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    Structured Review

    New England Biolabs hhai
    Methylation status of the CpG island stipulates Sipa1 transcription in breast cancer cells. (A) Sipa1 promoter plasmid construction. In the map of the Sipa1 promoter proximal elements, the CpG dinucleotide is indicated by short vertical lines and the CpG island is represented by a rectangle. Three pGL4 plasmids containing different fragments of the possible Sipa1 promoter-proximal elements: SP-1203 contains the entire promoter sequence, SP-895 lacks the CpG island and SP-326 contains the CpG island only. (B) Methylation status of each plasmid examined by digestion with methylation-sensitive restriction enzymes. Lane 1 is a negative control; lanes 2 and 3 represent plasmids treated with M . Sss I without SAM as methylation substrates and digested with methylation-sensitive restriction enzyme <t>HpaI</t> ( Hp ) or <t>HhaI</t> ( Hp ); lanes 4 and 5 represent plasmids treated with M . Sss I and SAM, and digested with HpaI ( Hp ) or HhaI ( Hp ). Blue arrows indicate the digested DNA bands that were sensitive to HpaI or HhaI digestion; red arrows indicate the DNA bands that were more resistant to HpaI or HhaI digestion. (C) Transcriptional activity of the possible Sipa1 promoter-proximal elements with varying degrees of methylation status. Each plasmid was transfected into MDA-MB-231 cells, and the luciferase activity was measured 36 h later. The pGL4-Basic plasmid was used as a negative control. The columns represent the mean of triplicate experiments (*** P
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    Images

    1) Product Images from "5-Aza-2′-deoxycytidine advances the epithelial–mesenchymal transition of breast cancer cells by demethylating Sipa1 promoter-proximal elements"

    Article Title: 5-Aza-2′-deoxycytidine advances the epithelial–mesenchymal transition of breast cancer cells by demethylating Sipa1 promoter-proximal elements

    Journal: Journal of Cell Science

    doi: 10.1242/jcs.236125

    Methylation status of the CpG island stipulates Sipa1 transcription in breast cancer cells. (A) Sipa1 promoter plasmid construction. In the map of the Sipa1 promoter proximal elements, the CpG dinucleotide is indicated by short vertical lines and the CpG island is represented by a rectangle. Three pGL4 plasmids containing different fragments of the possible Sipa1 promoter-proximal elements: SP-1203 contains the entire promoter sequence, SP-895 lacks the CpG island and SP-326 contains the CpG island only. (B) Methylation status of each plasmid examined by digestion with methylation-sensitive restriction enzymes. Lane 1 is a negative control; lanes 2 and 3 represent plasmids treated with M . Sss I without SAM as methylation substrates and digested with methylation-sensitive restriction enzyme HpaI ( Hp ) or HhaI ( Hp ); lanes 4 and 5 represent plasmids treated with M . Sss I and SAM, and digested with HpaI ( Hp ) or HhaI ( Hp ). Blue arrows indicate the digested DNA bands that were sensitive to HpaI or HhaI digestion; red arrows indicate the DNA bands that were more resistant to HpaI or HhaI digestion. (C) Transcriptional activity of the possible Sipa1 promoter-proximal elements with varying degrees of methylation status. Each plasmid was transfected into MDA-MB-231 cells, and the luciferase activity was measured 36 h later. The pGL4-Basic plasmid was used as a negative control. The columns represent the mean of triplicate experiments (*** P
    Figure Legend Snippet: Methylation status of the CpG island stipulates Sipa1 transcription in breast cancer cells. (A) Sipa1 promoter plasmid construction. In the map of the Sipa1 promoter proximal elements, the CpG dinucleotide is indicated by short vertical lines and the CpG island is represented by a rectangle. Three pGL4 plasmids containing different fragments of the possible Sipa1 promoter-proximal elements: SP-1203 contains the entire promoter sequence, SP-895 lacks the CpG island and SP-326 contains the CpG island only. (B) Methylation status of each plasmid examined by digestion with methylation-sensitive restriction enzymes. Lane 1 is a negative control; lanes 2 and 3 represent plasmids treated with M . Sss I without SAM as methylation substrates and digested with methylation-sensitive restriction enzyme HpaI ( Hp ) or HhaI ( Hp ); lanes 4 and 5 represent plasmids treated with M . Sss I and SAM, and digested with HpaI ( Hp ) or HhaI ( Hp ). Blue arrows indicate the digested DNA bands that were sensitive to HpaI or HhaI digestion; red arrows indicate the DNA bands that were more resistant to HpaI or HhaI digestion. (C) Transcriptional activity of the possible Sipa1 promoter-proximal elements with varying degrees of methylation status. Each plasmid was transfected into MDA-MB-231 cells, and the luciferase activity was measured 36 h later. The pGL4-Basic plasmid was used as a negative control. The columns represent the mean of triplicate experiments (*** P

    Techniques Used: Methylation, Plasmid Preparation, Sequencing, Negative Control, Activity Assay, Transfection, Multiple Displacement Amplification, Luciferase

    2) Product Images from "Fidelity Index Determination of DNA Methyltransferases"

    Article Title: Fidelity Index Determination of DNA Methyltransferases

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0063866

    Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.
    Figure Legend Snippet: Radioactive methylation assay using E. coli DNA-comparison between restriction enzyme digested substrate versus undigested substrate. The dilution series and presentation of data are the same as in Figure 3 . A) H 3 -methyl incorporation by M.EcoKDam. At a dilution of 1/2 4 , the substrate is fully methylated and after a dilution of 1/2 2 , the CPMs start to increase indicating star activity. This results in an FI of 4. B) H 3 -methyl incorporation by M.EcoKDam with DNA that has been digested with MboI restriction enzyme to remove all M.EcoKDam cognate sites. After a dilution of 1/2 2 , the CPMs start to increase, indicating methylation at non-cognate sites. C) H 3 -methyl incorporation by M.HhaI. At a dilution of 1/2 8 , the substrate is fully methylated and after a dilution of 1/2 3 , the CPMs start to increase indicating star activity. This results in an FI of 32. D) H 3 -methyl incorporation by M.HhaI with DNA that has been digested with HhaI restriction enzyme to remove all M.HhaI cognate sites. After a dilution of 1/2 3 , the CPMs start to increase, indicating methylation at non-cognate sites.

    Techniques Used: Methylation, Activity Assay

    3) Product Images from "Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer"

    Article Title: Methylation of Wnt7a Is Modulated by DNMT1 and Cigarette Smoke Condensate in Non-Small Cell Lung Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0032921

    Wnt7a is methylated in NSCLC cell lines and tissues. A) B2B and HBEC cells were treated with 10 uM NNK for 2 hours and Wnt7a expression was measured by QPCR compared to an untreated control. Error bars are the SEM of triplicate experiments. Percent methylation in B2B and HBEC cells was analyzed using the Methyl Profiler system after 2 hr 10 uM NNK treatment compared to untreated cells. B) Mean percent methylation of the Wnt7a promoter was measured by pyrosequencing in NSCLC tissue and compared to matched normal adjacent lung tissue by paired t-test. Mean percent methylation of B2B, H157, and H1299 cell lines was measured by pyrosequencing. C) A Wnt7a promoter luciferase was modified by SssI, HpaII, and HhaI methylating enzymes and transiently transfected into B2B and HBEC cell lines. Fold-change in luciferase activity was measured compared to unmodified Wnt7a promoter luciferase activity. Error bars are the SE of triplicate experiments.
    Figure Legend Snippet: Wnt7a is methylated in NSCLC cell lines and tissues. A) B2B and HBEC cells were treated with 10 uM NNK for 2 hours and Wnt7a expression was measured by QPCR compared to an untreated control. Error bars are the SEM of triplicate experiments. Percent methylation in B2B and HBEC cells was analyzed using the Methyl Profiler system after 2 hr 10 uM NNK treatment compared to untreated cells. B) Mean percent methylation of the Wnt7a promoter was measured by pyrosequencing in NSCLC tissue and compared to matched normal adjacent lung tissue by paired t-test. Mean percent methylation of B2B, H157, and H1299 cell lines was measured by pyrosequencing. C) A Wnt7a promoter luciferase was modified by SssI, HpaII, and HhaI methylating enzymes and transiently transfected into B2B and HBEC cell lines. Fold-change in luciferase activity was measured compared to unmodified Wnt7a promoter luciferase activity. Error bars are the SE of triplicate experiments.

    Techniques Used: Methylation, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Modification, Transfection, Activity Assay

    4) Product Images from "GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2?-Deoxycytidine Efficacy in Human Prostate Cancer Cells"

    Article Title: GSTP1 DNA Methylation and Expression Status Is Indicative of 5-aza-2?-Deoxycytidine Efficacy in Human Prostate Cancer Cells

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0025634

    GSTP1 DNA methylation and protein expression in LNCaP cells after daily 5-aza-CdR treatment. DNA and proteins were extracted from LNCaP cells treated with increasing doses of 5-aza-CdR (0.005–2.5 µM). Cells were treated daily and DNA and protein harvested after 6 days of treatment. (A) MSP was performed on bisulfite-modified DNA with primers targeting bisulfite-modified methylated GSTP1 promoter or unmethylated GSTP1 promoter. (B–C) the relative methylation status of the GSTP1 promoter following 5-aza-CdR treatment was further assessed by COBRA using two restriction enzymes, BstUI and HhaI. MDA-MB-231 breast cancer cells were used as a control for unmethylated GSTP1 promoter. (D) Immunoblot was performed to analyze GSTP1 protein expression. Detection of Hsp90 was used as a loading control.
    Figure Legend Snippet: GSTP1 DNA methylation and protein expression in LNCaP cells after daily 5-aza-CdR treatment. DNA and proteins were extracted from LNCaP cells treated with increasing doses of 5-aza-CdR (0.005–2.5 µM). Cells were treated daily and DNA and protein harvested after 6 days of treatment. (A) MSP was performed on bisulfite-modified DNA with primers targeting bisulfite-modified methylated GSTP1 promoter or unmethylated GSTP1 promoter. (B–C) the relative methylation status of the GSTP1 promoter following 5-aza-CdR treatment was further assessed by COBRA using two restriction enzymes, BstUI and HhaI. MDA-MB-231 breast cancer cells were used as a control for unmethylated GSTP1 promoter. (D) Immunoblot was performed to analyze GSTP1 protein expression. Detection of Hsp90 was used as a loading control.

    Techniques Used: DNA Methylation Assay, Expressing, Modification, Methylation, Combined Bisulfite Restriction Analysis Assay, Multiple Displacement Amplification

    DNA methylation status and protein expression of GSTP1 in LNCaP cells after Zebularine treatment. DNA and proteins were extracted from LNCaP cells after 8 days of treatment with increasing doses of Zebularine (0–400 µM). (A) DNA was bisulfite-modified and MSP was performed with primers targeting bisulfite-modified methylated GSTP1 or unmethylated GSTP1 . (B) The relative DNA methylation status of the GSTP1 promoter following Zebularine treatment was assessed by COBRA using two restriction enzymes, BstUI or HhaI. (C) Immunoblot was performed to analyse GSTP1 protein expression in LNCaP cells after 8 days of Zebularine treatment. Proteins were extracted from LNCaP cells treated with increasing doses of Zebularine (0–400 µM). PC3 cells express endogenous GSTP1 protein and were used as positive control.
    Figure Legend Snippet: DNA methylation status and protein expression of GSTP1 in LNCaP cells after Zebularine treatment. DNA and proteins were extracted from LNCaP cells after 8 days of treatment with increasing doses of Zebularine (0–400 µM). (A) DNA was bisulfite-modified and MSP was performed with primers targeting bisulfite-modified methylated GSTP1 or unmethylated GSTP1 . (B) The relative DNA methylation status of the GSTP1 promoter following Zebularine treatment was assessed by COBRA using two restriction enzymes, BstUI or HhaI. (C) Immunoblot was performed to analyse GSTP1 protein expression in LNCaP cells after 8 days of Zebularine treatment. Proteins were extracted from LNCaP cells treated with increasing doses of Zebularine (0–400 µM). PC3 cells express endogenous GSTP1 protein and were used as positive control.

    Techniques Used: DNA Methylation Assay, Expressing, Modification, Methylation, Combined Bisulfite Restriction Analysis Assay, Positive Control

    5) Product Images from "Helicobacter pullorum Outbreak in C57BL/6NTac and C3H/HeNTac Barrier-Maintained Mice ▿"

    Article Title: Helicobacter pullorum Outbreak in C57BL/6NTac and C3H/HeNTac Barrier-Maintained Mice ▿

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.02531-09

    Restriction fragment length polymorphism analysis patterns of the Helicobacter 16S rRNA gene with AluI and HhaI digestion. Lanes 1 to 3, Helicobacter isolates from Taconic mice; lane 4, H. pullorum human isolate MIT 98-5489; lane 5, H. typhlonius ; lane 6, H. bilis ; lane 7, H. muridarum ; lane 8, H. hepaticus ; lane 9, H. rodentium ; M, 100-bp DNA ladder.
    Figure Legend Snippet: Restriction fragment length polymorphism analysis patterns of the Helicobacter 16S rRNA gene with AluI and HhaI digestion. Lanes 1 to 3, Helicobacter isolates from Taconic mice; lane 4, H. pullorum human isolate MIT 98-5489; lane 5, H. typhlonius ; lane 6, H. bilis ; lane 7, H. muridarum ; lane 8, H. hepaticus ; lane 9, H. rodentium ; M, 100-bp DNA ladder.

    Techniques Used: Mouse Assay

    6) Product Images from "Expansion of epigenetic alterations in EFEMP1 promoter predicts malignant formation in pancreatobiliary intraductal papillary mucinous neoplasms"

    Article Title: Expansion of epigenetic alterations in EFEMP1 promoter predicts malignant formation in pancreatobiliary intraductal papillary mucinous neoplasms

    Journal: Journal of Cancer Research and Clinical Oncology

    doi: 10.1007/s00432-016-2164-x

    Methylation analysis of EFEMP1 . a Results of EFEMP1 methylation by fluorescent Hi-SA. Blue arrows represent PCR fragments with non-methylation in HhaI sites. Red arrows represent methylated PCR fragments cleaved by HhaI. b Frequencies of EFEMP1 methylation according to pathological findings. Kaplan–Meier survival curves for disease-free survival ( c ), excluding a patient with remaining cancer at the resected margin, and overall survival ( d ) according to EFEMP1 promoter methylation status
    Figure Legend Snippet: Methylation analysis of EFEMP1 . a Results of EFEMP1 methylation by fluorescent Hi-SA. Blue arrows represent PCR fragments with non-methylation in HhaI sites. Red arrows represent methylated PCR fragments cleaved by HhaI. b Frequencies of EFEMP1 methylation according to pathological findings. Kaplan–Meier survival curves for disease-free survival ( c ), excluding a patient with remaining cancer at the resected margin, and overall survival ( d ) according to EFEMP1 promoter methylation status

    Techniques Used: Methylation, Polymerase Chain Reaction

    7) Product Images from "AP-2? Induces Epigenetic Silencing of Tumor Suppressive Genes and Microsatellite Instability in Head and Neck Squamous Cell Carcinoma"

    Article Title: AP-2? Induces Epigenetic Silencing of Tumor Suppressive Genes and Microsatellite Instability in Head and Neck Squamous Cell Carcinoma

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0006931

    Decreased AP-2α expression reduces proliferation; and AP-2α is required for target remethylation following demethylation. a) AP-2α western blot analysis of SCC22B cells with AP-2α overexpression. b) Cell cycle analysis via flow cytometry comparing WT, AP-2α-overexpressing, and AP-2α-downregulated SCC22B cells. * P-value = 0.0003. c) Methylation sensitive HhaI digests were performed on DNAs from SCC22B cells with and without AP-2α downregulation, revealing no distinguishable difference in global DNA methylation. “M” = methylated; “U” = unmethylated. d) 1 µM 5-aza-2′deoxycytidine was applied to the cells for 96 hours, changing the media daily. After 96 hours, the 5-aza-2′deoxycytidine was removed and the cells were continued in culture for 10 days, collecting cells at 0, 7, and 10 days post-treatment. Combined bisulfite restriction analysis on bisulfite-treated DNA obtained from these cells at 0, 7, and 10 days. Increased methylation at 10 days is seen in the AP-2α-expressing cells (i.e. the digested band indicated by the arrow).
    Figure Legend Snippet: Decreased AP-2α expression reduces proliferation; and AP-2α is required for target remethylation following demethylation. a) AP-2α western blot analysis of SCC22B cells with AP-2α overexpression. b) Cell cycle analysis via flow cytometry comparing WT, AP-2α-overexpressing, and AP-2α-downregulated SCC22B cells. * P-value = 0.0003. c) Methylation sensitive HhaI digests were performed on DNAs from SCC22B cells with and without AP-2α downregulation, revealing no distinguishable difference in global DNA methylation. “M” = methylated; “U” = unmethylated. d) 1 µM 5-aza-2′deoxycytidine was applied to the cells for 96 hours, changing the media daily. After 96 hours, the 5-aza-2′deoxycytidine was removed and the cells were continued in culture for 10 days, collecting cells at 0, 7, and 10 days post-treatment. Combined bisulfite restriction analysis on bisulfite-treated DNA obtained from these cells at 0, 7, and 10 days. Increased methylation at 10 days is seen in the AP-2α-expressing cells (i.e. the digested band indicated by the arrow).

    Techniques Used: Expressing, Western Blot, Over Expression, Cell Cycle Assay, Flow Cytometry, Cytometry, Methylation, DNA Methylation Assay

    8) Product Images from "Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae"

    Article Title: Generation and Molecular Characterization of New Temperature-Sensitive Plasmids Intended for Genetic Engineering of Pasteurellaceae

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.11.7187-7195.2005

    Protection of DNA against HsoI and HhaI cleavage by HhaI methyltransferase. Plasmid DNAs in lanes 1 to 3 were protected by in vitro methylation with HhaI methyltransferase. Plasmid DNAs in lanes 4 to 6 were not methylated. The plasmids in lanes 1 and
    Figure Legend Snippet: Protection of DNA against HsoI and HhaI cleavage by HhaI methyltransferase. Plasmid DNAs in lanes 1 to 3 were protected by in vitro methylation with HhaI methyltransferase. Plasmid DNAs in lanes 4 to 6 were not methylated. The plasmids in lanes 1 and

    Techniques Used: Plasmid Preparation, In Vitro, Methylation

    Determination of the HsoI cleavage site alongside that of isoschisomer HhaI. The cleavage sites were determined by digestion of a primed-synthesis reaction containing the recognition site on pBluescript SK. A standard dideoxy DNA sequencing reaction
    Figure Legend Snippet: Determination of the HsoI cleavage site alongside that of isoschisomer HhaI. The cleavage sites were determined by digestion of a primed-synthesis reaction containing the recognition site on pBluescript SK. A standard dideoxy DNA sequencing reaction

    Techniques Used: DNA Sequencing

    9) Product Images from "Defining characteristics of Tn5 Transposase non-specific DNA binding"

    Article Title: Defining characteristics of Tn5 Transposase non-specific DNA binding

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkl179

    Tn 5 Tnp interacts with supercoiled pUC19. REBA of Tnp bound to supercoiled pUC19 is shown in lanes 4–9 and DNA size markers are shown in lanes 1–3. Increasing concentrations of Tnp (lanes 5–9) were incubated with pUC19 followed by addition of frequent cutting restriction enzymes, HhaI, Fnu4HI and Sau3AI. Blockage of restriction sites by Tnp results in the appearance of larger bands and decrease in some smaller bands as Tnp concentration is increased. This is most evident when examining the overlay of the traces from a no Tnp control reaction (blue, lane 4) and 900 µM Tnp lanes (red, lane 9), seen to the right of the gel. Bands increasing in intensity are marked with a plus sign (+). Bands decreasing in intensity are marked with a minus sign (−).
    Figure Legend Snippet: Tn 5 Tnp interacts with supercoiled pUC19. REBA of Tnp bound to supercoiled pUC19 is shown in lanes 4–9 and DNA size markers are shown in lanes 1–3. Increasing concentrations of Tnp (lanes 5–9) were incubated with pUC19 followed by addition of frequent cutting restriction enzymes, HhaI, Fnu4HI and Sau3AI. Blockage of restriction sites by Tnp results in the appearance of larger bands and decrease in some smaller bands as Tnp concentration is increased. This is most evident when examining the overlay of the traces from a no Tnp control reaction (blue, lane 4) and 900 µM Tnp lanes (red, lane 9), seen to the right of the gel. Bands increasing in intensity are marked with a plus sign (+). Bands decreasing in intensity are marked with a minus sign (−).

    Techniques Used: Incubation, Concentration Assay

    10) Product Images from "Inhibition of chromatin remodeling by Polycomb Group protein Posterior Sex Combs is mechanistically distinct from nucleosome binding 1"

    Article Title: Inhibition of chromatin remodeling by Polycomb Group protein Posterior Sex Combs is mechanistically distinct from nucleosome binding 1

    Journal: Biochemistry

    doi: 10.1021/bi100532a

    PSC inhibits remodeling of mononucleosomes poorly Restriction enzyme accessibility (REA) assays were carried out with PSC at the indicated concentrations and hSwi/Snf. A,B) Representative gels of REAs with 6N (A) and 2N (B) templates. (N refers to nucleosomes, so 6N is a 6-nucleosome template; all of these templates are composed of repeats of the 5S nucleosome positioning sequence.). C) Summary of REAs with 6N and 2N templates. D, E) Representative gel of REA with mononucleosomes assembled on a 157-bp (10-1N) 5S template at 1nM (D) or 5nM (E). Note that PstI was used for digestion in the experiment with 1nM nucleosomes, while HhaI was used with 5nM nucleosomes. Both enzymes have single digestion sites in the template, but produce slightly different digestion patterns (HhaI digests the template into 77 and 80 bp fragments, which are not resolved, while PstI digests it into 99 and 52 bp fragments). Both enzymes were used in these assays and they produce very similar results. D) Summary of REAs with mononucleosome template. Error bars in are SEM.
    Figure Legend Snippet: PSC inhibits remodeling of mononucleosomes poorly Restriction enzyme accessibility (REA) assays were carried out with PSC at the indicated concentrations and hSwi/Snf. A,B) Representative gels of REAs with 6N (A) and 2N (B) templates. (N refers to nucleosomes, so 6N is a 6-nucleosome template; all of these templates are composed of repeats of the 5S nucleosome positioning sequence.). C) Summary of REAs with 6N and 2N templates. D, E) Representative gel of REA with mononucleosomes assembled on a 157-bp (10-1N) 5S template at 1nM (D) or 5nM (E). Note that PstI was used for digestion in the experiment with 1nM nucleosomes, while HhaI was used with 5nM nucleosomes. Both enzymes have single digestion sites in the template, but produce slightly different digestion patterns (HhaI digests the template into 77 and 80 bp fragments, which are not resolved, while PstI digests it into 99 and 52 bp fragments). Both enzymes were used in these assays and they produce very similar results. D) Summary of REAs with mononucleosome template. Error bars in are SEM.

    Techniques Used: Sequencing

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    New England Biolabs hha i
    PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by <t>Alu</t> I (a) or <t>Hha</t> I (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588
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    PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by Alu I (a) or Hha I (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588

    Journal: Journal of Medical Microbiology

    Article Title: Helicobactermarmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs

    doi: 10.1099/jmm.0.032144-0

    Figure Lengend Snippet: PCR products of 1.2 kb produced using Helicobacter genus-specific primers were digested by Alu I (a) or Hha I (b) and analysed by electrophoresis on a 6 % Visigel matrix. Lanes 1–4, prairie dog isolates MIT 07-5168, MIT 07-5167, MIT 04-8588

    Article Snippet: The PCR-amplified 1.2 kb fragment of the 16S rRNA gene (20 µl) was digested with 10 U Alu I and Hha I (New England BioLabs) in appropriate buffer, as recommended by the manufacturer, at 37 °C for 3 h ( ).

    Techniques: Polymerase Chain Reaction, Produced, Electrophoresis

    Fig. 1. Methylation analysis of Lκ single cells. ( A ) Map of the Jκ 3–4 region showing the location of primers used for MSRE-PCR analysis and relevant restriction enzyme sites. Ava I was used to detect DNA methylation on genomic DNA, whereas Hha I was employed on the PCR product to distinguish between the two alleles. ( B ) Genomic DNA from Mus musculus BALB/c (M), SJL or F 1 mice were PCR amplified with primers P8 and P9, digested with Hha I and analyzed on a 4% agarose gel. The BALB/c gene contains the polymorphic Hha I site, while the SJL gene product is uncut. F 1 DNA yields a product that shows 50% digestion. DNA from individual κ + B cells was digested with Ava I and amplified using two rounds of PCR. Both examples (cells 1 and 2) shown were found to be methylated monoallelically (BALB/c and SJL allele, respectively). ( C ) Summary of the number of PCR-positive cells that show a monoallelic PCR product in the MSRE-PCR assay, using either Ava I or Hin dIII (control) digestion. The percentage of biallelic single cells observed following Hin ). The distribution (%) of single cells containing either 0, 1 or 2 methylated alleles (pie diagram) was derived as follows: 24% of PCR positive cells have a biallelic methylation pattern. Previous experiments had shown that even normal κ + B cells, which always have at least one allele unmethylated, yield a biallelic pattern in 14% of the cells using this assay, and this is probably due to inefficient Ava ). Subtracting this background (24–14), we conclude that 10% of the PCR positive cells actually have two methylated alleles. In order to calculate the percentage of biallelically methylated cells in the full Lκ B cell population, we utilized Southern blot data showing that only 40% of the κ alleles are methylated (see Figure 2 for an example). Using simple algebra, these data are translated into the numbers shown in the diagram. A simple check shows that out of 100 cells, with seven having two methylated alleles and 66 having one, the fraction of methylated alleles is indeed 40% (80/200). The distribution of methylated alleles can also be ascertained by an alternate method based on first determining the percentage of cells that have two unmethylated alleles. This can be determined by counting the number of single cells that yield no PCR product and correcting for the efficiency of the amplification. After digestion with Hin dIII alone, 75% of the cells yield a PCR product, but only 54% gave a product when digested with Ava I. As an additional control, we also amplified another region that lacks Ava I sites, and this yielded a product from 73% of the single cells. Thus, in (75–54)/75 = 28% of the Lκ B cells that should have yielded a product, none was obtained, implying that in 28% of the cells, both alleles are unmethylated.

    Journal: The EMBO Journal

    Article Title: Differential accessibility at the ? chain locus plays a role in allelic exclusion

    doi: 10.1093/emboj/cdf518

    Figure Lengend Snippet: Fig. 1. Methylation analysis of Lκ single cells. ( A ) Map of the Jκ 3–4 region showing the location of primers used for MSRE-PCR analysis and relevant restriction enzyme sites. Ava I was used to detect DNA methylation on genomic DNA, whereas Hha I was employed on the PCR product to distinguish between the two alleles. ( B ) Genomic DNA from Mus musculus BALB/c (M), SJL or F 1 mice were PCR amplified with primers P8 and P9, digested with Hha I and analyzed on a 4% agarose gel. The BALB/c gene contains the polymorphic Hha I site, while the SJL gene product is uncut. F 1 DNA yields a product that shows 50% digestion. DNA from individual κ + B cells was digested with Ava I and amplified using two rounds of PCR. Both examples (cells 1 and 2) shown were found to be methylated monoallelically (BALB/c and SJL allele, respectively). ( C ) Summary of the number of PCR-positive cells that show a monoallelic PCR product in the MSRE-PCR assay, using either Ava I or Hin dIII (control) digestion. The percentage of biallelic single cells observed following Hin ). The distribution (%) of single cells containing either 0, 1 or 2 methylated alleles (pie diagram) was derived as follows: 24% of PCR positive cells have a biallelic methylation pattern. Previous experiments had shown that even normal κ + B cells, which always have at least one allele unmethylated, yield a biallelic pattern in 14% of the cells using this assay, and this is probably due to inefficient Ava ). Subtracting this background (24–14), we conclude that 10% of the PCR positive cells actually have two methylated alleles. In order to calculate the percentage of biallelically methylated cells in the full Lκ B cell population, we utilized Southern blot data showing that only 40% of the κ alleles are methylated (see Figure 2 for an example). Using simple algebra, these data are translated into the numbers shown in the diagram. A simple check shows that out of 100 cells, with seven having two methylated alleles and 66 having one, the fraction of methylated alleles is indeed 40% (80/200). The distribution of methylated alleles can also be ascertained by an alternate method based on first determining the percentage of cells that have two unmethylated alleles. This can be determined by counting the number of single cells that yield no PCR product and correcting for the efficiency of the amplification. After digestion with Hin dIII alone, 75% of the cells yield a PCR product, but only 54% gave a product when digested with Ava I. As an additional control, we also amplified another region that lacks Ava I sites, and this yielded a product from 73% of the single cells. Thus, in (75–54)/75 = 28% of the Lκ B cells that should have yielded a product, none was obtained, implying that in 28% of the cells, both alleles are unmethylated.

    Article Snippet: The reaction mixture was then heated to 65°C for 15 min. Half of the ligation volume (1 µg of DNA) was then subjected to digestion overnight at 37°C with 200 U of Hha I (NEB).

    Techniques: Methylation, Polymerase Chain Reaction, Antiviral Assay, DNA Methylation Assay, Mouse Assay, Amplification, Agarose Gel Electrophoresis, Derivative Assay, Southern Blot

    Fig. 2. DNase I sensitivity of methylated and unmethylated κ alleles. A map of the Jκ locus showing the J1–5 segments (black rectangles), the relevant restriction enzyme sites and the probes for Southern hybridization. ( A ) Nuclei were prepared from splenic B cells isolated from Lκ mice treated for 3 days with LPS (10 µg/ml) and reacted with DNase I (0.1–3 µg/ml). Remaining DNA was extracted and digested with Hin dIII/ Sac II to distinguish between the 2.7 kb methylated (Me) and 2.5 kb unmethylated (Un) alleles, and subjected to blot hybridization using probe 2. Similar results were obtained using unstimulated ex vivo κ + or B220 + B cells from Lκ mice. ( B ) Genomic DNA was isolated from DNase I-treated nuclei purified from splenic B cells taken from wild-type (wt) mice. DNA was digested with Bgl II/ Hin dIII/ Hha I to distinguish between the 1.0 kb methylated and 0.8 kb unmethylated alleles originating from productive and non-productive molecules, respectively, and hybridized with probe 1.

    Journal: The EMBO Journal

    Article Title: Differential accessibility at the ? chain locus plays a role in allelic exclusion

    doi: 10.1093/emboj/cdf518

    Figure Lengend Snippet: Fig. 2. DNase I sensitivity of methylated and unmethylated κ alleles. A map of the Jκ locus showing the J1–5 segments (black rectangles), the relevant restriction enzyme sites and the probes for Southern hybridization. ( A ) Nuclei were prepared from splenic B cells isolated from Lκ mice treated for 3 days with LPS (10 µg/ml) and reacted with DNase I (0.1–3 µg/ml). Remaining DNA was extracted and digested with Hin dIII/ Sac II to distinguish between the 2.7 kb methylated (Me) and 2.5 kb unmethylated (Un) alleles, and subjected to blot hybridization using probe 2. Similar results were obtained using unstimulated ex vivo κ + or B220 + B cells from Lκ mice. ( B ) Genomic DNA was isolated from DNase I-treated nuclei purified from splenic B cells taken from wild-type (wt) mice. DNA was digested with Bgl II/ Hin dIII/ Hha I to distinguish between the 1.0 kb methylated and 0.8 kb unmethylated alleles originating from productive and non-productive molecules, respectively, and hybridized with probe 1.

    Article Snippet: The reaction mixture was then heated to 65°C for 15 min. Half of the ligation volume (1 µg of DNA) was then subjected to digestion overnight at 37°C with 200 U of Hha I (NEB).

    Techniques: Methylation, Hybridization, Isolation, Mouse Assay, Ex Vivo, Purification

    CAPS procedure for the detection of a thymine to cysteine change (C2088R mutation) at nucleotide position 6262 in Lolium spp. The Hha I digested fragment (126 bp) correspond to the resistant R2088 allele and the Hha 1 undigested fragment (161 bp) correspond to the C2088 allele. Heterozygous plants display both the 126 bp and 161 resistant and sensitive alleles respectively. Lanes 1 and 10: DNA ladder, lanes 2, 3 and 4: homozygous mutant RR2088, lanes 4, 5, 6: heterozygous CR2088 plants, lanes 7, 8, 9: homozygous wild CC2088 plants.

    Journal: PLoS ONE

    Article Title: Broad Resistance to ACCase Inhibiting Herbicides in a Ryegrass Population Is Due Only to a Cysteine to Arginine Mutation in the Target Enzyme

    doi: 10.1371/journal.pone.0039759

    Figure Lengend Snippet: CAPS procedure for the detection of a thymine to cysteine change (C2088R mutation) at nucleotide position 6262 in Lolium spp. The Hha I digested fragment (126 bp) correspond to the resistant R2088 allele and the Hha 1 undigested fragment (161 bp) correspond to the C2088 allele. Heterozygous plants display both the 126 bp and 161 resistant and sensitive alleles respectively. Lanes 1 and 10: DNA ladder, lanes 2, 3 and 4: homozygous mutant RR2088, lanes 4, 5, 6: heterozygous CR2088 plants, lanes 7, 8, 9: homozygous wild CC2088 plants.

    Article Snippet: Two µl aliquots of neat PCR products were digested with 2 units of Hha I (New England Biolabs, Hertfordshire, UK) in a total volume of 20 µL according to the manufacturer's recommendations and analysed on 2% agarose/1 X TBE buffer gels containing 0.5 µg mL−1 ethidium bromide.

    Techniques: Mutagenesis

    Alu I and Hha I restriction sites of Pvmsp - 3α gene using in silico digestion of 20 isolates from Indian sub-continent (drawn according to scale)

    Journal: Malaria Journal

    Article Title: Plasmodium vivax msp-3α polymorphisms: analysis in the Indian subcontinent

    doi: 10.1186/s12936-016-1524-y

    Figure Lengend Snippet: Alu I and Hha I restriction sites of Pvmsp - 3α gene using in silico digestion of 20 isolates from Indian sub-continent (drawn according to scale)

    Article Snippet: To determine the level of Pvmsp -3α polymorphism, RFLP analysis was carried out using the Hha I and Alu I restriction enzymes (NEB, Inc, Beverly, MA, USA) in a 10-µL reaction volume.

    Techniques: In Silico