hgai  (New England Biolabs)


Bioz Verified Symbol New England Biolabs is a verified supplier
Bioz Manufacturer Symbol New England Biolabs manufactures this product  
  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    Name:
    HgaI
    Description:
    HgaI 500 units
    Catalog Number:
    r0154l
    Price:
    282
    Size:
    500 units
    Category:
    Restriction Enzymes
    Buy from Supplier


    Structured Review

    New England Biolabs hgai
    HgaI
    HgaI 500 units
    https://www.bioz.com/result/hgai/product/New England Biolabs
    Average 95 stars, based on 80 article reviews
    Price from $9.99 to $1999.99
    hgai - by Bioz Stars, 2020-09
    95/100 stars

    Images

    1) Product Images from "Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis"

    Article Title: Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis

    Journal: Veterinary Journal (London, England : 1997)

    doi: 10.1016/j.tvjl.2007.07.007

    RFLP analysis of PEDV N gene PCR products. The 691 bp PCR products were digested with AspLEI (a), HgaI (b), MspR9I (c) or Tru9I (d) and analysed in 1.5% agarose gel. Lane 1, 100 bp DNA marker; lanes 2–14, field PEDV isolates; lane 15, Japanese vaccine virus; lane 16, PEDV CV777. 1.5% agarose gel electrophoresis.
    Figure Legend Snippet: RFLP analysis of PEDV N gene PCR products. The 691 bp PCR products were digested with AspLEI (a), HgaI (b), MspR9I (c) or Tru9I (d) and analysed in 1.5% agarose gel. Lane 1, 100 bp DNA marker; lanes 2–14, field PEDV isolates; lane 15, Japanese vaccine virus; lane 16, PEDV CV777. 1.5% agarose gel electrophoresis.

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

    2) Product Images from "Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu"

    Article Title: Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa319

    DNA modification activity and co-factor-interactions of Mom mutants. ( A ) Analysis of in vivo DNA modification activity of Mom mutants. Plasmid DNA was isolated from E. coli C41(DE3) cells expressing wild type or mutant Mom proteins, as indicated. VC denotes vector control and ΔCTD denotes a 175 aa long Mom construct with 56 residues from the C-terminus deleted. M represents 1kb ladder. 1 μg DNA was digested with the restriction endonuclease HgaI and products were analyzed on a 1% agarose ethidium bromide gel. Different mutations exert variable degrees of effects on the activity of Mom, with S114 and R101A compromising Mom activity most severely. Lower panel shows the undigested plasmids corresponding to those in the upper panel. ( B ) Co-factor-binding defects in Mom mutants analyzed using microscale thermophoresis. His-tagged wild-type Mom (WT) and mutants were fluorescently labeled and titrated against indicated concentrations of ligands acetyl CoA (upper panel) and Fe 2+/3+ (lower panel). Normalized fluorescence is plotted for analysis of thermophoresis. Data were fitted using the law of mass action to determine dissociation constants (K D ). Data shown are representative of three independent experiments. Error bars represent standard deviations of n = 3 measurements. All mutants except R101A and D139A are defective in acetyl CoA binding, whereas Y49A, D139A and Y149A are compromised for Fe 2+/3+ binding. ( C ) Active site of Mom. Residues found to be important for the DNA modification activity of Mom (side chains shown) map onto the active site pocket of Mom, with the splayed strands β4 and β5 which form the acetyl CoA-binding cleft colored blue. A Fe 2+/3+ ion is colocalized near the acetyl arm of acetyl CoA within the active site of Mom. The tentative location of the Fe 2+/3+ ion is depicted based on mutagenesis data, with residues critical for Fe 2+/3+ -binding highlighted. ( D ) R101 residue of Mom. Of all the point mutations introduced in the Mom active site, the effect of R101A substitution was the most detrimental to the modification activity, despite having no effect on acetyl CoA- and Fe 2+/3+ -binding. The active site cleft viewed from the acetyl end of acetyl CoA shows the acetyl group emerging into a grove, likely to be the substrate adenine-binding cleft. The residue R101 is positioned at the opening of the cleft and appears poised for substrate recognition.
    Figure Legend Snippet: DNA modification activity and co-factor-interactions of Mom mutants. ( A ) Analysis of in vivo DNA modification activity of Mom mutants. Plasmid DNA was isolated from E. coli C41(DE3) cells expressing wild type or mutant Mom proteins, as indicated. VC denotes vector control and ΔCTD denotes a 175 aa long Mom construct with 56 residues from the C-terminus deleted. M represents 1kb ladder. 1 μg DNA was digested with the restriction endonuclease HgaI and products were analyzed on a 1% agarose ethidium bromide gel. Different mutations exert variable degrees of effects on the activity of Mom, with S114 and R101A compromising Mom activity most severely. Lower panel shows the undigested plasmids corresponding to those in the upper panel. ( B ) Co-factor-binding defects in Mom mutants analyzed using microscale thermophoresis. His-tagged wild-type Mom (WT) and mutants were fluorescently labeled and titrated against indicated concentrations of ligands acetyl CoA (upper panel) and Fe 2+/3+ (lower panel). Normalized fluorescence is plotted for analysis of thermophoresis. Data were fitted using the law of mass action to determine dissociation constants (K D ). Data shown are representative of three independent experiments. Error bars represent standard deviations of n = 3 measurements. All mutants except R101A and D139A are defective in acetyl CoA binding, whereas Y49A, D139A and Y149A are compromised for Fe 2+/3+ binding. ( C ) Active site of Mom. Residues found to be important for the DNA modification activity of Mom (side chains shown) map onto the active site pocket of Mom, with the splayed strands β4 and β5 which form the acetyl CoA-binding cleft colored blue. A Fe 2+/3+ ion is colocalized near the acetyl arm of acetyl CoA within the active site of Mom. The tentative location of the Fe 2+/3+ ion is depicted based on mutagenesis data, with residues critical for Fe 2+/3+ -binding highlighted. ( D ) R101 residue of Mom. Of all the point mutations introduced in the Mom active site, the effect of R101A substitution was the most detrimental to the modification activity, despite having no effect on acetyl CoA- and Fe 2+/3+ -binding. The active site cleft viewed from the acetyl end of acetyl CoA shows the acetyl group emerging into a grove, likely to be the substrate adenine-binding cleft. The residue R101 is positioned at the opening of the cleft and appears poised for substrate recognition.

    Techniques Used: Modification, Activity Assay, In Vivo, Plasmid Preparation, Isolation, Expressing, Mutagenesis, Construct, Binding Assay, Microscale Thermophoresis, Labeling, Fluorescence

    DNA modification activity of Mom. ( A ) Structure of the modified base N 6 -methylcarbamoyl adenine. ( B ) Analysis of the in vivo DNA modification activity of Mom using the restriction endonuclease HgaI. Plasmid DNA was isolated from E. coli C41(DE3) cells harbouring the empty vector (vector control) or expressing Mom. DNA modification is depicted by red dots in the schematic. To examine if cellular DNA was modified by Mom, 1 μg plasmid DNA was digested with HgaI and products were analyzed on a 1% agarose ethidium bromide gel. While plasmids from vector control cells get digested into expected sized products, plasmids from mom- expressing cells remain unaffected by HgaI.
    Figure Legend Snippet: DNA modification activity of Mom. ( A ) Structure of the modified base N 6 -methylcarbamoyl adenine. ( B ) Analysis of the in vivo DNA modification activity of Mom using the restriction endonuclease HgaI. Plasmid DNA was isolated from E. coli C41(DE3) cells harbouring the empty vector (vector control) or expressing Mom. DNA modification is depicted by red dots in the schematic. To examine if cellular DNA was modified by Mom, 1 μg plasmid DNA was digested with HgaI and products were analyzed on a 1% agarose ethidium bromide gel. While plasmids from vector control cells get digested into expected sized products, plasmids from mom- expressing cells remain unaffected by HgaI.

    Techniques Used: Modification, Activity Assay, In Vivo, Plasmid Preparation, Isolation, Expressing

    3) Product Images from "Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression"

    Article Title: Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

    Journal: bioRxiv

    doi: 10.1101/859892

    Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.
    Figure Legend Snippet: Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.

    Techniques Used: Methylation, Incubation, Next-Generation Sequencing, Methylation Sequencing

    4) Product Images from "Germline and somatic mosaicism for FGFR2 mutation in the mother of a child with Crouzon syndrome: Implications for genetic testing in "paternal age-effect" syndromes"

    Article Title: Germline and somatic mosaicism for FGFR2 mutation in the mother of a child with Crouzon syndrome: Implications for genetic testing in "paternal age-effect" syndromes

    Journal: American Journal of Medical Genetics. Part a

    doi: 10.1002/ajmg.a.33513

    FGFR2 exon IIIc sequence and restriction enzyme digestion A : Pedigree indicating the segregation of the microsatellite D10S1483 located 6.7 kb away from the site of the FGFR2 mutation in the proposita (II-1), showing that the two sibs have inherited opposite maternal FGFR2 alleles. B–F: DNA sequencing chromatograms around the mutation site (p.Asp336 codon corresponding to the GAC boxed sequence) in FGFR2 exon IIIc ( B , C : reverse complementary sequence of the minus strand; D – F : forward sequence of the plus strand); the proposita's blood DNA (B) shows a heterozygous c.1007A > G mutation encoding a p.Asp336Gly substitution; (C) the same change is absent from the father's blood DNA. D–F: Chromatograms from the mother's blood (D), saliva (E) and hair roots (F) genomic DNA, revealing a variable amount of the c.1007A > G mutation in these tissues (arrowheads). G : Hga I restriction digest of a 132 bp PCR product for the different genomic DNA samples (as indicated), shows that the normal allele (c.1007A), is cut into two fragments (65 and 67 bp), while the c.1007A > G mutation abolishes the Hga I restriction site (GACGCN 5 ). Comparison of the ratio of mutant (upper undigested fragment) to normal (lower fragments) alleles in the maternal samples to that of the heterozygous proposita, shows that the relative amount of undigested fragment is reduced in the mother's blood and saliva samples and is barely visible in the hair root sample (right and left lanes are 100 bp ladders). [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]
    Figure Legend Snippet: FGFR2 exon IIIc sequence and restriction enzyme digestion A : Pedigree indicating the segregation of the microsatellite D10S1483 located 6.7 kb away from the site of the FGFR2 mutation in the proposita (II-1), showing that the two sibs have inherited opposite maternal FGFR2 alleles. B–F: DNA sequencing chromatograms around the mutation site (p.Asp336 codon corresponding to the GAC boxed sequence) in FGFR2 exon IIIc ( B , C : reverse complementary sequence of the minus strand; D – F : forward sequence of the plus strand); the proposita's blood DNA (B) shows a heterozygous c.1007A > G mutation encoding a p.Asp336Gly substitution; (C) the same change is absent from the father's blood DNA. D–F: Chromatograms from the mother's blood (D), saliva (E) and hair roots (F) genomic DNA, revealing a variable amount of the c.1007A > G mutation in these tissues (arrowheads). G : Hga I restriction digest of a 132 bp PCR product for the different genomic DNA samples (as indicated), shows that the normal allele (c.1007A), is cut into two fragments (65 and 67 bp), while the c.1007A > G mutation abolishes the Hga I restriction site (GACGCN 5 ). Comparison of the ratio of mutant (upper undigested fragment) to normal (lower fragments) alleles in the maternal samples to that of the heterozygous proposita, shows that the relative amount of undigested fragment is reduced in the mother's blood and saliva samples and is barely visible in the hair root sample (right and left lanes are 100 bp ladders). [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]

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

    Related Articles

    Methylation:

    Article Title: Epigenetic impact of simulated maternal grooming on estrogen receptor alpha within the developing amygdala
    Article Snippet: .. HgaI (Cat. #R0154, New England BioLabs) recognizes the sequence GACGC and cleaves five nucleotides downstream of the recognition site if the CG is not methylated ( ). .. HhaI (Cat. #R0139, New England BioLabs) recognizes GCGC sequences and cleaves the C away from GCG if the CG is not methylated ( ).

    Isolation:

    Article Title: Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu
    Article Snippet: .. Plasmid DNA was isolated from the cultures using Qiagen miniprep kits and 1 μg DNA was digested with HgaI (2 units/μl, NEB). ..

    Polymerase Chain Reaction:

    Article Title: Is the rs1801282 (G/C) Polymorphism of PPAR - Gamma Gene Associated with T2DM in Iraqi People?
    Article Snippet: .. The recognition site for HgaI is the following: Ten-µl of PCR products were digested with 3 units of HgaI (New England Biolabs, UK) in a total reaction volume of 50 µl containing 5 µl of 10X 1.1 NEBuffer (10 mM Bis Tris Propane-HCl, 10 mM MgCl2 , 1 mM dithiothreitol, pH 7.0 at 25°C) at 37C° for 60 minutes. .. The reaction was terminated by enzyme inactivation at 65°C for 20 min. DNA fragments were separated on 2% agarose gel stained with SYBR® Safe DNA Gel Stain (Promega, USA) following standard gel electrophoresis, visualised on the UV transilluminator and photographed.

    Article Title: Germline and somatic mosaicism for FGFR2 mutation in the mother of a child with Crouzon syndrome: Implications for genetic testing in "paternal age-effect" syndromes
    Article Snippet: .. Restriction digest with Hga I (New England Biolabs, Ipswich, CA) was performed on 15 µl of the pyrosequencing PCR product (see below) in a 30 µl reaction containing 1× Buffer 1 and 5 U of Hga I for 4 hr at 37°C. .. The resulting digest was run on a 4% TBE agarose gel.

    Incubation:

    Article Title: Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression
    Article Snippet: .. 100ng DNA sample was incubated with HgaI (2U/μl) and appurtenant 10X NEBuffer 1.1 (both from New England Biolabs, Bioké, Leiden, The Netherlands) for 60 minutes at 37°C and 65°C for 20 minutes. .. To assess the MF of a CpG adjacent to cg11625005, located in 1,295,731, the MSRE AvaI (10U/μl; New England Biolabs) was employed, which recognises this CpG and cleaves it when unmethylated.

    Sequencing:

    Article Title: Epigenetic impact of simulated maternal grooming on estrogen receptor alpha within the developing amygdala
    Article Snippet: .. HgaI (Cat. #R0154, New England BioLabs) recognizes the sequence GACGC and cleaves five nucleotides downstream of the recognition site if the CG is not methylated ( ). .. HhaI (Cat. #R0139, New England BioLabs) recognizes GCGC sequences and cleaves the C away from GCG if the CG is not methylated ( ).

    Article Title: Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis
    Article Snippet: .. After preliminary examination of a number of candidate restriction enzymes (REs) selected on the basis of the N gene sequence of CV777, AspLEI, HgaI, MspR9I and Tru9I (New England Biolabs) were studied further. ..

    Plasmid Preparation:

    Article Title: The Pattern of Disulfide Linkages in the Extracellular Loop Regions of Connexin 32 Suggests a Model for the Docking Interface of Gap Junctions
    Article Snippet: .. Combinations of cysteine shifts within E1 were prepared by HgaI ( New England Biolabs Inc. , Beverly, MA) digestion of the Cx32 insert that was first excised from the vector by HindIII/SacI to avoid confusion from HgaI sites in the vector. .. HgaI separates the cysteine 63 and 74 mutagenesis sites into fragments that could be separated on 1% agarose gels before elution and purification (Gene Clean; Bio101, La Jolla, CA) and then followed by religation with the vector.

    Article Title: Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu
    Article Snippet: .. Plasmid DNA was isolated from the cultures using Qiagen miniprep kits and 1 μg DNA was digested with HgaI (2 units/μl, NEB). ..

    Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 95
    New England Biolabs hgai
    RFLP analysis of PEDV N gene PCR products. The 691 bp PCR products were digested with AspLEI (a), <t>HgaI</t> (b), MspR9I (c) or Tru9I (d) and analysed in 1.5% agarose gel. Lane 1, 100 bp DNA marker; lanes 2–14, field PEDV isolates; lane 15, Japanese vaccine virus; lane 16, PEDV <t>CV777.</t> 1.5% agarose gel electrophoresis.
    Hgai, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 15 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hgai/product/New England Biolabs
    Average 95 stars, based on 15 article reviews
    Price from $9.99 to $1999.99
    hgai - by Bioz Stars, 2020-09
    95/100 stars
      Buy from Supplier

    Image Search Results


    RFLP analysis of PEDV N gene PCR products. The 691 bp PCR products were digested with AspLEI (a), HgaI (b), MspR9I (c) or Tru9I (d) and analysed in 1.5% agarose gel. Lane 1, 100 bp DNA marker; lanes 2–14, field PEDV isolates; lane 15, Japanese vaccine virus; lane 16, PEDV CV777. 1.5% agarose gel electrophoresis.

    Journal: Veterinary Journal (London, England : 1997)

    Article Title: Genetic differentiation of the nucleocapsid protein of Korean isolates of porcine epidemic diarrhoea virus by RT-PCR based restriction fragment length polymorphism analysis

    doi: 10.1016/j.tvjl.2007.07.007

    Figure Lengend Snippet: RFLP analysis of PEDV N gene PCR products. The 691 bp PCR products were digested with AspLEI (a), HgaI (b), MspR9I (c) or Tru9I (d) and analysed in 1.5% agarose gel. Lane 1, 100 bp DNA marker; lanes 2–14, field PEDV isolates; lane 15, Japanese vaccine virus; lane 16, PEDV CV777. 1.5% agarose gel electrophoresis.

    Article Snippet: After preliminary examination of a number of candidate restriction enzymes (REs) selected on the basis of the N gene sequence of CV777, AspLEI, HgaI, MspR9I and Tru9I (New England Biolabs) were studied further.

    Techniques: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Marker

    DNA modification activity and co-factor-interactions of Mom mutants. ( A ) Analysis of in vivo DNA modification activity of Mom mutants. Plasmid DNA was isolated from E. coli C41(DE3) cells expressing wild type or mutant Mom proteins, as indicated. VC denotes vector control and ΔCTD denotes a 175 aa long Mom construct with 56 residues from the C-terminus deleted. M represents 1kb ladder. 1 μg DNA was digested with the restriction endonuclease HgaI and products were analyzed on a 1% agarose ethidium bromide gel. Different mutations exert variable degrees of effects on the activity of Mom, with S114 and R101A compromising Mom activity most severely. Lower panel shows the undigested plasmids corresponding to those in the upper panel. ( B ) Co-factor-binding defects in Mom mutants analyzed using microscale thermophoresis. His-tagged wild-type Mom (WT) and mutants were fluorescently labeled and titrated against indicated concentrations of ligands acetyl CoA (upper panel) and Fe 2+/3+ (lower panel). Normalized fluorescence is plotted for analysis of thermophoresis. Data were fitted using the law of mass action to determine dissociation constants (K D ). Data shown are representative of three independent experiments. Error bars represent standard deviations of n = 3 measurements. All mutants except R101A and D139A are defective in acetyl CoA binding, whereas Y49A, D139A and Y149A are compromised for Fe 2+/3+ binding. ( C ) Active site of Mom. Residues found to be important for the DNA modification activity of Mom (side chains shown) map onto the active site pocket of Mom, with the splayed strands β4 and β5 which form the acetyl CoA-binding cleft colored blue. A Fe 2+/3+ ion is colocalized near the acetyl arm of acetyl CoA within the active site of Mom. The tentative location of the Fe 2+/3+ ion is depicted based on mutagenesis data, with residues critical for Fe 2+/3+ -binding highlighted. ( D ) R101 residue of Mom. Of all the point mutations introduced in the Mom active site, the effect of R101A substitution was the most detrimental to the modification activity, despite having no effect on acetyl CoA- and Fe 2+/3+ -binding. The active site cleft viewed from the acetyl end of acetyl CoA shows the acetyl group emerging into a grove, likely to be the substrate adenine-binding cleft. The residue R101 is positioned at the opening of the cleft and appears poised for substrate recognition.

    Journal: Nucleic Acids Research

    Article Title: Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

    doi: 10.1093/nar/gkaa319

    Figure Lengend Snippet: DNA modification activity and co-factor-interactions of Mom mutants. ( A ) Analysis of in vivo DNA modification activity of Mom mutants. Plasmid DNA was isolated from E. coli C41(DE3) cells expressing wild type or mutant Mom proteins, as indicated. VC denotes vector control and ΔCTD denotes a 175 aa long Mom construct with 56 residues from the C-terminus deleted. M represents 1kb ladder. 1 μg DNA was digested with the restriction endonuclease HgaI and products were analyzed on a 1% agarose ethidium bromide gel. Different mutations exert variable degrees of effects on the activity of Mom, with S114 and R101A compromising Mom activity most severely. Lower panel shows the undigested plasmids corresponding to those in the upper panel. ( B ) Co-factor-binding defects in Mom mutants analyzed using microscale thermophoresis. His-tagged wild-type Mom (WT) and mutants were fluorescently labeled and titrated against indicated concentrations of ligands acetyl CoA (upper panel) and Fe 2+/3+ (lower panel). Normalized fluorescence is plotted for analysis of thermophoresis. Data were fitted using the law of mass action to determine dissociation constants (K D ). Data shown are representative of three independent experiments. Error bars represent standard deviations of n = 3 measurements. All mutants except R101A and D139A are defective in acetyl CoA binding, whereas Y49A, D139A and Y149A are compromised for Fe 2+/3+ binding. ( C ) Active site of Mom. Residues found to be important for the DNA modification activity of Mom (side chains shown) map onto the active site pocket of Mom, with the splayed strands β4 and β5 which form the acetyl CoA-binding cleft colored blue. A Fe 2+/3+ ion is colocalized near the acetyl arm of acetyl CoA within the active site of Mom. The tentative location of the Fe 2+/3+ ion is depicted based on mutagenesis data, with residues critical for Fe 2+/3+ -binding highlighted. ( D ) R101 residue of Mom. Of all the point mutations introduced in the Mom active site, the effect of R101A substitution was the most detrimental to the modification activity, despite having no effect on acetyl CoA- and Fe 2+/3+ -binding. The active site cleft viewed from the acetyl end of acetyl CoA shows the acetyl group emerging into a grove, likely to be the substrate adenine-binding cleft. The residue R101 is positioned at the opening of the cleft and appears poised for substrate recognition.

    Article Snippet: Plasmid DNA was isolated from the cultures using Qiagen miniprep kits and 1 μg DNA was digested with HgaI (2 units/μl, NEB).

    Techniques: Modification, Activity Assay, In Vivo, Plasmid Preparation, Isolation, Expressing, Mutagenesis, Construct, Binding Assay, Microscale Thermophoresis, Labeling, Fluorescence

    DNA modification activity of Mom. ( A ) Structure of the modified base N 6 -methylcarbamoyl adenine. ( B ) Analysis of the in vivo DNA modification activity of Mom using the restriction endonuclease HgaI. Plasmid DNA was isolated from E. coli C41(DE3) cells harbouring the empty vector (vector control) or expressing Mom. DNA modification is depicted by red dots in the schematic. To examine if cellular DNA was modified by Mom, 1 μg plasmid DNA was digested with HgaI and products were analyzed on a 1% agarose ethidium bromide gel. While plasmids from vector control cells get digested into expected sized products, plasmids from mom- expressing cells remain unaffected by HgaI.

    Journal: Nucleic Acids Research

    Article Title: Emergence of a novel immune-evasion strategy from an ancestral protein fold in bacteriophage Mu

    doi: 10.1093/nar/gkaa319

    Figure Lengend Snippet: DNA modification activity of Mom. ( A ) Structure of the modified base N 6 -methylcarbamoyl adenine. ( B ) Analysis of the in vivo DNA modification activity of Mom using the restriction endonuclease HgaI. Plasmid DNA was isolated from E. coli C41(DE3) cells harbouring the empty vector (vector control) or expressing Mom. DNA modification is depicted by red dots in the schematic. To examine if cellular DNA was modified by Mom, 1 μg plasmid DNA was digested with HgaI and products were analyzed on a 1% agarose ethidium bromide gel. While plasmids from vector control cells get digested into expected sized products, plasmids from mom- expressing cells remain unaffected by HgaI.

    Article Snippet: Plasmid DNA was isolated from the cultures using Qiagen miniprep kits and 1 μg DNA was digested with HgaI (2 units/μl, NEB).

    Techniques: Modification, Activity Assay, In Vivo, Plasmid Preparation, Isolation, Expressing

    Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.

    Journal: bioRxiv

    Article Title: Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

    doi: 10.1101/859892

    Figure Lengend Snippet: Methylation fraction (MF) analysed through ddPCR. MF was plotted through RoodCom WebAnalysis (version 1.4.2., Rogier J. Nell, Leiden). MDNA and UDNA are commercially available methylated and unmethylated DNA. A. Calibration curve using different expected ratios (25%, 50% and 75%) of methylated DNA and F332 to demonstrate the quantitative capacity of ddPCR. B. MF of cg11625005 in a subset of healthy primary skin samples – fibroblasts (F332 and F537) and keratinocytes (K060 and K409) and cutaneous melanoma cell lines (A375, 94.07 and 518A2) incubated with MSRE HgaI. C D . Correlation plots between MF obtained through golden standard NGS-based deep bisulfite sequencing versus ddPCR using either the MSRE HgaI ( C. ) or AvaI ( D. ), which digest unmethylated CpG in position 1,295,737 and 1,295,731, respectively.

    Article Snippet: 100ng DNA sample was incubated with HgaI (2U/μl) and appurtenant 10X NEBuffer 1.1 (both from New England Biolabs, Bioké, Leiden, The Netherlands) for 60 minutes at 37°C and 65°C for 20 minutes.

    Techniques: Methylation, Incubation, Next-Generation Sequencing, Methylation Sequencing

    FGFR2 exon IIIc sequence and restriction enzyme digestion A : Pedigree indicating the segregation of the microsatellite D10S1483 located 6.7 kb away from the site of the FGFR2 mutation in the proposita (II-1), showing that the two sibs have inherited opposite maternal FGFR2 alleles. B–F: DNA sequencing chromatograms around the mutation site (p.Asp336 codon corresponding to the GAC boxed sequence) in FGFR2 exon IIIc ( B , C : reverse complementary sequence of the minus strand; D – F : forward sequence of the plus strand); the proposita's blood DNA (B) shows a heterozygous c.1007A > G mutation encoding a p.Asp336Gly substitution; (C) the same change is absent from the father's blood DNA. D–F: Chromatograms from the mother's blood (D), saliva (E) and hair roots (F) genomic DNA, revealing a variable amount of the c.1007A > G mutation in these tissues (arrowheads). G : Hga I restriction digest of a 132 bp PCR product for the different genomic DNA samples (as indicated), shows that the normal allele (c.1007A), is cut into two fragments (65 and 67 bp), while the c.1007A > G mutation abolishes the Hga I restriction site (GACGCN 5 ). Comparison of the ratio of mutant (upper undigested fragment) to normal (lower fragments) alleles in the maternal samples to that of the heterozygous proposita, shows that the relative amount of undigested fragment is reduced in the mother's blood and saliva samples and is barely visible in the hair root sample (right and left lanes are 100 bp ladders). [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]

    Journal: American Journal of Medical Genetics. Part a

    Article Title: Germline and somatic mosaicism for FGFR2 mutation in the mother of a child with Crouzon syndrome: Implications for genetic testing in "paternal age-effect" syndromes

    doi: 10.1002/ajmg.a.33513

    Figure Lengend Snippet: FGFR2 exon IIIc sequence and restriction enzyme digestion A : Pedigree indicating the segregation of the microsatellite D10S1483 located 6.7 kb away from the site of the FGFR2 mutation in the proposita (II-1), showing that the two sibs have inherited opposite maternal FGFR2 alleles. B–F: DNA sequencing chromatograms around the mutation site (p.Asp336 codon corresponding to the GAC boxed sequence) in FGFR2 exon IIIc ( B , C : reverse complementary sequence of the minus strand; D – F : forward sequence of the plus strand); the proposita's blood DNA (B) shows a heterozygous c.1007A > G mutation encoding a p.Asp336Gly substitution; (C) the same change is absent from the father's blood DNA. D–F: Chromatograms from the mother's blood (D), saliva (E) and hair roots (F) genomic DNA, revealing a variable amount of the c.1007A > G mutation in these tissues (arrowheads). G : Hga I restriction digest of a 132 bp PCR product for the different genomic DNA samples (as indicated), shows that the normal allele (c.1007A), is cut into two fragments (65 and 67 bp), while the c.1007A > G mutation abolishes the Hga I restriction site (GACGCN 5 ). Comparison of the ratio of mutant (upper undigested fragment) to normal (lower fragments) alleles in the maternal samples to that of the heterozygous proposita, shows that the relative amount of undigested fragment is reduced in the mother's blood and saliva samples and is barely visible in the hair root sample (right and left lanes are 100 bp ladders). [Color figure can be viewed in the online issue, which is available at http://www.interscience.wiley.com .]

    Article Snippet: Restriction digest with Hga I (New England Biolabs, Ipswich, CA) was performed on 15 µl of the pyrosequencing PCR product (see below) in a 30 µl reaction containing 1× Buffer 1 and 5 U of Hga I for 4 hr at 37°C.

    Techniques: Sequencing, Mutagenesis, DNA Sequencing, Polymerase Chain Reaction