Cells were cultured in DMEM either in hypoxic (1%O 2 ) ( H ) or normoxic ( N ) conditions for 48 hrs. Cells were then used for protein isolation. Proteins were separated by 10% SDS-PAGE, and transferred to a membrane that was then immunoblotted with Rp anti - PHD3 Ab and incubated with goat anti-rabbit HRP-conjugated Ab. The membrane was then stripped and reblotted with Rp anti-GAPDH Ab, followed by incubation with goat anti-rabbit HRP-conjugated Ab. The band densitometry readings were normalized to GAPDH loading control. The ratio of PHD3 to GAPDH for DLD-1 in normoxic conditions was assumed to be 1.

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

    Techniques: DNA Methylation Assay, Expressing, Cell Culture, DNA Extraction, Modification, Methylation, Real-time Polymerase Chain Reaction, Amplification, Incubation, Isolation, Polymerase Chain Reaction, SDS Page

    DNA methylation assessment of PHD3 gene regulatory region by bisulfite sequencing and HRM analysis in primary tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1-P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The PHD3 regions containing 60 CpG dinucleotides (chr14: 34 419 929-34 420 563) (Top panel A ) and 44 CpG dinucleotides (ch14: 34 419 346-34 419 943) (Top panel B ) were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server [ 23 , 24 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. Red rectangles correspond to regions amplified in HRM analysis by specific primers PHD3.1 (chr14: 34 419 922-34 420 080), PHD3.2 (chr14: 34 419 795- 34 419 935) and PHD3.3 (chr14: 34 419 400-34 419 538) (Additional file 1 , Additional file 2 ). Bottom panels A and B represent HRM profiles of standard and example of patient DNA (patient P2 from bisulfite sequencing) PCR product. Methylation percentage of three DNA fragments within the PHD3 CpG island was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite treated DNA. HRM methylation analysis was performed using Light Cycler®480 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate.

    Journal: BMC Cancer

    Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

    doi: 10.1186/1471-2407-13-526

    Figure Lengend Snippet: DNA methylation assessment of PHD3 gene regulatory region by bisulfite sequencing and HRM analysis in primary tissue samples from patients with CRC. Primary cancerous and histopathologically unchanged tissues from the same patients with CRC (P1-P5) were used for genomic DNA isolation followed by bisulfite conversion of cytosine to uracil. The PHD3 regions containing 60 CpG dinucleotides (chr14: 34 419 929-34 420 563) (Top panel A ) and 44 CpG dinucleotides (ch14: 34 419 346-34 419 943) (Top panel B ) were then amplified by a pair of primers complementary to the bisulfite-DNA modified sequence (Additional file 1 , Additional file 2 ). The PCR products were purified with subsequent cloning into a plasmid vector. Plasmid DNA isolated from five positive bacterial clones was used for commercial sequencing. The results of bisulfite sequencing were assessed and presented using BiQ analyzer software and BDPC web server [ 23 , 24 ]. Black and grey boxes represent methylated and unmethylated CpG dinucleotide, respectively. Red rectangles correspond to regions amplified in HRM analysis by specific primers PHD3.1 (chr14: 34 419 922-34 420 080), PHD3.2 (chr14: 34 419 795- 34 419 935) and PHD3.3 (chr14: 34 419 400-34 419 538) (Additional file 1 , Additional file 2 ). Bottom panels A and B represent HRM profiles of standard and example of patient DNA (patient P2 from bisulfite sequencing) PCR product. Methylation percentage of three DNA fragments within the PHD3 CpG island was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. DNA standards were prepared by mixing different ratios of methylated and non-methylated bisulfite treated DNA. HRM methylation analysis was performed using Light Cycler®480 Gene Scanning software, Roche Diagnostics GmbH (Mannheim, Germany). Each PCR amplification and HRM profile analysis was performed in triplicate.

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

    Techniques: DNA Methylation Assay, Methylation Sequencing, DNA Extraction, Amplification, Modification, Sequencing, Polymerase Chain Reaction, Purification, Clone Assay, Plasmid Preparation, Isolation, Software, Methylation, Real-time Polymerase Chain Reaction

    Ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level in three ranges of PHD3 methylation status: 0–1%; 1–10% and 10–100%. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. The methylation for each patient was calculated as an average percentage of methylation in amplified fragments located in the CpG island of PHD3. The samples were divided into three groups for statistical analysis: 0–1% methylation, 1–10% methylation and 10–100% methylation (Table 2 ) [ 28 - 30 ]. To evaluate the statistically significant difference in the ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level between the three DNA methylation ranges (0–1% methylation, 1–10% methylation and 10–100% methylation), the non-parametric Kruskal-Wallis test was employed.

    Journal: BMC Cancer

    Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

    doi: 10.1186/1471-2407-13-526

    Figure Lengend Snippet: Ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level in three ranges of PHD3 methylation status: 0–1%; 1–10% and 10–100%. Methylation percentage of three DNA fragments within the PHD3 CpG island (Additional file 1 , Additional file 2 ) was determined by Real Time PCR amplification of bisulfite treated standard and patient DNA, followed by comparison of their HRM profiles. The methylation for each patient was calculated as an average percentage of methylation in amplified fragments located in the CpG island of PHD3. The samples were divided into three groups for statistical analysis: 0–1% methylation, 1–10% methylation and 10–100% methylation (Table 2 ) [ 28 - 30 ]. To evaluate the statistically significant difference in the ratio of cancerous PHD3 mRNA level to histopathologically unchanged tissue PHD3 mRNA level between the three DNA methylation ranges (0–1% methylation, 1–10% methylation and 10–100% methylation), the non-parametric Kruskal-Wallis test was employed.

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

    Techniques: Methylation, Real-time Polymerase Chain Reaction, Amplification, DNA Methylation Assay

    Effect of 5-dAzaC on PHD3 gene DNA methylation in HCT116 and DLD-1 CRC cells. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions either in the absence or in the presence of 5-dAzaC at a concentration of 5.00 μM for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island: A (chr14: 34 419 922–34 420 080), B (chr14: 34 419 795–34 419 935) and C (chr14: 34 419 400–34 419 538) (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles.

    Journal: BMC Cancer

    Article Title: Expression and DNA methylation levels of prolyl hydroxylases PHD1, PHD2, PHD3 and asparaginyl hydroxylase FIH in colorectal cancer

    doi: 10.1186/1471-2407-13-526

    Figure Lengend Snippet: Effect of 5-dAzaC on PHD3 gene DNA methylation in HCT116 and DLD-1 CRC cells. HCT116 and DLD-1 cells were cultured under normoxic or hypoxic (1% O 2 ) conditions either in the absence or in the presence of 5-dAzaC at a concentration of 5.00 μM for 48 hrs. Cells were then used for DNA isolation followed by bisulfite modification. Methylation percentage of three DNA fragments within the PHD3 CpG island: A (chr14: 34 419 922–34 420 080), B (chr14: 34 419 795–34 419 935) and C (chr14: 34 419 400–34 419 538) (Additional file 1 , Additional file 2 ) in HCT116 and DLD-1 cells under hypoxic and normoxic conditions was determined by Real Time PCR amplification of bisulfite treated standard and cell line DNA, followed by comparison of their HRM profiles.

    Article Snippet: DNA methylation assessment by high resolution melting (HRM) analysis Methylation levels of DNA fragments located within the CpG island of the PHD1 , PHD2 , PHD3 and FIH genes (Additional file ) were determined by Real Time PCR amplification of bisulfite treated DNA followed by HRM profile analysis by Light Cycler®480 Real-Time PCR System, Roche Diagnostics GmbH (Mannheim, Germany).

    Techniques: DNA Methylation Assay, Cell Culture, Concentration Assay, DNA Extraction, Modification, Methylation, Real-time Polymerase Chain Reaction, Amplification

    Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.

    Journal: PLoS ONE

    Article Title: Dissection of Structure and Function of the N-Terminal Domain of Mouse DNMT1 Using Regional Frame-Shift Mutagenesis

    doi: 10.1371/journal.pone.0009831

    Figure Lengend Snippet: Genomic methylation assay for IAP LTR and α-actin sequences. (A) Southern blots of total DNA extracted from wild-type (R1), Dnmt1 c/c (c/c), Dnmt1 c/c cells expressing DNMT1 RFM mutants and Dnmt1 c/c cells expressing wild-type DNMT1 (WT). Genomic DNA was digested with the methylation-sensitive enzyme Hpa II (H) and its methylation-insensitive isoschizomer, Msp I (M) and hybridized on a Southern blot with an IAP LTR probe. Hypomethylation of IAP LTR sequences in the Dnmt1 c/c cells is indicated by hybridization to low-molecular weight DNA (1.1-kb band) in the Hpa II digests. (B) Methylation analysis of IAP LTR by COBRA. (C) Methylation analysis of α-actin by COBRA. PCR amplification products represent unmethylated (U) genomic DNA sequences and their digested products represent methylated (M) genomic sequences; sizes are indicated.

    Article Snippet: Bisulfite genomic sequencing IAP sequences were amplified from bisulfite treated DNA , cloned into TOPO TA vector (Invitrogen) and sequenced.

    Techniques: Methylation, Expressing, Southern Blot, Hybridization, Molecular Weight, Combined Bisulfite Restriction Analysis Assay, Polymerase Chain Reaction, Amplification, Genomic Sequencing

    Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: Amplification of 24 individual human genomic loci. Comparison of PCR performance of Taq (top) panel, with two different Taq/5D4 blends (5/1 (middle panel); 10/1 (bottom panel)) on the amplification of 24 different promotor regions in bisulfite-treated and fully desulphonated human genomic DNA. Both blends are able to amplify a significantly larger number of loci than Taq alone and together enable amplification of 18 out of 24 loci (75%).

    Article Snippet: Here we describe the characterization of the utility of one such polymerase (5D4), originally selected for bypass of hydrophobic base analogues ( ) for PCR amplification of bisulfite-treated DNA with potential applications in epigenomics.

    Techniques: Amplification, Polymerase Chain Reaction

    PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Journal: Nucleic Acids Research

    Article Title: A polymerase engineered for bisulfite sequencing

    doi: 10.1093/nar/gkv798

    Figure Lengend Snippet: PCR amplification of bisulfite-treated plasmid templates. PCR amplification of bisulfite-treated high GC and low GC content templates ranging from 200–600 bp in size using fully desulphonated templates and three different 5D4/Taq blends (1/10, 1/5, 1/1) with progressively lower Taq content and Taq alone on low dC content plasmid regions (top panel) and high dC content plasmid regions (bottom panel). On templates with low dC content (and hence lower levels of dU and residual dhU6S adducts post bisulfite treatment and desulphonation) either Taq or Taq/5D4 polymerase blends with a high amount of Taq perform best. In contrast on the higher dC content templates only blends containing 5D4 yield amplicons with Taq/5D4 blends (10/1; 5/1) superior to 5D4/Taq 1/1 blend, while Taq alone does not yield any amplification products. Thus only 5D4/Taq blends are able to copy the high GC content templates indicating that the blended enzymes are more efficient at copying templates containing sporadic dUs (and dhU6S adducts) and dU homopolymer stretches. Low molecular weight bands result from primer-dimer formation. (M: E-Gel ® Low Range Quantitative DNA Ladder).

    Article Snippet: Here we describe the characterization of the utility of one such polymerase (5D4), originally selected for bypass of hydrophobic base analogues ( ) for PCR amplification of bisulfite-treated DNA with potential applications in epigenomics.

    Techniques: Polymerase Chain Reaction, Amplification, Plasmid Preparation, Molecular Weight

    HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated DNA indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and LCRed640-labeled detection probes specific for methylated bisulfite-treated DNA.

    Journal: Nucleic Acids Research

    Article Title: A real-time PCR assay for DNA-methylation using methylation-specific blockers

    doi: 10.1093/nar/gnh008

    Figure Lengend Snippet: HeavyMethyl real-time assays for GSTP1 exon and promoter. (A–C) HeavyMethyl GSTP1 exon assay. Amplification plots of 15 ng SssI-methylated DNA indicated by solid or broken lines and 50 ng of unmethylated DNA indicated by lines with circles ( A ). ( B ) 50 ng of unmethylated DNA and 30 pg methylated DNA spiked into 50 ng unmethylated DNA representing a relative sensitivity of 1:1600 ( C ), which were bisulfite treated and subsequently used as template for the HeavyMethyl assay. The LightCycler detection probes were specific for bisulfite-treated unmethylated (B) or methylated DNA (A, C). HeavyMethyl assays performed with and without blocker are indicated by solid and broken lines, respectively. In (A), the HeavyMethyl assay with and without blocker is also indicated by filled and open circles, respectively. ( D ) HeavyMethyl GSTP1 promoter assay. The GSTP1 promoter fragment was amplified on 400 ng unmethylated (five replicates indicated by broken lines), 100 pg SssI-methylated DNA (black diamonds) and mixtures containing 100 pg (six replicates indicated by solid lines) or 50 pg (six replicates indicated by lines labeled with open diamonds) methylated DNA in 400 ng unmethylated DNA. All template DNA samples were bisulfite treated prior to real-time PCR experiments. The products were specifically detected via the FRET signal generated by the Fluorescein- and LCRed640-labeled detection probes specific for methylated bisulfite-treated DNA.

    Article Snippet: The 20 µl PCR reactions contained 0.31 µM GSTP1Exon forward primer, 0.31 µM GSTP1Exon reverse primer, 0.25 g/l bovine serum albumin (BSA, Sigma), 0.25 mM dNTPs, 0.25 µM GSTP1Exon anchor probe (GTTTAGAGTTTTTAGTATGGGGTTAATT-Fluo; where Fluo = fluorescein), 0.25 µM GSTP1Exon probe I, specific for methylated bisulfite-treated DNA (LCRed640-TAGTATTAGGTTCGGGTTTTCGG-phos) or probe II, specific for unmethylated bisulfite-treated DNA (LCRed705-TAGTATTAGGTTTGGGTTTTTGG-phos; all probes obtained from TIB-Molbiol), 10 µM GSTP1Exon blocker 3 oligonucleotide ( GTGAGT ATGTGTGGTTTGTGTT-phos), 1× Qiagen reaction buffer, 1.5 mM MgCl2 and 1 U Hotstar polymerase.

    Techniques: Amplification, Methylation, Promoter Assay, Labeling, Real-time Polymerase Chain Reaction, Generated