hha i  (New England Biolabs)


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    Structured Review

    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
    Hha I, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Helicobactermarmotae and novel Helicobacter and Campylobacter species isolated from the livers and intestines of prairie dogs"

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

    Journal: Journal of Medical Microbiology

    doi: 10.1099/jmm.0.032144-0

    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
    Figure Legend 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

    Techniques Used: Polymerase Chain Reaction, Produced, Electrophoresis

    2) Product Images from "Differential accessibility at the ? chain locus plays a role in allelic exclusion"

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

    Journal: The EMBO Journal

    doi: 10.1093/emboj/cdf518

    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.
    Figure Legend 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.

    Techniques Used: 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.
    Figure Legend 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.

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

    3) Product Images from "Broad Resistance to ACCase Inhibiting Herbicides in a Ryegrass Population Is Due Only to a Cysteine to Arginine Mutation in the Target Enzyme"

    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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0039759

    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.
    Figure Legend 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.

    Techniques Used: Mutagenesis

    4) Product Images from "Plasmodium vivax msp-3α polymorphisms: analysis in the Indian subcontinent"

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

    Journal: Malaria Journal

    doi: 10.1186/s12936-016-1524-y

    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)
    Figure Legend 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)

    Techniques Used: In Silico

    5) Product Images from "Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece"

    Article Title: Biodiversity and ITS-RFLP Characterisation of Aspergillus Section Nigri Isolates in Grapes from Four Traditional Grape-Producing Areas in Greece

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0093923

    Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus grape isolates. Restriction digestion patterns (designated as A , B and C ) of ribosomal 5.8S-ITS DNA amplicons from various Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I and Hinf I . 50 bp ( l ) and 100 bp (L) DNA ladders are also shown.
    Figure Legend Snippet: Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus grape isolates. Restriction digestion patterns (designated as A , B and C ) of ribosomal 5.8S-ITS DNA amplicons from various Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I and Hinf I . 50 bp ( l ) and 100 bp (L) DNA ladders are also shown.

    Techniques Used:

    Alignment of the ribosomal 5.8S-ITS amplicon sequences of representative Aspergilli isolates. A part of the amplicon sequences (nucleotides 101 to 200) alignment is presented. Restriction sites are presented as bold-underlined, and variable nucleotides are highlighted. The extra Hha I restriction site present in the Ai-1 sequence is also shown.
    Figure Legend Snippet: Alignment of the ribosomal 5.8S-ITS amplicon sequences of representative Aspergilli isolates. A part of the amplicon sequences (nucleotides 101 to 200) alignment is presented. Restriction sites are presented as bold-underlined, and variable nucleotides are highlighted. The extra Hha I restriction site present in the Ai-1 sequence is also shown.

    Techniques Used: Amplification, Sequencing

    Restriction digestion patterns of sequenced ribosomal 5.8S-ITS region amplicons. Restriction digestion patterns (designated as A , B , C and D ) of five sequenced ribosomal 5.8S-ITS DNA amplicons from five different Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I , Hinf I and Rsa I . Each isolate is a representative of the five different Aspergillus species characterized in this study. L : DNA ladder.
    Figure Legend Snippet: Restriction digestion patterns of sequenced ribosomal 5.8S-ITS region amplicons. Restriction digestion patterns (designated as A , B , C and D ) of five sequenced ribosomal 5.8S-ITS DNA amplicons from five different Aspergilli grape isolates (presented as isolate designations), after digestion with the restriction endonucleases Hha I , Hinf I and Rsa I . Each isolate is a representative of the five different Aspergillus species characterized in this study. L : DNA ladder.

    Techniques Used:

    Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus reference strains. Restriction digestion patterns (designated as A and B ) of ribosomal 5.8S-ITS DNA amplicons from Aspergillus reference strains, after digestion with the restriction endonucleases Hinf I , Hha I and Rsa I . Ac : Aspergillus carbonarius , An : Aspergillus niger , At : Aspergillus tubingensis , Aw : Aspergillus westerdijkiae , Ao : Aspergillus ochraceus , L : Low molecular weight DNA ladder (molecular sizes are 766, 500, 350, 300, 250, 200, 150, 100, 75, 50 and 25 bp respectively).
    Figure Legend Snippet: Ribosomal 5.8S-ITS region restriction digestion patterns of Aspergillus reference strains. Restriction digestion patterns (designated as A and B ) of ribosomal 5.8S-ITS DNA amplicons from Aspergillus reference strains, after digestion with the restriction endonucleases Hinf I , Hha I and Rsa I . Ac : Aspergillus carbonarius , An : Aspergillus niger , At : Aspergillus tubingensis , Aw : Aspergillus westerdijkiae , Ao : Aspergillus ochraceus , L : Low molecular weight DNA ladder (molecular sizes are 766, 500, 350, 300, 250, 200, 150, 100, 75, 50 and 25 bp respectively).

    Techniques Used: Molecular Weight

    6) Product Images from "Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma"

    Article Title: Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma

    Journal: Journal of Medical Microbiology

    doi: 10.1099/jmm.0.072462-0

    RFLP patterns of  Helicobacter  sp. monkey taxon 4,  H. macacae  and  Helicobacter  sp. monkey taxon 2 generated by  Alu I and  Hha I.
    Figure Legend Snippet: RFLP patterns of Helicobacter sp. monkey taxon 4, H. macacae and Helicobacter sp. monkey taxon 2 generated by Alu I and Hha I.

    Techniques Used: Generated

    7) Product Images from "Modulation of DNA Binding Protein Affinity Directly Affects Target Site Demethylation"

    Article Title: Modulation of DNA Binding Protein Affinity Directly Affects Target Site Demethylation

    Journal: Molecular and Cellular Biology

    doi:

    Modulation of demethylation by the IPTG concentration. The 293/ElacI cells were treated with a final IPTG concentration of 5 mM for 4 h before transfection with Hha I-methylated pOLucOriP. The transfected cells were divided among several plates and treated with lower concentrations of IPTG. The fraction of plasmids becoming demethylated in the lacO sites was analyzed as described previously. A linear and inversely correlated relationship between the IPTG concentration and demethylation of lacO was observed.
    Figure Legend Snippet: Modulation of demethylation by the IPTG concentration. The 293/ElacI cells were treated with a final IPTG concentration of 5 mM for 4 h before transfection with Hha I-methylated pOLucOriP. The transfected cells were divided among several plates and treated with lower concentrations of IPTG. The fraction of plasmids becoming demethylated in the lacO sites was analyzed as described previously. A linear and inversely correlated relationship between the IPTG concentration and demethylation of lacO was observed.

    Techniques Used: Concentration Assay, Transfection, Methylation

    Demethylation of lacO sites. (A) Illustration of the SV40 intron harboring the three lacO sites and the sizes of Hin dIII and Hha I restriction fragments in the region. (B) Southern blot of Hin dIII- Hha I-double-digested plasmid DNA harvested 15 days after transfection into cell lines with (+ lacI , 293/ElacI) and without (− lacI , 293/EBNA1) lacI expression. The unmethylated plasmid can be digested to completion by Hin dIII and Hha I; therefore, a 338-bp band is detected. The Hha I- or Sss I-methylated plasmids remain methylated at the three Hha I sites within lacO when LacI is absent, so that a 467-bp band is detected. In contrast, these Hha I sites are demethylated when LacI is present, and they are digestable by Hha I to give rise to the 338-bp band. (C) Lack of demethylation in the vector backbone. There is no detectable difference in the Hha I-digested vector backbone with or without lacI .
    Figure Legend Snippet: Demethylation of lacO sites. (A) Illustration of the SV40 intron harboring the three lacO sites and the sizes of Hin dIII and Hha I restriction fragments in the region. (B) Southern blot of Hin dIII- Hha I-double-digested plasmid DNA harvested 15 days after transfection into cell lines with (+ lacI , 293/ElacI) and without (− lacI , 293/EBNA1) lacI expression. The unmethylated plasmid can be digested to completion by Hin dIII and Hha I; therefore, a 338-bp band is detected. The Hha I- or Sss I-methylated plasmids remain methylated at the three Hha I sites within lacO when LacI is absent, so that a 467-bp band is detected. In contrast, these Hha I sites are demethylated when LacI is present, and they are digestable by Hha I to give rise to the 338-bp band. (C) Lack of demethylation in the vector backbone. There is no detectable difference in the Hha I-digested vector backbone with or without lacI .

    Techniques Used: Southern Blot, Plasmid Preparation, Transfection, Expressing, Methylation

    The presence of a high concentration of IPTG can prevent lacO demethylation. The 293/ElacI cells were treated with a final IPTG concentration of 5 mM beginning 4 h before transfection. The cells were divided evenly between two plates 3 days after transfection. The cells in one plate were treated with 10 μM IPTG, and the cells in the other plate were not treated with IPTG. Plasmid DNA was harvested for probing multiple times during a 19-day interval for Southern blot analysis using the 467-bp Hin dIII fragment as a probe. When IPTG was present, the Hha I-methylated plasmid remained methylated at the Hha I sites within the lacO sequence at 19 days after transfection (lanes 1 and 2). In contrast, after IPTG was withdrawn from the tissue culture medium 15 days after transfection, demethylation within the lacO sites could be detected 4 days later (lanes 3 and 4).
    Figure Legend Snippet: The presence of a high concentration of IPTG can prevent lacO demethylation. The 293/ElacI cells were treated with a final IPTG concentration of 5 mM beginning 4 h before transfection. The cells were divided evenly between two plates 3 days after transfection. The cells in one plate were treated with 10 μM IPTG, and the cells in the other plate were not treated with IPTG. Plasmid DNA was harvested for probing multiple times during a 19-day interval for Southern blot analysis using the 467-bp Hin dIII fragment as a probe. When IPTG was present, the Hha I-methylated plasmid remained methylated at the Hha I sites within the lacO sequence at 19 days after transfection (lanes 1 and 2). In contrast, after IPTG was withdrawn from the tissue culture medium 15 days after transfection, demethylation within the lacO sites could be detected 4 days later (lanes 3 and 4).

    Techniques Used: Concentration Assay, Transfection, Plasmid Preparation, Southern Blot, Methylation, Sequencing

    8) Product Images from "Analysis of Polymorphisms in the Merozoite Surface Protein-3? Gene and Two Microsatellite Loci in Sri Lankan Plasmodium vivax: Evidence of Population Substructure in Sri Lanka"

    Article Title: Analysis of Polymorphisms in the Merozoite Surface Protein-3? Gene and Two Microsatellite Loci in Sri Lankan Plasmodium vivax: Evidence of Population Substructure in Sri Lanka

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.2011.11-0338

    Restriction fragment length polymorphism (RFLP) with restriction enzyme Hha I of selected pvmsp -3α alleles in eight different Plasmodium vivax samples from Sri Lanka. With exclusion of bands above 600 bp, and below 150 bp, the estimated allele
    Figure Legend Snippet: Restriction fragment length polymorphism (RFLP) with restriction enzyme Hha I of selected pvmsp -3α alleles in eight different Plasmodium vivax samples from Sri Lanka. With exclusion of bands above 600 bp, and below 150 bp, the estimated allele

    Techniques Used:

    9) Product Images from "Analysis of C43G mutation in the promoter region of the XIST gene in patients with idiopathic primary ovarian insufficiency"

    Article Title: Analysis of C43G mutation in the promoter region of the XIST gene in patients with idiopathic primary ovarian insufficiency

    Journal: Clinical and Experimental Reproductive Medicine

    doi: 10.5653/cerm.2015.42.2.58

    C43G variant site and the Hha I recognition site in the promoter region of the XIST gene.
    Figure Legend Snippet: C43G variant site and the Hha I recognition site in the promoter region of the XIST gene.

    Techniques Used: Variant Assay

    10) Product Images from "Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain"

    Article Title: Hybrid mouse-prokaryotic DNA (cytosine-5) methyltransferases retain the specificity of the parental C-terminal domain

    Journal: The EMBO Journal

    doi: 10.1093/emboj/19.9.2103

    Fig. 4. De novo methylation catalyzed by the hybrid MTases. ( A ) Total DNA from High Five cells expressing Dnmt1– Hha I, Dnmt1– Hpa II, Dnmt1– Sss I or Dnmt1 was extracted 48 h post-infection. Each lane represents DNA digested with the restriction enzymes indicated (+) above each lane. Lane M contains a Hin dIII digest of λ DNA molecular weight marker. ( B ) De novo methylation of the hybrid MTase genes. Southern blots of total DNA probed with the Bam HI 2100 bp N-terminal fragment of the mouse Dnmt1 cDNA. Each lane represents either a single or double restriction digest as indicated (+). The 2100 bp band is indicated by an arrow. ( C ) De novo methylation of viral upstream and polyhedrin promoter sequences. Southern blots of total DNA probed with a 2200 bp Nsi I fragment consisting of the viral upstream sequences, polyhedrin promoter and a part of the N-terminus of the mouse Dnmt1. Each lane represents either a single or double restriction digest as indicated (+). The 2200 bp band is shown by an arrow. ( D ) Key features of the hybrid MTase and conceptual restriction maps of the Bam HI (2100 bp) and Nsi I (2200 bp) fragments. Features of the hybrid MTase are presented schematically. A 3400 bp cDNA containing the mouse Dnmt1, indicated by black shading, was fused to the N-terminus of the prokaryotic MTase, as shown in grey. The hatched line represents the upstream baculovirus ORF. pPH marks the polyhedrin promoter. Fragments analyzed by restriction enzyme digests [ Bam HI (∼2100 bp) and Nsi I (∼2200 bp)] are indicated by B and N. Restriction enzymes used for methylation analysis are listed on the left. The numbers of target sites and sequences are on the right. Restriction sites are indicated by vertical black lines.
    Figure Legend Snippet: Fig. 4. De novo methylation catalyzed by the hybrid MTases. ( A ) Total DNA from High Five cells expressing Dnmt1– Hha I, Dnmt1– Hpa II, Dnmt1– Sss I or Dnmt1 was extracted 48 h post-infection. Each lane represents DNA digested with the restriction enzymes indicated (+) above each lane. Lane M contains a Hin dIII digest of λ DNA molecular weight marker. ( B ) De novo methylation of the hybrid MTase genes. Southern blots of total DNA probed with the Bam HI 2100 bp N-terminal fragment of the mouse Dnmt1 cDNA. Each lane represents either a single or double restriction digest as indicated (+). The 2100 bp band is indicated by an arrow. ( C ) De novo methylation of viral upstream and polyhedrin promoter sequences. Southern blots of total DNA probed with a 2200 bp Nsi I fragment consisting of the viral upstream sequences, polyhedrin promoter and a part of the N-terminus of the mouse Dnmt1. Each lane represents either a single or double restriction digest as indicated (+). The 2200 bp band is shown by an arrow. ( D ) Key features of the hybrid MTase and conceptual restriction maps of the Bam HI (2100 bp) and Nsi I (2200 bp) fragments. Features of the hybrid MTase are presented schematically. A 3400 bp cDNA containing the mouse Dnmt1, indicated by black shading, was fused to the N-terminus of the prokaryotic MTase, as shown in grey. The hatched line represents the upstream baculovirus ORF. pPH marks the polyhedrin promoter. Fragments analyzed by restriction enzyme digests [ Bam HI (∼2100 bp) and Nsi I (∼2200 bp)] are indicated by B and N. Restriction enzymes used for methylation analysis are listed on the left. The numbers of target sites and sequences are on the right. Restriction sites are indicated by vertical black lines.

    Techniques Used: Methylation, Expressing, Infection, Molecular Weight, Marker

    Fig. 6. Time course of methylation catalyzed by the hybrid Dnmt1– Hha I. ( A ) Reactions contained 30 nM hybrid Dnmt1– Hha I, 50 µM CG [poly (dG-dC)·poly (dG-dC)] and 1, 5 or 10 µM AdoMet and were incubated at 37°C. Duplicate 25 µl samples of each reaction were spotted onto DE81 paper, processed as described in Materials and methods, and 3 H incorporation was measured. The mean values, with standard deviation, for the time points are plotted as filled diamonds (1 µM AdoMet), open circles (5 µM AdoMet) or filled circles (10 µM AdoMet). ( B ) Linearity of methyltransfer reaction as a function of enzyme concentration. Duplicate reactions in 25 µl contained 50 µM CG, 5 µM AdoMet and various mouse Dnmt1– Hha I concentrations (5, 10, 20, 30, 40 and 50 µM) and were incubated at 37°C for 30 min and processed. The solid circles depict the mean values of the [ 3 H]methyl group incorporation.
    Figure Legend Snippet: Fig. 6. Time course of methylation catalyzed by the hybrid Dnmt1– Hha I. ( A ) Reactions contained 30 nM hybrid Dnmt1– Hha I, 50 µM CG [poly (dG-dC)·poly (dG-dC)] and 1, 5 or 10 µM AdoMet and were incubated at 37°C. Duplicate 25 µl samples of each reaction were spotted onto DE81 paper, processed as described in Materials and methods, and 3 H incorporation was measured. The mean values, with standard deviation, for the time points are plotted as filled diamonds (1 µM AdoMet), open circles (5 µM AdoMet) or filled circles (10 µM AdoMet). ( B ) Linearity of methyltransfer reaction as a function of enzyme concentration. Duplicate reactions in 25 µl contained 50 µM CG, 5 µM AdoMet and various mouse Dnmt1– Hha I concentrations (5, 10, 20, 30, 40 and 50 µM) and were incubated at 37°C for 30 min and processed. The solid circles depict the mean values of the [ 3 H]methyl group incorporation.

    Techniques Used: Methylation, Incubation, Standard Deviation, Concentration Assay

    Fig. 2. Expression and purification of hybrid MTases. ( A ) Western blot analysis of High Five cell extracts expressing the hybrid methyltransferases using a mouse Dnmt1 specific Ab 334. The relative positions of biotinylated mol. wt markers (165, 105, 76) are in kDa. His-MMT3 is a His 6 ). An uninfected High Five cell extract was used as a control (C). The MTase bands are indicated by the arrow. The hybrid proteins are indicated above each lane. ( B ) Two purified hybrid MTases, Dnmt1– Hpa II and Dnmt1– Hha I resolved on SDS–PAGE and stained with Coomassie Blue. The broad range molecular weight markers are indicated on the left in kDa. The hybrid MTases are indicated by the arrow.
    Figure Legend Snippet: Fig. 2. Expression and purification of hybrid MTases. ( A ) Western blot analysis of High Five cell extracts expressing the hybrid methyltransferases using a mouse Dnmt1 specific Ab 334. The relative positions of biotinylated mol. wt markers (165, 105, 76) are in kDa. His-MMT3 is a His 6 ). An uninfected High Five cell extract was used as a control (C). The MTase bands are indicated by the arrow. The hybrid proteins are indicated above each lane. ( B ) Two purified hybrid MTases, Dnmt1– Hpa II and Dnmt1– Hha I resolved on SDS–PAGE and stained with Coomassie Blue. The broad range molecular weight markers are indicated on the left in kDa. The hybrid MTases are indicated by the arrow.

    Techniques Used: Expressing, Purification, Western Blot, SDS Page, Staining, Molecular Weight

    11) Product Images from "Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro"

    Article Title: Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.22.7919-7928.2002

    PRC1 efficiently represses templates challenged with Hha I. 5S array templates were cut with Hha I before incubation with PRC1 and then transcribed with T7 RNA polymerase. Templates that were nucleosomal at the Hha I site adjacent to the T7 start site were protected from digestion, while any residual naked templates were digested. Since transcription was initiated more often from templates in which the T7 start site was unprotected, digesting the template with Hha I resulted in a ninefold drop in transcription. Primer extension with the primer used for these reactions gave two products of approximately the expected size. The primer may have recognized two adjacent sequences. Quantitation of transcription products is relative to −PRC1 lane for each set of reactions.
    Figure Legend Snippet: PRC1 efficiently represses templates challenged with Hha I. 5S array templates were cut with Hha I before incubation with PRC1 and then transcribed with T7 RNA polymerase. Templates that were nucleosomal at the Hha I site adjacent to the T7 start site were protected from digestion, while any residual naked templates were digested. Since transcription was initiated more often from templates in which the T7 start site was unprotected, digesting the template with Hha I resulted in a ninefold drop in transcription. Primer extension with the primer used for these reactions gave two products of approximately the expected size. The primer may have recognized two adjacent sequences. Quantitation of transcription products is relative to −PRC1 lane for each set of reactions.

    Techniques Used: Incubation, Quantitation Assay

    12) Product Images from "Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma"

    Article Title: Pathogenic properties of enterohepatic Helicobacter spp. isolated from rhesus macaques with intestinal adenocarcinoma

    Journal: Journal of Medical Microbiology

    doi: 10.1099/jmm.0.072462-0

    RFLP patterns of Helicobacter sp. monkey taxon 4, H. macacae and Helicobacter sp. monkey taxon 2 generated by Alu I and Hha I.
    Figure Legend Snippet: RFLP patterns of Helicobacter sp. monkey taxon 4, H. macacae and Helicobacter sp. monkey taxon 2 generated by Alu I and Hha I.

    Techniques Used: Generated

    13) Product Images from "Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology of Bartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California"

    Article Title: Coyotes (Canis latrans) as the Reservoir for a Human Pathogenic Bartonella sp.: Molecular Epidemiology of Bartonella vinsonii subsp. berkhoffii Infection in Coyotes from Central Coastal California

    Journal: Journal of Clinical Microbiology

    doi:

    PCR (lanes 2 to 5) and PCR-RFLP (lanes 6 to 9, Taq I digestion; lanes 10 to 13, Hha I digestion) analyses of the gltA ). Lanes 1 and 14, standard 100-bp molecular ladder; lanes 2, 6, and 10, coyote isolates; lanes 3, 7, and 11, B. vinsonii subsp. berkhoffii ATCC 51672; lanes 4, 8, and 12, B. vinsonii ATCC VR152; lanes 5, 9 and 13, B. henselae (strain U-4, University of California, Davis).
    Figure Legend Snippet: PCR (lanes 2 to 5) and PCR-RFLP (lanes 6 to 9, Taq I digestion; lanes 10 to 13, Hha I digestion) analyses of the gltA ). Lanes 1 and 14, standard 100-bp molecular ladder; lanes 2, 6, and 10, coyote isolates; lanes 3, 7, and 11, B. vinsonii subsp. berkhoffii ATCC 51672; lanes 4, 8, and 12, B. vinsonii ATCC VR152; lanes 5, 9 and 13, B. henselae (strain U-4, University of California, Davis).

    Techniques Used: Polymerase Chain Reaction

    14) Product Images from "Neutrophilic Fe-Oxidizing Bacteria Are Abundant at the Loihi Seamount Hydrothermal Vents and Play a Major Role in Fe Oxide Deposition"

    Article Title: Neutrophilic Fe-Oxidizing Bacteria Are Abundant at the Loihi Seamount Hydrothermal Vents and Play a Major Role in Fe Oxide Deposition

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.6.3085-3093.2002

    T-RFLP profile of isolates JV-1 and PV-1 along with four hydrothermal vent bacterial communities sampled from Loihi Seamount with the Pisces V submersible. Terminal fragments were generated from SSU rDNA amplicons digested with Hha I. (A) Isolate JV-1; (B) isolate PV-1; (C) Pit of Death site, dive 244, September 1993; (D) Pele's vents “Boulder Patch” site, dive 247, September 1993; (E) Lohiau vents site, dive 311, October 1996; (F) Ikaika vents lower jets site, dive 393, October 1998. Terminal fragments of 332 bp are congruous with the JV-1-PV-1 phylotype and are prevalent in all of the environmental samples, suggesting that the presence of this Fe-oxidizing phylotype is common throughout this hydrothermal system.
    Figure Legend Snippet: T-RFLP profile of isolates JV-1 and PV-1 along with four hydrothermal vent bacterial communities sampled from Loihi Seamount with the Pisces V submersible. Terminal fragments were generated from SSU rDNA amplicons digested with Hha I. (A) Isolate JV-1; (B) isolate PV-1; (C) Pit of Death site, dive 244, September 1993; (D) Pele's vents “Boulder Patch” site, dive 247, September 1993; (E) Lohiau vents site, dive 311, October 1996; (F) Ikaika vents lower jets site, dive 393, October 1998. Terminal fragments of 332 bp are congruous with the JV-1-PV-1 phylotype and are prevalent in all of the environmental samples, suggesting that the presence of this Fe-oxidizing phylotype is common throughout this hydrothermal system.

    Techniques Used: Generated, Environmental Sampling

    15) Product Images from "Identification of Novel Helicobacter spp. from a Beluga Whale †"

    Article Title: Identification of Novel Helicobacter spp. from a Beluga Whale †

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.4.2040-2043.2002

    PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from beluga gastric fluid and feces. Lane MW, molecular weight standards; lane 1, MIT 00-7128 PCR product from beluga fecal isolate digested by Hha I; lane 2, MIT 00-7129 PCR product from beluga gastric fluid digested by Hha I; lane 3, MIT 00-7128 PCR product from beluga fecal isolate digested by Alu I; lane 4, MIT 00-7129 PCR product from beluga gastric fluid digested by Alu I.
    Figure Legend Snippet: PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from beluga gastric fluid and feces. Lane MW, molecular weight standards; lane 1, MIT 00-7128 PCR product from beluga fecal isolate digested by Hha I; lane 2, MIT 00-7129 PCR product from beluga gastric fluid digested by Hha I; lane 3, MIT 00-7128 PCR product from beluga fecal isolate digested by Alu I; lane 4, MIT 00-7129 PCR product from beluga gastric fluid digested by Alu I.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight

    16) Product Images from "Genome Sequence-Based Fluorescent Amplified Fragment Length Polymorphism of Campylobacter jejuni, Its Relationship to Serotyping, and Its Implications for Epidemiological Analysis"

    Article Title: Genome Sequence-Based Fluorescent Amplified Fragment Length Polymorphism of Campylobacter jejuni, Its Relationship to Serotyping, and Its Implications for Epidemiological Analysis

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.11.3823-3829.2001

    FAFLP genome map of C. jejuni strain NCTC 11168. Relative positions and predicted sizes (in base pairs) of AFs in the C. jejuni ) modeled for the primer pair Hin dIII+A and Hha I+A and experimentally validated by FAFLP analysis. The genes that AFs represent are indicated on the outer circle; AFs in the inner circle represent putative genes.
    Figure Legend Snippet: FAFLP genome map of C. jejuni strain NCTC 11168. Relative positions and predicted sizes (in base pairs) of AFs in the C. jejuni ) modeled for the primer pair Hin dIII+A and Hha I+A and experimentally validated by FAFLP analysis. The genes that AFs represent are indicated on the outer circle; AFs in the inner circle represent putative genes.

    Techniques Used:

    17) Product Images from "Helicobacter cetorum sp. nov., a Urease-Positive Helicobacter Species Isolated from Dolphins and Whales"

    Article Title: Helicobacter cetorum sp. nov., a Urease-Positive Helicobacter Species Isolated from Dolphins and Whales

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.12.4536-4543.2002

    PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from cultures. Lanes 1 through 5 show results for DNA digested by the restriction enzymes Hha I and Alu I as indicated. Lanes 1 and 2, MIT 99-5656 and MIT 99-5657 DNA from Atlantic white-sided dolphin stomach tissues; lanes 3 to 5, MIT 00-7128 (beluga whale), MIT 01-5903 (Pacific white-sided dolphin), and MIT 01-6201 (Atlantic bottlenose dolphin) DNA, respectively, obtained from fecal isolates.
    Figure Legend Snippet: PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from cultures. Lanes 1 through 5 show results for DNA digested by the restriction enzymes Hha I and Alu I as indicated. Lanes 1 and 2, MIT 99-5656 and MIT 99-5657 DNA from Atlantic white-sided dolphin stomach tissues; lanes 3 to 5, MIT 00-7128 (beluga whale), MIT 01-5903 (Pacific white-sided dolphin), and MIT 01-6201 (Atlantic bottlenose dolphin) DNA, respectively, obtained from fecal isolates.

    Techniques Used: Polymerase Chain Reaction

    18) Product Images from "Active Repression of Methylated Genes by the Chromosomal Protein MBD1"

    Article Title: Active Repression of Methylated Genes by the Chromosomal Protein MBD1

    Journal: Molecular and Cellular Biology

    doi:

    Repression of a methylated reporter gene by MBD1 depends on the TRD and methyl-CpG binding domains and is sensitive to TSA. (A) Mouse L929 cells were transfected with an SV40-luciferase reporter (2 μg) that was nonmethylated (M−) or methylated at 25 Hha I (GCGC) sites (M+). Levels of M+ and M− reporter expression (luciferase activity) were normalised to the expression level of cotransfected CMV β-galactosidase control reporter (1 μg). Transcription levels were expressed as the ratio of M+ to M− normalised expression levels. The low density of methylation in the pGL2 control reporter had a negligible effect on transcription in the absence of cotransfected MBD1 but caused repression relative to the nonmethylated reporter in the presence of 0.5 μg of intact MBD1 fused to a Myc epitope tag (5MT-MBD1). N-terminal or C-terminal deletions prevented repression. (B) Diagram of the pGL2 SV40-luciferase reporter, showing the locations of methylated Hha I sites. (C) Diagram of the 5MT-MBD1 proteins used to obtain data shown in panel A. (D) Western blots of proteins extracted from cells transfected by each of the 5MT-MBD1 constructs showing equivalent expression. The blots were probed with anti-Myc monoclonal antibody 9e10. (E) TSA (100 ng/ml) overcomes repression by MBD1 of a methylated reporter gene. The results shown are based on three independent transfections.
    Figure Legend Snippet: Repression of a methylated reporter gene by MBD1 depends on the TRD and methyl-CpG binding domains and is sensitive to TSA. (A) Mouse L929 cells were transfected with an SV40-luciferase reporter (2 μg) that was nonmethylated (M−) or methylated at 25 Hha I (GCGC) sites (M+). Levels of M+ and M− reporter expression (luciferase activity) were normalised to the expression level of cotransfected CMV β-galactosidase control reporter (1 μg). Transcription levels were expressed as the ratio of M+ to M− normalised expression levels. The low density of methylation in the pGL2 control reporter had a negligible effect on transcription in the absence of cotransfected MBD1 but caused repression relative to the nonmethylated reporter in the presence of 0.5 μg of intact MBD1 fused to a Myc epitope tag (5MT-MBD1). N-terminal or C-terminal deletions prevented repression. (B) Diagram of the pGL2 SV40-luciferase reporter, showing the locations of methylated Hha I sites. (C) Diagram of the 5MT-MBD1 proteins used to obtain data shown in panel A. (D) Western blots of proteins extracted from cells transfected by each of the 5MT-MBD1 constructs showing equivalent expression. The blots were probed with anti-Myc monoclonal antibody 9e10. (E) TSA (100 ng/ml) overcomes repression by MBD1 of a methylated reporter gene. The results shown are based on three independent transfections.

    Techniques Used: Methylation, Binding Assay, Transfection, Luciferase, Expressing, Activity Assay, Western Blot, Construct

    19) Product Images from "Isolation and Characterization of a Helicobacter sp. from the Gastric Mucosa of Dolphins, Lagenorhynchus acutus and Delphinus delphis"

    Article Title: Isolation and Characterization of a Helicobacter sp. from the Gastric Mucosa of Dolphins, Lagenorhynchus acutus and Delphinus delphis

    Journal: Applied and Environmental Microbiology

    doi:

    PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from the dolphin isolates and from stomach tissue. Lanes 1 and 2, 99-5660 and 99-5659 DNA from dolphin stomach tissue digested by Hha I; lanes 3 to 5, 99-5656, 99-5657, and 99-5665 DNA from dolphin isolates digested by Hha I; lanes 6 and 7, 99-5660 and 99-5659 DNA from dolphin stomach tissue digested by Alu I; lanes 8 to 10, 99-5656, 99-5657, and 99-5665 DNA from dolphin isolates digested by Alu I. Two patterns were observed in the Hha I digests, and two patterns were observed in the Alu I digests.
    Figure Legend Snippet: PCR-RFLP patterns of the 1,200-bp species-specific Helicobacter PCR product from the dolphin isolates and from stomach tissue. Lanes 1 and 2, 99-5660 and 99-5659 DNA from dolphin stomach tissue digested by Hha I; lanes 3 to 5, 99-5656, 99-5657, and 99-5665 DNA from dolphin isolates digested by Hha I; lanes 6 and 7, 99-5660 and 99-5659 DNA from dolphin stomach tissue digested by Alu I; lanes 8 to 10, 99-5656, 99-5657, and 99-5665 DNA from dolphin isolates digested by Alu I. Two patterns were observed in the Hha I digests, and two patterns were observed in the Alu I digests.

    Techniques Used: Polymerase Chain Reaction

    20) Product Images from "Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum"

    Article Title: Molecular Typing of Australian Scedosporium Isolates Showing Genetic Variability and Numerous S. aurantiacum

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid1402.070920

    Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).
    Figure Legend Snippet: Internal transcribed spacer–restriction fragment length polymorphism (ITS-RFLP) patterns obtained by double digestion with the enzymes Sau 96I and Hha I (A) and of the PCR fingerprinting profiles obtained with the microsatellite specific primer M13 (B) for Scedosporium prolificans : lane 1, WM 06.457; lane 2, WM 06.458; lane 3, WM 06.503; lane 4, WM 06.502; lane 5, WM 06.399; lane 6, WM 06.434. S. aurantiacum : lane 7, WM 06.495; lane 8, WM 06.496; lane 9, WM 06.386; lane 10, WM 06.385; lane 11, WM 06.482; lane 12, WM 06.390. S. apiospermum : lane 13, WM 06.475; lane 14, WM 06.474; lane 15, WM 06.472; lane 16, WM 06.471; lane 17, WM 06.424; lane 18, WM 06.443; lane M, 1-kb marker (GIBCO-BRL, Gaithersburg, MD, USA).

    Techniques Used: Polymerase Chain Reaction, Marker

    21) Product Images from "The molecular basis for stability of heterochromatin-mediated silencing in mammals"

    Article Title: The molecular basis for stability of heterochromatin-mediated silencing in mammals

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-2-14

    The DNA methylation status of the hCD2 transgene in hCD2+ and hCD2- T cells from the CD2 1.3 transgenic lines . (A) Schematic transgene map showing the restriction sites for Bgl II ( B ) and Hha I ( H ) and location of the probe used in B. (B) The CpG methylation analysis of the proximal promoter region by methylation-sensitive restriction enzyme digest and Southern blot. The bar charts show the ratios between the unmethylated and methylated Hha I sites. (C) Bisulfite sequencing analysis of the 3' regulatory regions of the hCD2 transgene. Methylated and unmethylated CpGs are shown as filled and open circles, respectively. Each line represents the sequence from a single clone. Similar results were obtained from two independent experiments. Comparisons between numbers of methylated CpGs were done using the Mann-Whitney U-test and Fisher's exact test (see text).
    Figure Legend Snippet: The DNA methylation status of the hCD2 transgene in hCD2+ and hCD2- T cells from the CD2 1.3 transgenic lines . (A) Schematic transgene map showing the restriction sites for Bgl II ( B ) and Hha I ( H ) and location of the probe used in B. (B) The CpG methylation analysis of the proximal promoter region by methylation-sensitive restriction enzyme digest and Southern blot. The bar charts show the ratios between the unmethylated and methylated Hha I sites. (C) Bisulfite sequencing analysis of the 3' regulatory regions of the hCD2 transgene. Methylated and unmethylated CpGs are shown as filled and open circles, respectively. Each line represents the sequence from a single clone. Similar results were obtained from two independent experiments. Comparisons between numbers of methylated CpGs were done using the Mann-Whitney U-test and Fisher's exact test (see text).

    Techniques Used: DNA Methylation Assay, Transgenic Assay, CpG Methylation Assay, Methylation, Southern Blot, Methylation Sequencing, Sequencing, MANN-WHITNEY

    22) Product Images from "C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD"

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-014-1286-y

    C9orf72 promoter demethylation promotes toxic RNA accumulation. a Hha I resistance shown as mean + SE ( n = 5) from control ( left ) or expanded ( right ) lymphoblast cells treated with 5-aza-dC ( open ) or untreated ( filled ). Two-way ANOVA: genotype p
    Figure Legend Snippet: C9orf72 promoter demethylation promotes toxic RNA accumulation. a Hha I resistance shown as mean + SE ( n = 5) from control ( left ) or expanded ( right ) lymphoblast cells treated with 5-aza-dC ( open ) or untreated ( filled ). Two-way ANOVA: genotype p

    Techniques Used:

    23) Product Images from "Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans"

    Article Title: Identification, Characterization, and Distribution of a Shiga Toxin 1 Gene Variant (stx1c) in Escherichia coli Strains Isolated from Humans

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.4.1441-1446.2002

    Agarose gel electrophoresis of KS7-KS8 PCR products digested with Fsp I (A) or Hha I (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 ( stx 1c + stx 2d ; ONT:H8); 2, 3115/97 ( stx 1c + stx 2d ; O128:H2); 3, 521/99 ( stx 1c + stx 2d ; O rough :H19); 4, 4756/98 ( stx 1c + stx 2d ; O70:H − ); 5, 273/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 6, 295/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 7, EDL 933 ( stx 1 + stx 2 ; O157:H7); 8, 2544/00 ( stx 1 ; O145:H − ); 9, 3385/00 ( stx 1 + stx 2 ; O111:H − ); 10, 4424/99 ( stx 1 + stx 2 ; O157:H7); 11, 2049/98 ( stx 1 + stx 2c ; O157:H − ); 12, 2050/98 ( stx 1 + stx 2 + stx 2c ; O157:H − ).
    Figure Legend Snippet: Agarose gel electrophoresis of KS7-KS8 PCR products digested with Fsp I (A) or Hha I (B) and of PCR amplification products with primers Stx1c-1 and Stx1c-2 (C). M, molecular weight marker (1-kb DNA ladder; Gibco BRL, Eggenstein, Germany). In lanes 1 to 12, the following STEC strains (genotypes, serotypes, and origins, if not human, in parentheses) are shown: 1, 808/97 ( stx 1c + stx 2d ; ONT:H8); 2, 3115/97 ( stx 1c + stx 2d ; O128:H2); 3, 521/99 ( stx 1c + stx 2d ; O rough :H19); 4, 4756/98 ( stx 1c + stx 2d ; O70:H − ); 5, 273/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 6, 295/00 ( stx 1c + stx 2d ; O128:H − ; sheep); 7, EDL 933 ( stx 1 + stx 2 ; O157:H7); 8, 2544/00 ( stx 1 ; O145:H − ); 9, 3385/00 ( stx 1 + stx 2 ; O111:H − ); 10, 4424/99 ( stx 1 + stx 2 ; O157:H7); 11, 2049/98 ( stx 1 + stx 2c ; O157:H − ); 12, 2050/98 ( stx 1 + stx 2 + stx 2c ; O157:H − ).

    Techniques Used: Agarose Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Molecular Weight, Marker

    24) Product Images from "Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5"

    Article Title: Skin Characteristics in Patients with Pityriasis Versicolor Using Non-Invasive Method, MPA5

    Journal: Annals of Dermatology

    doi: 10.5021/ad.2012.24.4.444

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hyperpigmented lesions of 21 patients. Lanes: M: molecular Marker, 1: Malassezia globosa (CBS7966), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. globosa (CBS7966), 5: M. restricta (KCTC7848), 6: M. restricta (KCTC7848), 7: M. restricta (KCTC7848), 8: M. slooffiae (KCTC17431), 9: M. globosa (CBS7966), 10: M. globosa (CBS7966), 11: M. restricta (KCTC7848), 12: M. globosa (CBS7966), 13: M. globosa (CBS7966), 14: M. furfur (KCTC7743), 15: M. slooffiae (KCTC17431), 16: M. globosa (CBS7966), 17: M. globosa (CBS7966), 18: M. restricta (KCTC7848), 19: M. restricta (KCTC7848), 20: M. furfur (KCTC7743), 21: M. sympodialis (KCTC7985). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.
    Figure Legend Snippet: PCR-RFLP patterns of 26S rDNA PCR digested with restriction enzymes (A) Hha I, (B) BtsC I of 11 Malassezia standard strains in hypopigmented lesions of 9 patients. Lanes: M: molecular Marker , 1: Malassezia slooffiae (KCTC17431), 2: M. globosa (CBS7966), 3: M. restricta (KCTC7848), 4: M. restricta (KCTC7848), 5: M. globosa (CBS7966), 6: M. globosa (CBS7966), 7: M. globosa (CBS7966), 8: M. sympodialis (KCTC7985), 9: M. furfur (KCTC7743). PCR: polymerase chain reaction, RFLP: restriction fragment length polymorphism.

    Techniques Used: Polymerase Chain Reaction, Marker

    25) Product Images from "The intronic G13964C variant in p53 is not a high-risk mutation in familial breast cancer in Australia"

    Article Title: The intronic G13964C variant in p53 is not a high-risk mutation in familial breast cancer in Australia

    Journal: Breast Cancer Research : BCR

    doi:

    Detection of the G13964C variant by PCR-RFLP assay. The C→G nucleotide substitution creates a Hha I site, and the G allele is detected by Hha I digestion of the 131-bp product to 98-bp and 33-bp products (the 33-bp product is not adequately resolved on this gel). Lanes 1 and 2, GG; lane 3, CG; lane 4, CC; lanes 5 and 6, no template control; and lane 7, BRL 1-kb ladder size marker.
    Figure Legend Snippet: Detection of the G13964C variant by PCR-RFLP assay. The C→G nucleotide substitution creates a Hha I site, and the G allele is detected by Hha I digestion of the 131-bp product to 98-bp and 33-bp products (the 33-bp product is not adequately resolved on this gel). Lanes 1 and 2, GG; lane 3, CG; lane 4, CC; lanes 5 and 6, no template control; and lane 7, BRL 1-kb ladder size marker.

    Techniques Used: Variant Assay, Polymerase Chain Reaction, RFLP Assay, Marker

    26) Product Images from "Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan"

    Article Title: Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan

    Journal: Malaria Journal

    doi: 10.1186/1475-2875-9-112

    Major merozoite surface protein-3α alleles identified by PCR and digestion with Alu I and Hha I restriction enzymes in the Plasmodium vivax population from Bannu district, Pakistan . Lane 1 = 50 bp, 2 = 1 Kb, and 3 = 100 bp DNA marker. A, B, C = Undigested PCR products. A1, A2 ... are allele types revealed by digestion of the respective PCR products with Alu I, while a1, a2 ... are the allele types obtained by digestion with Hha I, and M = mixed genotype. Frequencies of these alleles are presented in Table 2.
    Figure Legend Snippet: Major merozoite surface protein-3α alleles identified by PCR and digestion with Alu I and Hha I restriction enzymes in the Plasmodium vivax population from Bannu district, Pakistan . Lane 1 = 50 bp, 2 = 1 Kb, and 3 = 100 bp DNA marker. A, B, C = Undigested PCR products. A1, A2 ... are allele types revealed by digestion of the respective PCR products with Alu I, while a1, a2 ... are the allele types obtained by digestion with Hha I, and M = mixed genotype. Frequencies of these alleles are presented in Table 2.

    Techniques Used: Polymerase Chain Reaction, Marker

    27) Product Images from "C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD"

    Article Title: C9orf72 hypermethylation protects against repeat expansion-associated pathology in ALS/FTD

    Journal: Acta neuropathologica

    doi: 10.1007/s00401-014-1286-y

    C9orf72 promoter demethylation promotes toxic RNA accumulation. a Hha I resistance shown as mean + SE ( n = 5) from control ( left ) or expanded ( right ) lymphoblast cells treated with 5-aza-dC ( open ) or untreated ( filled ). Two-way ANOVA: genotype p
    Figure Legend Snippet: C9orf72 promoter demethylation promotes toxic RNA accumulation. a Hha I resistance shown as mean + SE ( n = 5) from control ( left ) or expanded ( right ) lymphoblast cells treated with 5-aza-dC ( open ) or untreated ( filled ). Two-way ANOVA: genotype p

    Techniques Used:

    28) Product Images from "Spread of Cryptococcus gattii in British Columbia, Canada, and Detection in the Pacific Northwest, USA"

    Article Title: Spread of Cryptococcus gattii in British Columbia, Canada, and Detection in the Pacific Northwest, USA

    Journal: Emerging Infectious Diseases

    doi: 10.3201/eid1301.060827

    URA5–restriction fragment length polymorphism (RFLP) profiles for selected human, animal, and environmental Cryptococcus gattii isolates. A) URA5-RFLP to determine the molecular type using Hha I and Sau96 I endonucleases ( 14 ). B) URA5-RFLP to confirm molecular type and determine VGII subtype, using Hha I, Dde I, and BsrG I endonucleases.
    Figure Legend Snippet: URA5–restriction fragment length polymorphism (RFLP) profiles for selected human, animal, and environmental Cryptococcus gattii isolates. A) URA5-RFLP to determine the molecular type using Hha I and Sau96 I endonucleases ( 14 ). B) URA5-RFLP to confirm molecular type and determine VGII subtype, using Hha I, Dde I, and BsrG I endonucleases.

    Techniques Used:

    29) Product Images from "Molecular Evidence of Bartonella spp. in Questing Adult Ixodes pacificus Ticks in California"

    Article Title: Molecular Evidence of Bartonella spp. in Questing Adult Ixodes pacificus Ticks in California

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.4.1221-1226.2001

    PCR-RFLP of the gltA gene of tick samples. Assays were performed by digestion with Taq I, Hha I, and Mse I. Lane M, 100-bp molecular size ladder; lanes 1, 11, and 21, tick 22; lanes 2, 12, and 22, tick 70; lanes 3, 13, and 23, tick 75; lanes 4, 14, and 24, tick 81; lanes 5, 15, and 25, tick 12; lanes 6, 16, and 26, tick 143; lanes 7, 17, and 27, B. henselae ; lanes 8, 18, and 28, B. vinsonii subsp. berkhoffii ; lanes 9, 19, and 29, Bartonella strain cattle-1; lanes 10, 20, and 30, B. quintana .
    Figure Legend Snippet: PCR-RFLP of the gltA gene of tick samples. Assays were performed by digestion with Taq I, Hha I, and Mse I. Lane M, 100-bp molecular size ladder; lanes 1, 11, and 21, tick 22; lanes 2, 12, and 22, tick 70; lanes 3, 13, and 23, tick 75; lanes 4, 14, and 24, tick 81; lanes 5, 15, and 25, tick 12; lanes 6, 16, and 26, tick 143; lanes 7, 17, and 27, B. henselae ; lanes 8, 18, and 28, B. vinsonii subsp. berkhoffii ; lanes 9, 19, and 29, Bartonella strain cattle-1; lanes 10, 20, and 30, B. quintana .

    Techniques Used: Polymerase Chain Reaction

    30) Product Images from "Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro"

    Article Title: Native and Recombinant Polycomb Group Complexes Establish a Selective Block to Template Accessibility To Repress Transcription In Vitro

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.22.22.7919-7928.2002

    PRC1 efficiently represses templates challenged with Hha I. 5S array templates were cut with Hha I before incubation with PRC1 and then transcribed with T7 RNA polymerase. Templates that were nucleosomal at the Hha I site adjacent to the T7 start site were protected from digestion, while any residual naked templates were digested. Since transcription was initiated more often from templates in which the T7 start site was unprotected, digesting the template with Hha I resulted in a ninefold drop in transcription. Primer extension with the primer used for these reactions gave two products of approximately the expected size. The primer may have recognized two adjacent sequences. Quantitation of transcription products is relative to −PRC1 lane for each set of reactions.
    Figure Legend Snippet: PRC1 efficiently represses templates challenged with Hha I. 5S array templates were cut with Hha I before incubation with PRC1 and then transcribed with T7 RNA polymerase. Templates that were nucleosomal at the Hha I site adjacent to the T7 start site were protected from digestion, while any residual naked templates were digested. Since transcription was initiated more often from templates in which the T7 start site was unprotected, digesting the template with Hha I resulted in a ninefold drop in transcription. Primer extension with the primer used for these reactions gave two products of approximately the expected size. The primer may have recognized two adjacent sequences. Quantitation of transcription products is relative to −PRC1 lane for each set of reactions.

    Techniques Used: Incubation, Quantitation Assay

    31) Product Images from "Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus"

    Article Title: Rapid Detection of Epidemic Strains of Methicillin-Resistant Staphylococcus aureus

    Journal: Journal of Clinical Microbiology

    doi:

    Agarose gel electrophoresis of mecA PCR product. Lanes: M, size markers; 1, mecA -positive S. aureus strain; 2, restriction of amplified DNA with Hha I; 3, mecA -negative S. aureus reference strain ATCC 25923.
    Figure Legend Snippet: Agarose gel electrophoresis of mecA PCR product. Lanes: M, size markers; 1, mecA -positive S. aureus strain; 2, restriction of amplified DNA with Hha I; 3, mecA -negative S. aureus reference strain ATCC 25923.

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

    32) Product Images from "Analysis of Polymorphisms in the Merozoite Surface Protein-3? Gene and Two Microsatellite Loci in Sri Lankan Plasmodium vivax: Evidence of Population Substructure in Sri Lanka"

    Article Title: Analysis of Polymorphisms in the Merozoite Surface Protein-3? Gene and Two Microsatellite Loci in Sri Lankan Plasmodium vivax: Evidence of Population Substructure in Sri Lanka

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.2011.11-0338

    Restriction fragment length polymorphism (RFLP) with restriction enzyme Hha I of selected pvmsp -3α alleles in eight different Plasmodium vivax samples from Sri Lanka. With exclusion of bands above 600 bp, and below 150 bp, the estimated allele
    Figure Legend Snippet: Restriction fragment length polymorphism (RFLP) with restriction enzyme Hha I of selected pvmsp -3α alleles in eight different Plasmodium vivax samples from Sri Lanka. With exclusion of bands above 600 bp, and below 150 bp, the estimated allele

    Techniques Used:

    33) Product Images from "Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome"

    Article Title: Osteoponin Promoter Controlled by DNA Methylation: Aberrant Methylation in Cloned Porcine Genome

    Journal: BioMed Research International

    doi: 10.1155/2014/327538

    Methylation and deletion analysis of OPN promoter in 293T cells. The match-like bar with black circle represents the methylation CpG site; white circle of match bar indicated the unmethylation CpG sites. PGL3 vector as standard; in 293T cell line; PGL3-enhance vector as negative control; cell lysate as the background; pCMV-b-gal as internal control. The relative value is adjusted by cell lysate; −495 M-luc indicated the methylation in PGL3 backbone with Hha I and Hpa II methyltransferase. (** P
    Figure Legend Snippet: Methylation and deletion analysis of OPN promoter in 293T cells. The match-like bar with black circle represents the methylation CpG site; white circle of match bar indicated the unmethylation CpG sites. PGL3 vector as standard; in 293T cell line; PGL3-enhance vector as negative control; cell lysate as the background; pCMV-b-gal as internal control. The relative value is adjusted by cell lysate; −495 M-luc indicated the methylation in PGL3 backbone with Hha I and Hpa II methyltransferase. (** P

    Techniques Used: Methylation, Plasmid Preparation, Negative Control

    The sequence and CpG sties distribution of pig OPN promoter. 20 CpG sites exist in the front region of OPN promoter. The underline indicates the predicted AP1-like binding site. The gray marker indicates two methyltransferase sites of Hha I (GCGC) and Hpa II (CCGG).
    Figure Legend Snippet: The sequence and CpG sties distribution of pig OPN promoter. 20 CpG sites exist in the front region of OPN promoter. The underline indicates the predicted AP1-like binding site. The gray marker indicates two methyltransferase sites of Hha I (GCGC) and Hpa II (CCGG).

    Techniques Used: Sequencing, Binding Assay, Marker

    34) Product Images from "Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils"

    Article Title: Diversity of Nitrite Reductase (nirK and nirS) Gene Fragments in Forested Upland and Wetland Soils

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.68.4.1893-1900.2002

    Rarefaction curves indicating diversity of denitrifying bacteria as revealed by RFLP analysis of cloned nitrite reductase gene fragments ( nirK and nirS ) from upland and marsh soil samples. nirK gene fragments were digested with the restriction enzymes Msp I and Hae III, and nirS gene fragments were digested with Msp I and Hha I. Data points represent average values from 26 replicate rarefaction curves, and thin lines are 95% confidence limits of the average values. The thick lines represent fit to an exponential model: y = a × (1 − e − bx ), where x = number of clones screened, y = cumulative number of RFLP patterns, and a and b are constants.
    Figure Legend Snippet: Rarefaction curves indicating diversity of denitrifying bacteria as revealed by RFLP analysis of cloned nitrite reductase gene fragments ( nirK and nirS ) from upland and marsh soil samples. nirK gene fragments were digested with the restriction enzymes Msp I and Hae III, and nirS gene fragments were digested with Msp I and Hha I. Data points represent average values from 26 replicate rarefaction curves, and thin lines are 95% confidence limits of the average values. The thick lines represent fit to an exponential model: y = a × (1 − e − bx ), where x = number of clones screened, y = cumulative number of RFLP patterns, and a and b are constants.

    Techniques Used: Clone Assay

    35) Product Images from "5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation"

    Article Title: 5meCpG Epigenetic Marks Neighboring a Primate-Conserved Core Promoter Short Tandem Repeat Indicate X-Chromosome Inactivation

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0103714

    Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).
    Figure Legend Snippet: Methylation statuses at CpG sites near the RP2 onshore tandem GAAA repeat. Random ( A ) and non-random ( B ) X-inactivation patterns generated for different CpG-containing 5 me CpG-sensitive restriction endonuclease sites obtained using the 5 me CpG-based PCR RP2 / AR biplex assay across the restriction sites. Electropherograms of alleles observed in either undigested genomic DNA or DNA digested with Hpa II, Hha I or Bst UI from females genotyped via quantitative fluorescent PCR are shown. The boxed numbers correspond to the areas under the allele peaks and the intensity of each peak is in relative fluorescence units (RFU).

    Techniques Used: Methylation, Generated, Polymerase Chain Reaction, Fluorescence

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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