Structured Review

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 ].
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Images

1) Product Images from "The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells"

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

Journal: Arthritis Research & Therapy

doi: 10.1186/ar3830

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 ].
Figure Legend 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 ].

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

2) Product Images from "DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells"

Article Title: DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2014.55.6.1656

DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests ( * p
Figure Legend Snippet: DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests ( * p

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Methylation

3) Product Images from "Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells"

Article Title: Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

Journal: Yonsei Medical Journal

doi: 10.3349/ymj.2014.55.5.1206

MUC13 expression changes following demethylation in SK-OV-3 cells. The SK-OV-3 cells were treated for 3 days with 0, 5, and 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, the DNA methylation status at the -64 CpG site of MUC13 was analyzed using qMSP (A), and MUC13 mRNA expression was measured by qRT-PCR (B). Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p
Figure Legend Snippet: MUC13 expression changes following demethylation in SK-OV-3 cells. The SK-OV-3 cells were treated for 3 days with 0, 5, and 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, the DNA methylation status at the -64 CpG site of MUC13 was analyzed using qMSP (A), and MUC13 mRNA expression was measured by qRT-PCR (B). Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

Techniques Used: Expressing, DNA Methylation Assay, Quantitative RT-PCR

DNA methylation is altered at CpG sites in the MUC13 promoter in metastatic implants from mouse xenografts. The DNA methylation status was analyzed using Bisulfite sequencing analysis (A). The MUC13 promoter region is located at positions 124653578-124653990 in the human GRCh37/hg19 assembly and contains six CpG residues within chromosome 3. The six CpGs are located at positions -273, -159, -123, -64, -37, and -21 from the transcription start site. Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles, and the total percentage of methylation at each site is indicated by a pie graph on the bottom line. The black segment of the pie graph indicates the methylated CpG percentage, whereas the white segment represents the unmethylated CpG percentage. The DNA methylation status at the -64 CpG site was analyzed using qMSP (B). Triangles above the circles in A indicate the specific CpG site used for qMSP. Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p
Figure Legend Snippet: DNA methylation is altered at CpG sites in the MUC13 promoter in metastatic implants from mouse xenografts. The DNA methylation status was analyzed using Bisulfite sequencing analysis (A). The MUC13 promoter region is located at positions 124653578-124653990 in the human GRCh37/hg19 assembly and contains six CpG residues within chromosome 3. The six CpGs are located at positions -273, -159, -123, -64, -37, and -21 from the transcription start site. Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles, and the total percentage of methylation at each site is indicated by a pie graph on the bottom line. The black segment of the pie graph indicates the methylated CpG percentage, whereas the white segment represents the unmethylated CpG percentage. The DNA methylation status at the -64 CpG site was analyzed using qMSP (B). Triangles above the circles in A indicate the specific CpG site used for qMSP. Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

Techniques Used: DNA Methylation Assay, Methylation Sequencing, Methylation

4) Product Images from "A vitamin C-derived DNA modification catalyzed by an algal TET homolog"

Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog

Journal: Nature

doi: 10.1038/s41586-019-1160-0

Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.
Figure Legend Snippet: Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.

Techniques Used: Purification, Recombinant, Staining, Filtration, Chromatography, Activity Assay, Incubation, High Performance Liquid Chromatography, Polymerase Chain Reaction, Mutagenesis

Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .
Figure Legend Snippet: Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .

Techniques Used: CRISPR, Selection, Mutagenesis, Introduce, Recombinant, Purification, Electroporation, Clone Assay, Binding Assay, Polymerase Chain Reaction, Southern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction

5) Product Images from "Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat"

Article Title: Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat

Journal: PLoS Genetics

doi: 10.1371/journal.pgen.1005142

Methylation-sensitive expression of ROS1 is evolutionarily conserved. ( A ) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata . Percentage DNA sequence identity between each paralog and A . thaliana ROS1 is shown for upstream, coding and downstream regions. ( B ) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A . thaliana and A . lyrata . Support values are Bayesian posterior probabilities. ( C ) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. ( D ) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. ( E ) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. ( F ) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p =
Figure Legend Snippet: Methylation-sensitive expression of ROS1 is evolutionarily conserved. ( A ) ROS1 is locally duplicated in the closely related species Arabidopsis lyrata . Percentage DNA sequence identity between each paralog and A . thaliana ROS1 is shown for upstream, coding and downstream regions. ( B ) Bayesian reconstruction of the phylogeny of ROS1 homologs within Brassicales shows AlROS1a and AlROS1b diverged prior to the divergence of A . thaliana and A . lyrata . Support values are Bayesian posterior probabilities. ( C ) Methylation of the sequence 5’ of AlROS1a was examined by BS-PCR. N represents the number of independent clones sequenced for each BS-PCR product. ( D ) Annotation of methylated cytosines in the AtROS1 and AlROS1a 5’ region. Blue lines annotate sequences matching sRNAs from their respective species. ( E ) Relative AlROS1a transcripts are significantly decreased when plants are grown on 5-azaC. ( F ) Relative AlROS1b transcripts levels are unaffected by growth on 5-azaC. Data represented as mean, error bars represent standard deviation. *p =

Techniques Used: Methylation, Expressing, Sequencing, Polymerase Chain Reaction, Clone Assay, Standard Deviation

6) Product Images from "A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution"

Article Title: A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution

Journal: Nucleic Acids Research

doi: 10.1093/nar/gkw994

( A ) A schematic of the EnIGMA method is shown. The black circles designate methylated cytosine, the red circles hydroxylmethylated cytosine and the white circles unmodified cytosine. The EnIGMA method analyzes the CpGs on one strand (orange line for top strand and green line for bottom strand in this figure). The DNA is digested by appropriate restriction endonuclease, and the hairpin DNA is ligated. Next the opposite strand DNA is synthesized in vitro (blue line). Resulted DNA is methylated by DNMT1 enzyme followed by bisulfite conversion and PCR by specific primers. ( B ) The decoding table for cytosine modification status of the CpGs shown in (A).
Figure Legend Snippet: ( A ) A schematic of the EnIGMA method is shown. The black circles designate methylated cytosine, the red circles hydroxylmethylated cytosine and the white circles unmodified cytosine. The EnIGMA method analyzes the CpGs on one strand (orange line for top strand and green line for bottom strand in this figure). The DNA is digested by appropriate restriction endonuclease, and the hairpin DNA is ligated. Next the opposite strand DNA is synthesized in vitro (blue line). Resulted DNA is methylated by DNMT1 enzyme followed by bisulfite conversion and PCR by specific primers. ( B ) The decoding table for cytosine modification status of the CpGs shown in (A).

Techniques Used: Methylation, Synthesized, In Vitro, Polymerase Chain Reaction, Modification

7) Product Images from "A vitamin C-derived DNA modification catalyzed by an algal TET homolog"

Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog

Journal: Nature

doi: 10.1038/s41586-019-1160-0

Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.
Figure Legend Snippet: Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.

Techniques Used: Purification, Recombinant, Staining, Filtration, Chromatography, Activity Assay, Incubation, High Performance Liquid Chromatography, Polymerase Chain Reaction, Mutagenesis

Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .
Figure Legend Snippet: Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .

Techniques Used: CRISPR, Selection, Mutagenesis, Introduce, Recombinant, Purification, Electroporation, Clone Assay, Binding Assay, Polymerase Chain Reaction, Southern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction

8) Product Images from "The SUMO E3 Ligase SIZ1 Positively Regulates Active DNA Demethylation by Stabilizing ROS1 in Arabidopsis thaliana"

Article Title: The SUMO E3 Ligase SIZ1 Positively Regulates Active DNA Demethylation by Stabilizing ROS1 in Arabidopsis thaliana

Journal: bioRxiv

doi: 10.1101/2020.03.05.978999

The siz1 mutant plants show DNA hyper-methylation at multiple loci. (A) Analysis of DNA methylation status at the 3’ region of At1g26400 by methylation-sensitive PCR (CHOP-PCR). Compared to that in wild type Col-0 plants, the methylation levels increased in ros1-4 , siz1-2 and siz1-3 mutants. Undigested genomic DNA was used as a control. (B) Analysis of DNA methylation status at the 3’ region of At1g26400 via locus-specific bisulfite sequencing. (C) Analysis of DNA methylation status at multiple loci by CHOP-PCR. Similar to those in ros1-4 mutant, the methylation levels increased at multiple loci in siz1-2 and siz1-3 mutants. (D) Analysis of DNA methylation status at the indicated loci via locus-specific bisulfite sequencing. The locus-specific bisulfite sequencing results confirmed the CHOP-PCR results.
Figure Legend Snippet: The siz1 mutant plants show DNA hyper-methylation at multiple loci. (A) Analysis of DNA methylation status at the 3’ region of At1g26400 by methylation-sensitive PCR (CHOP-PCR). Compared to that in wild type Col-0 plants, the methylation levels increased in ros1-4 , siz1-2 and siz1-3 mutants. Undigested genomic DNA was used as a control. (B) Analysis of DNA methylation status at the 3’ region of At1g26400 via locus-specific bisulfite sequencing. (C) Analysis of DNA methylation status at multiple loci by CHOP-PCR. Similar to those in ros1-4 mutant, the methylation levels increased at multiple loci in siz1-2 and siz1-3 mutants. (D) Analysis of DNA methylation status at the indicated loci via locus-specific bisulfite sequencing. The locus-specific bisulfite sequencing results confirmed the CHOP-PCR results.

Techniques Used: Mutagenesis, Methylation, DNA Methylation Assay, Polymerase Chain Reaction, Methylation Sequencing

9) Product Images from "Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus"

Article Title: Allele-specific locus binding and genome editing by CRISPR at the p16INK4a locus

Journal: Scientific Reports

doi: 10.1038/srep30485

Effects of CpG methylation of target sites on genome editing in vivo . ( a ) DNA sequences targeted by sgRNAs. Seed sequences and PAMs are shown in yellow and green, respectively. The single-guanine insertion in the Gx5 allele is shown in red. CpG sites in the Gx4 allele are underlin ed. ( b ) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S1 . Products of genotyping PCR were cloned, and 15 (sgRNA_mid2) or 18 (sgRNA_lef5 and sgRNA_rig3) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. ( c ) Evaluation of locus binding, as determined by DNA yields of enChIP. Error bars represent s.e.m. of three enChIP experiments (**t-test P-value
Figure Legend Snippet: Effects of CpG methylation of target sites on genome editing in vivo . ( a ) DNA sequences targeted by sgRNAs. Seed sequences and PAMs are shown in yellow and green, respectively. The single-guanine insertion in the Gx5 allele is shown in red. CpG sites in the Gx4 allele are underlin ed. ( b ) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S1 . Products of genotyping PCR were cloned, and 15 (sgRNA_mid2) or 18 (sgRNA_lef5 and sgRNA_rig3) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. ( c ) Evaluation of locus binding, as determined by DNA yields of enChIP. Error bars represent s.e.m. of three enChIP experiments (**t-test P-value

Techniques Used: CpG Methylation Assay, In Vivo, Polymerase Chain Reaction, Clone Assay, DNA Sequencing, Binding Assay

Allele-specific genome editing using an allele-specific single-nucleotide insertion in vivo . ( a ) DNA sequences targeted by sgRNAs. PAMs are shown in green. The inserted single guanine in the Gx5 allele is shown in red. ( b ) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S8 . Products of genotyping PCR were cloned, and 13 (sgRNA_Gx4#2) or 14 (sgRNA_Gx5#2) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. ( c ) Evaluation of locus binding, as determined by DNA yields of conventional in vivo enChIP. The error bar represents s.e.m. of three enChIP experiments (*t-test P-value
Figure Legend Snippet: Allele-specific genome editing using an allele-specific single-nucleotide insertion in vivo . ( a ) DNA sequences targeted by sgRNAs. PAMs are shown in green. The inserted single guanine in the Gx5 allele is shown in red. ( b ) Evaluation of genome editing. Schemes for genome editing and genotyping PCR are shown in Supplementary Fig. S8 . Products of genotyping PCR were cloned, and 13 (sgRNA_Gx4#2) or 14 (sgRNA_Gx5#2) independent clones were subjected to DNA sequencing analysis to identify the targeted alleles. ( c ) Evaluation of locus binding, as determined by DNA yields of conventional in vivo enChIP. The error bar represents s.e.m. of three enChIP experiments (*t-test P-value

Techniques Used: In Vivo, Polymerase Chain Reaction, Clone Assay, DNA Sequencing, Binding Assay

10) Product Images from "Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)"

Article Title: Quantitative and multiplexed DNA methylation analysis using long-read single-molecule real-time bisulfite sequencing (SMRT-BS)

Journal: BMC Genomics

doi: 10.1186/s12864-015-1572-7

SMRT-BS validation. (A) Methylation quantitation by SMRT-BS of 42 CpG sites was compared to available data on the same sample using the HumanMethylation450 BeadChip (450K Array), resulting in an overall correlation of 0.906 ± 0.052. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ). (B) Methylation quantitation by SMRT-BS of 174 CpG sites was compared to available data on the same sample using the SureSelect™ Human Methyl-Seq target enrichment next generation sequencing platform (MethylSeq), resulting in an overall correlation of 0.933 ± 0.031. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ).
Figure Legend Snippet: SMRT-BS validation. (A) Methylation quantitation by SMRT-BS of 42 CpG sites was compared to available data on the same sample using the HumanMethylation450 BeadChip (450K Array), resulting in an overall correlation of 0.906 ± 0.052. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ). (B) Methylation quantitation by SMRT-BS of 174 CpG sites was compared to available data on the same sample using the SureSelect™ Human Methyl-Seq target enrichment next generation sequencing platform (MethylSeq), resulting in an overall correlation of 0.933 ± 0.031. Correlation analyses were stratified by amplicon lengths, indicating a reduction in correlation with longer amplicons and possible PCR bias towards unmethylated DNA for the intermediate methylation region (see Results and discussion ).

Techniques Used: Methylation, Quantitation Assay, Amplification, Polymerase Chain Reaction, Next-Generation Sequencing, Methylation Sequencing

Bisulfite conversion and long-range amplification. (A) Six commercially available bisulfite conversion kits were tested and treated DNAs were examined using an Agilent 2100 Bioanalyzer to assess size distributions. (B) Two bisulfite-converted DNAs from each of the six kits were subjected to PCR with two amplicons (655 and 1109 bp) and three different extension temperatures (65°C, 68°C and 72°C) to assess capacity for long amplicon amplification. Bisulfite-converted DNA from two kits (Epigentek Methylamp and Qiagen EpiTect) with a PCR extension temperature of 65°C had the most robust amplification of the longer 1109 bp amplicon. (C) Eight amplicons ranging in size from 655–4027 bp (overlapping the MEST CpG island) were designed to determine the upper amplicon size limit of bisulfite PCR. (D) Agarose gel image of bisulfite PCR with the eight primer sets using DNA converted by the Epigentek Methylamp and Qiagen EpiTect kits revealing stable amplification of the 1631 bp amplicon with the Epigentek Methylamp converted DNA and the reported PCR conditions.
Figure Legend Snippet: Bisulfite conversion and long-range amplification. (A) Six commercially available bisulfite conversion kits were tested and treated DNAs were examined using an Agilent 2100 Bioanalyzer to assess size distributions. (B) Two bisulfite-converted DNAs from each of the six kits were subjected to PCR with two amplicons (655 and 1109 bp) and three different extension temperatures (65°C, 68°C and 72°C) to assess capacity for long amplicon amplification. Bisulfite-converted DNA from two kits (Epigentek Methylamp and Qiagen EpiTect) with a PCR extension temperature of 65°C had the most robust amplification of the longer 1109 bp amplicon. (C) Eight amplicons ranging in size from 655–4027 bp (overlapping the MEST CpG island) were designed to determine the upper amplicon size limit of bisulfite PCR. (D) Agarose gel image of bisulfite PCR with the eight primer sets using DNA converted by the Epigentek Methylamp and Qiagen EpiTect kits revealing stable amplification of the 1631 bp amplicon with the Epigentek Methylamp converted DNA and the reported PCR conditions.

Techniques Used: Amplification, Polymerase Chain Reaction, Agarose Gel Electrophoresis

11) Product Images from "APURINIC/APYRIMIDINIC ENDONUCLEASE2 and ZINC FINGER DNA 3′-PHOSPHOESTERASE Play Overlapping Roles in the Maintenance of Epigenome and Genome Stability [OPEN]"

Article Title: APURINIC/APYRIMIDINIC ENDONUCLEASE2 and ZINC FINGER DNA 3′-PHOSPHOESTERASE Play Overlapping Roles in the Maintenance of Epigenome and Genome Stability [OPEN]

Journal: The Plant Cell

doi: 10.1105/tpc.18.00287

APE2 Has Weak DNA 3′ Phosphatase Activity and Strong DNA 3′-5′ Exonuclease Activity in Vitro. (A) APE2 activity against DNA substrate with a 3′-P terminus. The wild-type or mutant form of APE2 proteins (10 nM) was incubated with 3′-P terminated DNA substrate (10 nM) for the indicated times. (B) APE2 activity against DNA substrate with a 3′-OH terminus. Reactions were performed as described in (A) with the exception that the 3′-P in the DNA substrate was replaced by a 3′-OH. (C) Stronger exonuclease activities of APE2. APE2 proteins at low concentrations (1 or 2 nM) were incubated with 3′-P terminated or 3′-OH terminated DNA substrate (10 nM) for 30 min. (D) Release of free phosphate residue from 3′- 32 P-labeled DNA substrate by APE2. APE2 proteins at different concentrations were incubated with 3′- 32 P-labeled DNA substrate (0.05 pM) for 3 h. CIP (5 and 10 units) was used as positive controls; np, no protein control. (E) Phenotypes of 30-d-old zdp-1ape2-2 seedlings complemented by wild-type or mutant forms of APE2. (F) Analysis of the DNA methylation level at the Chr2:17657100-17659549 locus by chop-PCR. Hap II is a methylation-sensitive restriction enzyme. DNA hypermethylation results in an increased level of the PCR product. Undigested DNA was amplified as a control.
Figure Legend Snippet: APE2 Has Weak DNA 3′ Phosphatase Activity and Strong DNA 3′-5′ Exonuclease Activity in Vitro. (A) APE2 activity against DNA substrate with a 3′-P terminus. The wild-type or mutant form of APE2 proteins (10 nM) was incubated with 3′-P terminated DNA substrate (10 nM) for the indicated times. (B) APE2 activity against DNA substrate with a 3′-OH terminus. Reactions were performed as described in (A) with the exception that the 3′-P in the DNA substrate was replaced by a 3′-OH. (C) Stronger exonuclease activities of APE2. APE2 proteins at low concentrations (1 or 2 nM) were incubated with 3′-P terminated or 3′-OH terminated DNA substrate (10 nM) for 30 min. (D) Release of free phosphate residue from 3′- 32 P-labeled DNA substrate by APE2. APE2 proteins at different concentrations were incubated with 3′- 32 P-labeled DNA substrate (0.05 pM) for 3 h. CIP (5 and 10 units) was used as positive controls; np, no protein control. (E) Phenotypes of 30-d-old zdp-1ape2-2 seedlings complemented by wild-type or mutant forms of APE2. (F) Analysis of the DNA methylation level at the Chr2:17657100-17659549 locus by chop-PCR. Hap II is a methylation-sensitive restriction enzyme. DNA hypermethylation results in an increased level of the PCR product. Undigested DNA was amplified as a control.

Techniques Used: Activity Assay, In Vitro, Mutagenesis, Incubation, Labeling, DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Amplification

Multiple Endogenous Loci Show DNA Hypermethylation upon APE2 Depletion or ZDP/APE2 Double Depletion. (A) Analysis of DNA methylation levels at loci DT-75 , DT-76 , DT-77 , and DT-78 by chop-PCR. Ava I, Hpa II, and Bst UI are DNA methylation-sensitive restriction enzymes. DNA hypermethylation results in increased levels of the PCR products. Undigested DNA was amplified as a control. DT-75 , DT-76 , DT-77 , and DT-78 reside in 3′ regions of genes AT1G26380 , AT1G26390 , AT1G26400 , and AT1G26410 for the precise genomic locations of these DT loci. (B) Analysis of DNA methylation levels at loci DT-75 , DT-77 , and DT-78 by locus-specific bisulfite sequencing. The overall percent methylation of cytosine sites in different sequence contexts is presented.
Figure Legend Snippet: Multiple Endogenous Loci Show DNA Hypermethylation upon APE2 Depletion or ZDP/APE2 Double Depletion. (A) Analysis of DNA methylation levels at loci DT-75 , DT-76 , DT-77 , and DT-78 by chop-PCR. Ava I, Hpa II, and Bst UI are DNA methylation-sensitive restriction enzymes. DNA hypermethylation results in increased levels of the PCR products. Undigested DNA was amplified as a control. DT-75 , DT-76 , DT-77 , and DT-78 reside in 3′ regions of genes AT1G26380 , AT1G26390 , AT1G26400 , and AT1G26410 for the precise genomic locations of these DT loci. (B) Analysis of DNA methylation levels at loci DT-75 , DT-77 , and DT-78 by locus-specific bisulfite sequencing. The overall percent methylation of cytosine sites in different sequence contexts is presented.

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Antiviral Assay, Amplification, Methylation Sequencing, Methylation, Sequencing

12) Product Images from "The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation"

Article Title: The cytosolic Fe-S cluster assembly component MET18 is required for the full enzymatic activity of ROS1 in active DNA demethylation

Journal: Scientific Reports

doi: 10.1038/srep26443

MET18 prevents the silencing of reporter genes and endogenous genes. ( a ) The MET18 mutation causes the silencing of 35S-LUC and 35S-NPTII reporter genes. The reporter genes were introduced into met18-2 by crossing. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. ( b ) Real-time PCR analysis of the expression levels of the LUC and NPTII reporter genes in the different genotypes. ( c ) Effect of MET18 mutation on the DNA methylation levels in WT control, ros1-4 and met18-2 , and the expression levels of endogenous genes At1g77780 and At1g77790. ( d ) Effect of MET18 mutation on the DNA methylation levels in WT control, ros1-4 and met18-2 , and the expression levels of endogenous genes At5g38540 and At5g38550 in the mutants. Snapshots in the Integrated Genome Browser show the DNA methylation levels, while the bar diagrams show the real-time PCR analysis of the expression levels of the hypermethylated genes or nearby genes in different genotypes. TUB8 was used as an internal control. Standard errors were calculated from three biological replications, * P
Figure Legend Snippet: MET18 prevents the silencing of reporter genes and endogenous genes. ( a ) The MET18 mutation causes the silencing of 35S-LUC and 35S-NPTII reporter genes. The reporter genes were introduced into met18-2 by crossing. Seedlings grown in MS plates were imaged after being sprayed with luciferase substrate. ( b ) Real-time PCR analysis of the expression levels of the LUC and NPTII reporter genes in the different genotypes. ( c ) Effect of MET18 mutation on the DNA methylation levels in WT control, ros1-4 and met18-2 , and the expression levels of endogenous genes At1g77780 and At1g77790. ( d ) Effect of MET18 mutation on the DNA methylation levels in WT control, ros1-4 and met18-2 , and the expression levels of endogenous genes At5g38540 and At5g38550 in the mutants. Snapshots in the Integrated Genome Browser show the DNA methylation levels, while the bar diagrams show the real-time PCR analysis of the expression levels of the hypermethylated genes or nearby genes in different genotypes. TUB8 was used as an internal control. Standard errors were calculated from three biological replications, * P

Techniques Used: Mutagenesis, Mass Spectrometry, Luciferase, Real-time Polymerase Chain Reaction, Expressing, DNA Methylation Assay

MET18 is required for the generation of Pol V-dependent noncoding RNAs and transcriptional gene silencing. ( a ) Detection of Pol V-dependent noncoding RNAs by real-time PCR, NP is a none NRPE1 enrichment region. ( b , c ) Snapshots in the Integrated Genome Browser showing the DNA methylation levels, and bar diagrams showing the real-time PCR-based expression analysis of the hypomethylated TEs in different genotypes. TUB8 serves as an internal control. Standard errors were calculated from three biological replications, * P
Figure Legend Snippet: MET18 is required for the generation of Pol V-dependent noncoding RNAs and transcriptional gene silencing. ( a ) Detection of Pol V-dependent noncoding RNAs by real-time PCR, NP is a none NRPE1 enrichment region. ( b , c ) Snapshots in the Integrated Genome Browser showing the DNA methylation levels, and bar diagrams showing the real-time PCR-based expression analysis of the hypomethylated TEs in different genotypes. TUB8 serves as an internal control. Standard errors were calculated from three biological replications, * P

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

Dysfunction of MET18 affects ROS1 activity. ( a ) Snapshots in the Integrated Genome Browser showing the DNA methylation levels at the ROS1 promoter. The specific region important for ROS1 regulation is highlighted with red box. ( b ) Real-time PCR analysis of ROS1 transcripts in different mutants. Standard errors were calculated from three biological repeats, * P
Figure Legend Snippet: Dysfunction of MET18 affects ROS1 activity. ( a ) Snapshots in the Integrated Genome Browser showing the DNA methylation levels at the ROS1 promoter. The specific region important for ROS1 regulation is highlighted with red box. ( b ) Real-time PCR analysis of ROS1 transcripts in different mutants. Standard errors were calculated from three biological repeats, * P

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

Iron-Sulfur cluster binding site is essential for ROS1 function. ( a ) Enzymatic activity of WT and mutated ROS1 in E. coli indicated by colony formation of WT and mutated (C1038S and C1045S) ROS1 transformants at 0 and 20 μM IPTG. ( b ) Complementation assay showing that WT, but not the mutant form of ROS1-3Flag, can restore LUC expression in ros1-1 . Leaves were collected for luminescence imaging after the treatment with 3% NaCl for 6 h. ( c ) DNA hypermethylation phenotype of ros1-1 plant transformed with the WT or mutant form (C1045S) of ROS1 . Methylation-sensitive restriction enzymes ( Hha I, Hpa II and BsmA I) were used for the chop-PCR analysis and undigested DNA was amplified as a control. ( d ) Detection of WT ROS1 protein and site-directed mutated (C1045S) ROS1 protein in transgenic Arabidopsis plants by Flag antibody. About 10 μg protein extracts were loaded in each well. The gel for loading control was stained with Coomassie blue.
Figure Legend Snippet: Iron-Sulfur cluster binding site is essential for ROS1 function. ( a ) Enzymatic activity of WT and mutated ROS1 in E. coli indicated by colony formation of WT and mutated (C1038S and C1045S) ROS1 transformants at 0 and 20 μM IPTG. ( b ) Complementation assay showing that WT, but not the mutant form of ROS1-3Flag, can restore LUC expression in ros1-1 . Leaves were collected for luminescence imaging after the treatment with 3% NaCl for 6 h. ( c ) DNA hypermethylation phenotype of ros1-1 plant transformed with the WT or mutant form (C1045S) of ROS1 . Methylation-sensitive restriction enzymes ( Hha I, Hpa II and BsmA I) were used for the chop-PCR analysis and undigested DNA was amplified as a control. ( d ) Detection of WT ROS1 protein and site-directed mutated (C1045S) ROS1 protein in transgenic Arabidopsis plants by Flag antibody. About 10 μg protein extracts were loaded in each well. The gel for loading control was stained with Coomassie blue.

Techniques Used: Binding Assay, Activity Assay, Mutagenesis, Expressing, Imaging, Transformation Assay, Methylation, Polymerase Chain Reaction, Amplification, Transgenic Assay, Staining

Dysfunction of MET18 causes DNA hypermethylation at the EPF2 promoter, At1g26390 and At1g26400 loci. ( a ) Analysis of DNA methylation levels at the EPF2 promoter, At1g26390 and At1g26400 loci using chop-PCR. The methylation-sensitive restriction enzymes used were Bst UI, Hpa II and Hha I. DNA hypermethylation results in no cleavage by the enzymes and increased levels of the PCR product. Undigested controls are shown in the lower panel. ( b – d ) Bisulfite sequencing data showing the effects of MET18 mutations on DNA methylation in different sequence contexts in the EPF2 promoter ( b ), At1g26390 ( c ) and At1g26400 ( d ). For each sample, at least 20 clones were analyzed and the percentage of DNA methylation was calculated from the indicated number of clones.
Figure Legend Snippet: Dysfunction of MET18 causes DNA hypermethylation at the EPF2 promoter, At1g26390 and At1g26400 loci. ( a ) Analysis of DNA methylation levels at the EPF2 promoter, At1g26390 and At1g26400 loci using chop-PCR. The methylation-sensitive restriction enzymes used were Bst UI, Hpa II and Hha I. DNA hypermethylation results in no cleavage by the enzymes and increased levels of the PCR product. Undigested controls are shown in the lower panel. ( b – d ) Bisulfite sequencing data showing the effects of MET18 mutations on DNA methylation in different sequence contexts in the EPF2 promoter ( b ), At1g26390 ( c ) and At1g26400 ( d ). For each sample, at least 20 clones were analyzed and the percentage of DNA methylation was calculated from the indicated number of clones.

Techniques Used: DNA Methylation Assay, Polymerase Chain Reaction, Methylation, Methylation Sequencing, Sequencing, Clone Assay

13) Product Images from "Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers"

Article Title: Loss of NDRG2 expression activates PI3K-AKT signalling via PTEN phosphorylation in ATLL and other cancers

Journal: Nature Communications

doi: 10.1038/ncomms4393

Downregulation of NDRG2 is associated with enhanced phosphorylation of PTEN-Ser380/Thr382/Thr383 and enhanced activation of PI3K-AKT in various cancers. ( a ) Bisulfite genomic sequencing of the NDRG2 promoter region in the KLM1 (pancreatic cancer), SKOV3 (ovarian cancer), HeLa (cervical cancer), HepG2 (hepatic cancer) and KatoIII (gastric cancer) cell lines. PCR products amplified from bisulfite-treated genomic DNA were subcloned, and ten clones in each cell line were sequenced. Open circles indicate unmethylated CpGs (Thy) and filled circles indicate methylated CpGs (Cyt). The region sequenced spans from −396 bp to −133 bp. ( b ) SKOV3, HepG2 and KLM1 cells were cultured with 10 μM 5-aza-dC for 72 h, with 1.2 μM TSA for 48 h, or with 1.2 μM of TSA for 48 h, followed by 10 μM of 5-aza-dC for 24 h. After treatments, total RNA was extracted and quantitative RT–PCR was performed with NDRG2 and β-actin. The relative amounts of mRNA were normalized against β-actin mRNA and expressed relative to the mRNA abundance in untreated cells. The mean±s.d. is shown; * P
Figure Legend Snippet: Downregulation of NDRG2 is associated with enhanced phosphorylation of PTEN-Ser380/Thr382/Thr383 and enhanced activation of PI3K-AKT in various cancers. ( a ) Bisulfite genomic sequencing of the NDRG2 promoter region in the KLM1 (pancreatic cancer), SKOV3 (ovarian cancer), HeLa (cervical cancer), HepG2 (hepatic cancer) and KatoIII (gastric cancer) cell lines. PCR products amplified from bisulfite-treated genomic DNA were subcloned, and ten clones in each cell line were sequenced. Open circles indicate unmethylated CpGs (Thy) and filled circles indicate methylated CpGs (Cyt). The region sequenced spans from −396 bp to −133 bp. ( b ) SKOV3, HepG2 and KLM1 cells were cultured with 10 μM 5-aza-dC for 72 h, with 1.2 μM TSA for 48 h, or with 1.2 μM of TSA for 48 h, followed by 10 μM of 5-aza-dC for 24 h. After treatments, total RNA was extracted and quantitative RT–PCR was performed with NDRG2 and β-actin. The relative amounts of mRNA were normalized against β-actin mRNA and expressed relative to the mRNA abundance in untreated cells. The mean±s.d. is shown; * P

Techniques Used: Activation Assay, Genomic Sequencing, Polymerase Chain Reaction, Amplification, Clone Assay, Methylation, Cell Culture, Quantitative RT-PCR

14) Product Images from "MGMT promoter methylation as a potential prognostic marker for acute leukemia"

Article Title: MGMT promoter methylation as a potential prognostic marker for acute leukemia

Journal: Archives of Medical Science : AMS

doi: 10.5114/aoms.2017.71067

Differences in global DNA hypomethylation in association with type of acute leukemia and methylation of p15
Figure Legend Snippet: Differences in global DNA hypomethylation in association with type of acute leukemia and methylation of p15

Techniques Used: Methylation

15) Product Images from "Association between DNA Methylation of the BDNF Promoter Region and Clinical Presentation in Alzheimer's Disease"

Article Title: Association between DNA Methylation of the BDNF Promoter Region and Clinical Presentation in Alzheimer's Disease

Journal: Dementia and Geriatric Cognitive Disorders EXTRA

doi: 10.1159/000375367

BDNF promoter region examined in the DNA methylation analysis. The target region used for the DNA methylation analysis contains 20 CpG sites, shown in bold type. The vector region has been shaded. The Pst_BDNF_P_F2 forward primer is shown in the box. The targeted regions, consisting of about 120 bp, were positioned from the terminal point of the forward primer (GenBank: AF411339; at position 53144) for direct nucleotide sequencing.
Figure Legend Snippet: BDNF promoter region examined in the DNA methylation analysis. The target region used for the DNA methylation analysis contains 20 CpG sites, shown in bold type. The vector region has been shaded. The Pst_BDNF_P_F2 forward primer is shown in the box. The targeted regions, consisting of about 120 bp, were positioned from the terminal point of the forward primer (GenBank: AF411339; at position 53144) for direct nucleotide sequencing.

Techniques Used: DNA Methylation Assay, Plasmid Preparation, Sequencing

16) Product Images from "A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q"

Article Title: A Comprehensive Analysis of Allelic Methylation Status of CpG Islands on Human Chromosome 21q

Journal: Genome Research

doi: 10.1101/gr.1351604

Mosaicism in maternal allele-specific methylation. ( A ) Maternal allele-specific methylation of CGI #59. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA isolated from PBLs. The arrowhead indicatesthe A/C SNP site. Note that the maternally inherited A allele wasdetected from both Hha I-treated (or methylated) DNA and McrBC-treated (or unmethylated) DNA. ( B ) Bisulfite genomic sequencing of CGI #59. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The A/C SNP site is also indicated. Note that the clonesfor the maternally inherited A allele are composed of two populations, one completely methylated and the other completely escaping methylation.
Figure Legend Snippet: Mosaicism in maternal allele-specific methylation. ( A ) Maternal allele-specific methylation of CGI #59. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA isolated from PBLs. The arrowhead indicatesthe A/C SNP site. Note that the maternally inherited A allele wasdetected from both Hha I-treated (or methylated) DNA and McrBC-treated (or unmethylated) DNA. ( B ) Bisulfite genomic sequencing of CGI #59. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The A/C SNP site is also indicated. Note that the clonesfor the maternally inherited A allele are composed of two populations, one completely methylated and the other completely escaping methylation.

Techniques Used: Methylation, Sequencing, Polymerase Chain Reaction, Isolation, Genomic Sequencing

Allele-specific, parental-origin-independent methylation of CGI #130. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA. In the pedigree shown in A , the paternally inherited C allele ismethylated. On the other hand, the maternally inherited C allele ismethylated in the pedigree shown in B .
Figure Legend Snippet: Allele-specific, parental-origin-independent methylation of CGI #130. Direct sequencing was performed using the PCR products from mock-treated ( bottom left ), HhaI- ( bottom center ), and McrBC-digested ( bottom right ) DNA. In the pedigree shown in A , the paternally inherited C allele ismethylated. On the other hand, the maternally inherited C allele ismethylated in the pedigree shown in B .

Techniques Used: Methylation, Sequencing, Polymerase Chain Reaction

Maternal allele-specific methylation pinpointed to tandem repeats. ( A ) Maternal allele-specific methylation of CGI #112. A map of CGI #112 (500 bp) isshown on the top with the positionsof A/T and C/T SNPs(i.e., SNP1 and SNP2). The arrowsin the map indicate the tandem repeats. The CGI wasPCR-amplified from untreated ( bottom left ), HhaI-digested ( bottom center ), and McrBC-digested ( bottom right ) genomic DNA isolated from PBLs of a C/T heterozygote at SNP2. The amplified productswere subjected to direct sequencing. The vertical arrowhead in each electropherogram denotes the SNP2 (C/T) sites. ( B ) Bisulfite genomic sequencing of CGI #112. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The SNP1 (A/T) site is indicated by the arrowhead.
Figure Legend Snippet: Maternal allele-specific methylation pinpointed to tandem repeats. ( A ) Maternal allele-specific methylation of CGI #112. A map of CGI #112 (500 bp) isshown on the top with the positionsof A/T and C/T SNPs(i.e., SNP1 and SNP2). The arrowsin the map indicate the tandem repeats. The CGI wasPCR-amplified from untreated ( bottom left ), HhaI-digested ( bottom center ), and McrBC-digested ( bottom right ) genomic DNA isolated from PBLs of a C/T heterozygote at SNP2. The amplified productswere subjected to direct sequencing. The vertical arrowhead in each electropherogram denotes the SNP2 (C/T) sites. ( B ) Bisulfite genomic sequencing of CGI #112. Each row of circles corresponds to each clone of bisulfite PCR products. Open and closed circles stand for unmethylated and methylated C residues, respectively. The SNP1 (A/T) site is indicated by the arrowhead.

Techniques Used: Methylation, Amplification, Isolation, Sequencing, Genomic Sequencing, Polymerase Chain Reaction

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Real-time Polymerase Chain Reaction:

Article Title: DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells
Article Snippet: .. For qMSP, 20 µL reaction mixture containing 2 µL (10-100 ng/µL) bisulfite-treated DNA, 10 µL SYBR Premix EX Taq (Takara Bio), 0.4 µL Rox reference dye (50x Takara Bio), and 200 nM each primer were reacted using a 7500 fast real-time PCR system (Applied Biosystems). .. The amplification reaction conditions were: 95℃ for 30 s, followed by 40 cycles of 95℃ for 3 s and 58℃ for 30 s. The PCR product was then reacted at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s to examine the specificity.

Article Title: Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells
Article Snippet: .. For qMSP, 20 µL reaction mixture containing 2 µL (10-100 ng/µL) bisulfite-treated DNA, 10 µL SYBR Premix EX Taq (Takara Bio), 0.4 µL Rox reference dye (50x Takara Bio), and 200 nM each primer were reacted using a 7500 fast real-time PCR system (Applied Biosystems). .. The amplification reaction conditions were: 95℃ for 30 s, followed by 40 cycles of 95℃ for 3 s and 58℃ for 30 s. The PCR product was then reacted at 95℃ for 15 s, 60℃ for 1 min, and 95℃ for 15 s to examine the specificity.

Polymerase Chain Reaction:

Article Title: A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution
Article Snippet: .. Bisulfite-treated DNA was amplified by PCR using EpiTaq HS (Takara) or KOD -Multi & Epi- (Toyobo) using specific primers with an Illumina sequence adaptor for 35 cycles. .. Then the PCR products were cleaned up with AMPure XP and the adaptor for sequencing was applied using the Nextera XT index kit to the resulting PCR products using five cycles of PCR.

Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog
Article Snippet: .. The bisulfite-treated DNA was subjected to PCR amplification using Taq HS polymerase (TAKARA). ..

Article Title: The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells
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. .. PCR products amplified by using Takara LA Taq HS were subcloned into T-Vector pMD20, and sequence analysis was performed.

Article Title: Methylation-Sensitive Expression of a DNA Demethylase Gene Serves As an Epigenetic Rheostat
Article Snippet: .. 2 μl bisulfite treated DNA was used in PCR reactions with 2.5 U ExTaq DNA polymerase (Takara) and 0.4 μM primers using the following cycling conditions (95 °C 3 minutes, 40 cycles of [95 °C for 15 seconds, 52 °C for 60 seconds, 72 °C for 60 seconds], 72 °C for 10 minutes). .. PCR products were cloned using TOPO-TA (Invitrogen) or CloneJet (Life Technologies) PCR cloning kit and individual colonies were sequenced.

Article Title: The SUMO E3 Ligase SIZ1 Positively Regulates Active DNA Demethylation by Stabilizing ROS1 in Arabidopsis thaliana
Article Snippet: .. One μl of bisulfite-treated DNA was used for PCR in 20 μl of reaction mixture using ExTag (Takara) with specific primers (Table S1). .. PCR products were cloned into the pMD-18T vector (Takara) following the manufacturer’s instructions.

Amplification:

Article Title: A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution
Article Snippet: .. Bisulfite-treated DNA was amplified by PCR using EpiTaq HS (Takara) or KOD -Multi & Epi- (Toyobo) using specific primers with an Illumina sequence adaptor for 35 cycles. .. Then the PCR products were cleaned up with AMPure XP and the adaptor for sequencing was applied using the Nextera XT index kit to the resulting PCR products using five cycles of PCR.

Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog
Article Snippet: .. The bisulfite-treated DNA was subjected to PCR amplification using Taq HS polymerase (TAKARA). ..

Article Title: The effects of oxygen tension and antiaging factor Klotho on Wnt signaling in nucleus pulposus cells
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. .. PCR products amplified by using Takara LA Taq HS were subcloned into T-Vector pMD20, and sequence analysis was performed.

Sequencing:

Article Title: A Novel method for the simultaneous identification of methylcytosine and hydroxymethylcytosine at a single base resolution
Article Snippet: .. Bisulfite-treated DNA was amplified by PCR using EpiTaq HS (Takara) or KOD -Multi & Epi- (Toyobo) using specific primers with an Illumina sequence adaptor for 35 cycles. .. Then the PCR products were cleaned up with AMPure XP and the adaptor for sequencing was applied using the Nextera XT index kit to the resulting PCR products using five cycles of PCR.

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    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 62 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    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

    DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests ( * p

    Journal: Yonsei Medical Journal

    Article Title: DNA Hypomethylation-Mediated Overexpression of Carbonic Anhydrase 9 Induces an Aggressive Phenotype in Ovarian Cancer Cells

    doi: 10.3349/ymj.2014.55.6.1656

    Figure Lengend Snippet: DNA methylation is altered at CpG sites in the CA9 promoter in metastatic implants from mouse xenografts. The DNA methylation status at the -6 CpG site was analyzed with the Illumina HumanMethylation 450 BeadChip (A) and qMSP (B). The DNA methylation status was analyzed by bisulfite sequencing (C). The CA9 promoter is located between positions 35673651 and 35674055 in the human GRCh37/hg19 assembly, and contains eight CpG residues on chromosome 9. The eight CpGs are at positions -197, -74, -19, -6, +4, +13, +40, and +86 relative to the transcription start site. Each circle represents a CpG dinucleotide. The methylation status of each CpG site is indicated with a black (methylated) or white (unmethylated) circle. The percentage of methylation at each site is indicated in a pie graph on the bottom line. The black segment of the pie graph indicates the percentage of methylated CpGs, whereas the white segment represents the percentage of unmethylated CpGs (C). Triangles above the circles in C indicate the specific CpG site that was used for qMSP. Statistical analyses were performed by one-way ANOVA and subsequent Bonferroni tests ( * p

    Article Snippet: For qMSP, 20 µL reaction mixture containing 2 µL (10-100 ng/µL) bisulfite-treated DNA, 10 µL SYBR Premix EX Taq (Takara Bio), 0.4 µL Rox reference dye (50x Takara Bio), and 200 nM each primer were reacted using a 7500 fast real-time PCR system (Applied Biosystems).

    Techniques: DNA Methylation Assay, Methylation Sequencing, Methylation

    MUC13 expression changes following demethylation in SK-OV-3 cells. The SK-OV-3 cells were treated for 3 days with 0, 5, and 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, the DNA methylation status at the -64 CpG site of MUC13 was analyzed using qMSP (A), and MUC13 mRNA expression was measured by qRT-PCR (B). Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

    Journal: Yonsei Medical Journal

    Article Title: Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

    doi: 10.3349/ymj.2014.55.5.1206

    Figure Lengend Snippet: MUC13 expression changes following demethylation in SK-OV-3 cells. The SK-OV-3 cells were treated for 3 days with 0, 5, and 10 µM 5-aza-2'-deoxycytidine, respectively. After treatment with 5-aza-2'-deoxycytidine, the DNA methylation status at the -64 CpG site of MUC13 was analyzed using qMSP (A), and MUC13 mRNA expression was measured by qRT-PCR (B). Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

    Article Snippet: For qMSP, 20 µL reaction mixture containing 2 µL (10-100 ng/µL) bisulfite-treated DNA, 10 µL SYBR Premix EX Taq (Takara Bio), 0.4 µL Rox reference dye (50x Takara Bio), and 200 nM each primer were reacted using a 7500 fast real-time PCR system (Applied Biosystems).

    Techniques: Expressing, DNA Methylation Assay, Quantitative RT-PCR

    DNA methylation is altered at CpG sites in the MUC13 promoter in metastatic implants from mouse xenografts. The DNA methylation status was analyzed using Bisulfite sequencing analysis (A). The MUC13 promoter region is located at positions 124653578-124653990 in the human GRCh37/hg19 assembly and contains six CpG residues within chromosome 3. The six CpGs are located at positions -273, -159, -123, -64, -37, and -21 from the transcription start site. Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles, and the total percentage of methylation at each site is indicated by a pie graph on the bottom line. The black segment of the pie graph indicates the methylated CpG percentage, whereas the white segment represents the unmethylated CpG percentage. The DNA methylation status at the -64 CpG site was analyzed using qMSP (B). Triangles above the circles in A indicate the specific CpG site used for qMSP. Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

    Journal: Yonsei Medical Journal

    Article Title: Overexpression of Mucin 13 due to Promoter Methylation Promotes Aggressive Behavior in Ovarian Cancer Cells

    doi: 10.3349/ymj.2014.55.5.1206

    Figure Lengend Snippet: DNA methylation is altered at CpG sites in the MUC13 promoter in metastatic implants from mouse xenografts. The DNA methylation status was analyzed using Bisulfite sequencing analysis (A). The MUC13 promoter region is located at positions 124653578-124653990 in the human GRCh37/hg19 assembly and contains six CpG residues within chromosome 3. The six CpGs are located at positions -273, -159, -123, -64, -37, and -21 from the transcription start site. Each circle represents CpG dinucleotides. The methylation status of each CpG site is illustrated by black (methylated) and white (unmethylated) circles, and the total percentage of methylation at each site is indicated by a pie graph on the bottom line. The black segment of the pie graph indicates the methylated CpG percentage, whereas the white segment represents the unmethylated CpG percentage. The DNA methylation status at the -64 CpG site was analyzed using qMSP (B). Triangles above the circles in A indicate the specific CpG site used for qMSP. Data are shown as the means±SD (n=3). Statistical analyses were performed using a one-way ANOVA and subsequent Bonferroni tests ( * p

    Article Snippet: For qMSP, 20 µL reaction mixture containing 2 µL (10-100 ng/µL) bisulfite-treated DNA, 10 µL SYBR Premix EX Taq (Takara Bio), 0.4 µL Rox reference dye (50x Takara Bio), and 200 nM each primer were reacted using a 7500 fast real-time PCR system (Applied Biosystems).

    Techniques: DNA Methylation Assay, Methylation Sequencing, Methylation

    Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.

    Journal: Nature

    Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog

    doi: 10.1038/s41586-019-1160-0

    Figure Lengend Snippet: Purification of recombinant CMD1 and determination of DNA substrate specificity. a, Coomassie blue staining of the untagged full-length CMD1 protein purified from E. coli . An image for fractions collected from gel filtration chromatography column (eluted between 14–17 min, 1 ml/min) is shown. Representative image is shown from at least three independent experiments. b, . c, CMD1 mutants had no or significantly reduced activity to convert 5mC into P1 and P2. Data shown are representative of two independent experiments. d, P1 and P2 nucleosides accumulate over a period of 2 h upon incubation of the 5mC-DNA substrate with CMD1 shown by HPLC analysis of nucleosides in DNA samples collected at the indicated time points. Data shown are representative of two independent experiments. e, Time-course of the relative amounts of 5mC, P1 and P2 during incubation of 5mC-DNA with CMD1. The amount was determined based on the peak area of each nucleoside in HPLC analysis in panel d . Data shown are representative of two independent experiments. f, 5mC-, but not C- or 5hmC-containing DNA, serves as a substrate for CMD1. DNA substrates containing C, 5hmC or 5mC were prepared by PCR, incubated with CMD1, and then subjected to nucleoside composition analysis using HPLC. Note that P1 and P2 nucleosides only appear in 5mC-DNA upon incubation with WT CMD1. Mut CMD1 is an inactive mutant carrying point mutations (H345Y/D347A). Data shown are representative of two independent experiments.

    Article Snippet: The bisulfite-treated DNA was subjected to PCR amplification using Taq HS polymerase (TAKARA).

    Techniques: Purification, Recombinant, Staining, Filtration, Chromatography, Activity Assay, Incubation, High Performance Liquid Chromatography, Polymerase Chain Reaction, Mutagenesis

    Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .

    Journal: Nature

    Article Title: A vitamin C-derived DNA modification catalyzed by an algal TET homolog

    doi: 10.1038/s41586-019-1160-0

    Figure Lengend Snippet: Generation of a cmd1 strain using a CRISPR/Cas9-based co-selection strategy and co-segregation of the high light-sensitive phenotype with the CMD1 mutation. a, The conversion of indole to tryptophan is catalyzed by the tryptophan (Trp) synthase β subunit encoded by the endogenous MAA7 gene in C. reinhardtii . When 5-fluoroindole (5-FI) is used in place of indole, it will be converted into 5-fluorotryptophan, which is lethally toxic to cells. b, The CRISPR/Cas9-mediated co-selection strategy to introduce mutation in C. reinhardtii . Recombinant Cas9 protein purified from E. coli was assembled with single guide RNA (sgRNA) for both the MAA7 gene and a target gene of interest to form RNP complexes. Upon electroporation of the mixture of the two RNP complexes into cells, 5-FI resistant colonies were selected and genotyped to identify clones with a desired mutation in the targeted gene. The mutant strains were then backcrossed with the wild-type strain to segregate the target gene mutation from the MAA7 mutation or other off-target mutations if any. c, The genomic loci of CMD1 (also known as CrTET1 ) and its close paralog CrTET2 . At the CMD1 locus of cmd1 cells, there is an insertion of 245 bp in the exon 3, thus generating a frame-shift mutation. Chromosome locations of the two paralogs are indicated on the top. DNA sequences from the targeted loci in wild-type and cmd1 strains are shown on the bottom. The 3-nt PAM and 20-nt sgRNA-binding sequences are distinctively colored. d, Genomic PCR genotyping of the cmd1 strain using two primer pairs as shown in panel c. Sizes expected for the PCR products are indicated. Note that the forward primer of primer pair 1 (panel c ) can binds to both the CMD1 and CrTET2 genomic loci. The forward primer of primer pair 2 is specific for a site upstream of CMD1 . Representative image is shown from at least three independent experiments. e, Southern blot analysis of the CMD1 genomic locus. The locations of the probe (dark blue bar) and the SalI and NheI restriction sites used for the digestion of the genomic DNA are indicated in panel c. Two bands detected in the lane of the cmd1 DNA sample arose from the mutant CMD1 locus with a 245-bp insert and the unaltered CrTET2 paralogous locus of almost identical sequence, respectively. Expected lengths of the detected restriction fragments are given in the brackets. Representative image is shown from two independent experiments. f, RT-PCR analysis of the region spanning the targeted site of exon 3. The expected lengths of PCR products from the wild-type and cmd1 cells are given in the brackets. Representative image is shown from two independent experiments. g, Co-segregation analysis of the CMD1 mutation in the progeny of a cross between wild-type CC124 with the cmd1 strain. Equal amounts of the cells were dripped on agar plates and exposed to low light (20 μmol photons·m −2 ·s −1 ) or high light (1000 μmol photons·m −2 ·s −1 ) for 66 h. A1 and A2 are the cmd1 and wild-type CC124 cells respectively. Red circles mark the clones of the parental cmd1 .

    Article Snippet: The bisulfite-treated DNA was subjected to PCR amplification using Taq HS polymerase (TAKARA).

    Techniques: CRISPR, Selection, Mutagenesis, Introduce, Recombinant, Purification, Electroporation, Clone Assay, Binding Assay, Polymerase Chain Reaction, Southern Blot, Sequencing, Reverse Transcription Polymerase Chain Reaction