Journal: PLoS ONE

Article Title: Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

doi: 10.1371/journal.pone.0194036

Figure Lengend Snippet: Schema of methylation-sensitive SNP array. If allele-specific DNA methylation (ASM) around heterozygous SNP A/C exists (which is hypermethylated around A allele and hypomethylated around C allele), SNP is called A/C in micro array before digestion and A/A after digestion by MSREs. Thus, the probes heterozygous in uncut genomic DNA and homozygous in MSREs-digested DNA indicate ASM around SNP. Because we expect methylation skew between two alleles, all heterozygous SNPs which the ratio of signal intensities given two alleles changed after digestion should be extracted. SNP, single-nucleotide polymorphisms; MSREs, methylation-sensitive restriction enzymes, which contain HpaII ( 5′-CˆCGG-3′ ), HhaI ( 5′-GCGˆC-3′ ), and AciI ( 5′-CˆCGC-3′ ).

Article Snippet: DNA from patients was digested using a cocktail of three MSREs: HpaII (5′-CˆCGG-3′), HhaI (5′-GCGˆC-3′), and AciI (5′-CˆCGC-3′) (New England BioLabs, Ipswich, USA), which in combination interrogate the methylation status of ~32.4% of CpG sites in the human genome [ ].

Techniques: Methylation, DNA Methylation Assay, Microarray

Journal: BMC Genomics

Article Title: Chromosome-wide mapping of DNA methylation patterns in normal and malignant prostate cells reveals pervasive methylation of gene-associated and conserved intergenic sequences

doi: 10.1186/1471-2164-12-313

Figure Lengend Snippet: Overview and pre-microarray performance of MBD-chip . A , Overview of MBD-chip. Genomic DNA is: i) fragmented (in this case with restriction enzymes), ii) enriched for methylated DNA using MBD2-MBD-magnetic beads, and iii) amplified, fragmented, labeled and hybridized to tiling microarrays. Comparison with a total input fraction allows identification of methylated regions. B , Degree of MBD2-MBD enrichment is non-linearly proportional to the number of methylated CpGs. WBC DNA was methylated at 0, 6, 10 or 37 CpG sites within an R.AluI restriction fragment within the GSTP1 promoter by treatment with M.HpaII +/- M.HhaI or with M.SssI or no enzymes. The degree of MBD2-MBD enrichment compared to mock (no MBD control), as measured by qPCR, was related to the number of methylated CpGs. ND, not detectable. C , The MBD-chip process enriches DNA with high density of methylated-CpGs. DNA from LNCaP and PrEC cells was completely methylated with M.SssI or left untreated. MBD2-MBD enrichment at regions in HBB and GSTP1 promoters, as examined by qPCR, are shown. The CpG density (Low, indicates

Article Snippet: Compare-MS DNA methylation analysis For COMPARE-MS analysis of DNA methylation at newly identified cancer hypermethylated regions in prostate cancer cell lines, tumor-normal paired tissues, and reference samples, DNA samples were digested with R.AluI and R.HhaI (NEB) and methylated fragments were enriched and analyzed by real-time PCR as described previously [ ].

Techniques: Microarray, Chromatin Immunoprecipitation, Methylation, Magnetic Beads, Amplification, Labeling, Real-time Polymerase Chain Reaction

Journal: Nucleic Acids Research

Article Title: Recognition and cleavage of 5-methylcytosine DNA by bacterial SRA-HNH proteins

doi: 10.1093/nar/gku1376

Figure Lengend Snippet: EMSA analysis of Sco5333 to methylated 219 bp derived from pUC18. ( A ) Sequence analysis of 219 bp which is PCR amplified from NT885–1103 of pUC18. Bases in shadow are modification sites of relative DNA MTases, and black rectangle designates the 5mC. C and G in bold represent the distribution of CpG and GpC modification sites. The underlined sequences are then synthesized and are named as 55ntDcm1 and 55ntDcm2. ( B ) Titration of Sco5333 on Dcm-modified and non-modified 219 bp fragment. Sco5333 shows high specificity to Dcm-modified 219 bp, and the two shifted bands in lane labelled 0.05 and 0.1 at right-half of the gel are due to the two Dcm modification sites which are centered at 219 bp. ( C ) EMSA of Sco5333 on 219 bp fragment modified by M.AluI, M.HhaI, M.HpaII, M.MspI, GpC, CpG or Dcm. Final concentration of Sco5333 is 0.1 μM. M.AluI, M.HhaI and Dcm have two modification sites on 219 bp, thus lanes 3, 4, and 9 show higher shift bands, whereas 219 bp modified by other MTases that have single modification site show lower shift bands. CpG and GpC have multiple modificaition sites (19 and 21 respectively), which result in multiple retarded bands. Statistics of Sco5333 binding sites on 219 bp shows poor sequence dependency.

Article Snippet: In addition, the 219 bp DNA fragment was methylated by commercially available methylases M.AluI, M.HhaI, M.HpaII, M.MspI, GpC(M.CviPI) and CpG(M.SssI) (NEB) respectively.

Techniques: Methylation, Derivative Assay, Sequencing, Polymerase Chain Reaction, Amplification, Modification, Gel Permeation Chromatography, Synthesized, Titration, Concentration Assay, Binding Assay

Journal: Infection and Immunity

Article Title: Colonization of the Cecal Mucosa by Helicobacter hepaticus Impacts the Diversity of the Indigenous Microbiota

doi: 10.1128/IAI.73.10.6852-6961.2005

Figure Lengend Snippet: Changes in diversity measures of the mucosa-associated microbiota of the murine cecum 30 days after experimental infection with H. hepaticus . The Shannon diversity index ( H ) and Shannon evenness ( H / H max ) were calculated for each mouse based on normalized T-RFLP profiles obtained with HhaI and MspI digestion. Compared to uninfected animals (“Uninfected” category), animals experimentally infected with H. hepaticus (“Infected” category) had significant decreases in both diversity and evenness. When the analysis was repeated, this time suppressing the H. hepaticus terminal restriction fragment before normalization of the profiles (“Infected [suppressed]” category), the change in diversity and evenness in infected animals was no longer apparent. Comparisons for all pairs of time points was performed by analysis of variance using Tukey-Kramer honestly significant difference. Categories within each plot not connected by the same roman numeral are significantly different with an alpha level set to 0.05.

Article Snippet: Terminal restriction fragments using the restriction enzymes HhaI and MspI were calculated for each of the 16S rRNA-encoding gene clones using a software tool designed to be integrated into the ARB program suite ( ).

Techniques: Infection

Journal: Scientific Reports

Article Title: Detection of Novel duck reovirus (NDRV) using visual reverse transcription loop-mediated isothermal amplification (RT-LAMP)

doi: 10.1038/s41598-018-32473-4

Figure Lengend Snippet: Agarose gel electrophoresis results for NDRV RT-LAMP reaction products and their HhaI restriction enzyme digestion results. M, 2000 bp DNA Marker; lane 1(HhaI restriction enzyme digestion results); lane 2 ladder-like pattern bands; lane 3 NC.

Article Snippet: Two microliters of RT-LAMP amplified products were digested with restriction enzyme HhaI (New England Biolabs, USA) at 37 °C for 2 h according to the manufacturer’s protocol and the restriction enzyme digestion products were analyzed on 2% agarose gel electrophoresis.

Techniques: Agarose Gel Electrophoresis, Marker

Journal: PLoS ONE

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

doi: 10.1371/journal.pone.0103714

Figure Lengend 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).

Article Snippet: Digestion with methylation-sensitive restriction enzymes Genomic DNA (500 ng) was digested with Hpa II (Invitrogen, Carlsbad, CA, USA), Bst UI and Hha I (New England Biolabs, Ipswich, MA, USA) for 6 h at 37°C (Hpa II and Hha I) or 60°C (Bst UI), or was mock-digested without the restriction enzymes.

Techniques: Methylation, Generated, Polymerase Chain Reaction, Fluorescence

Journal: Applied and Environmental Microbiology

Article Title: Chronic Helicobacter pylori Infection Does Not Significantly Alter the Microbiota of the Murine Stomach ▿ Infection Does Not Significantly Alter the Microbiota of the Murine Stomach ▿ †

doi: 10.1128/AEM.01675-06

Figure Lengend Snippet: Dendrograms generated from T-RFLP analyses using (a) a DraI/HhaI double digest or (b) a HaeIII digest of FAM-labeled PCR products. Scale bars indicate a 10% difference between individual samples. Numbers: 1 to 10, mice infected for 2 weeks; 11 to 19, mice infected for 3 months; 20 to 29, mice infected for 6 months; N1 to N10, naïve mice in the 2-week group; N11 to N20, naïve mice in the 3-month group; N21 to N29, naïve mice in the 6-month group. Dashed ovals indicate the apparent clustering of mice from the 6-month group.

Article Snippet: A double digest using DraI and HhaI (Promega, WI) allowed us to discriminate between the Lactobacillus species detected in our preliminary studies, and the second analysis used HaeIII (Promega).

Techniques: Generated, Labeling, Polymerase Chain Reaction, Mouse Assay, Infection

Journal: Applied and Environmental Microbiology

Article Title: Analysis of the Endophytic Actinobacterial Population in the Roots of Wheat (Triticum aestivum L.) by Terminal Restriction Fragment Length Polymorphism and Sequencing of 16S rRNA Clones

doi: 10.1128/AEM.70.3.1787-1794.2004

Figure Lengend Snippet: T-RFLP profile of the 16S rRNA genes of the actinobacterial population amplified from the roots of 6-week-old wheat grown in the field soil obtained from Swedes Flat and digested with Hin fI (a), Hha I (b), and Mbo I (c).

Article Snippet: From the restriction patterns generated with Hha I, plasmids that showed different restriction fragment patterns were further digested with the restriction enzyme Hin fI (Promega) to further identify plasmids with dissimilar 16S rRNA RFLPs.

Techniques: Amplification

Journal: Applied and Environmental Microbiology

Article Title: Anatoxin-a Synthetase Gene Cluster of the Cyanobacterium Anabaena sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ sp. Strain 37 and Molecular Methods To Detect Potential Producers ▿ †

doi: 10.1128/AEM.06022-11

Figure Lengend Snippet: RFLP analysis of anaC amplicons (366 bp) of cyanobacterial strains and environmental samples with the HhaI and HinfI restriction enzymes. The amplicons are indicated above the lanes according to the strain/sample used for amplification: Anabaena sp. 37

Article Snippet: In single-strain reactions, 200 ng of the purified PCR product was digested with 1 μl HhaI or HinfI FastDigest enzyme (Fermentas) in 0.67× FastDigest Green Buffer (Fermentas) in a total volume of 30 μl.

Techniques: Environmental Sampling, Amplification

Journal: Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences

Article Title: Polymorphism of Apo lipoprotein E gene and the risk of multiple sclerosis

doi:

Figure Lengend Snippet: Digestion of PCR amplified ApoE gene with HhaI restriction enzyme Lanes 1 and 4 show heterozygote E3/E4 genotype (91, 72, 48, and 53 bp bands). Lanes 2 and 3 show homozygote E3/E3 genotype (91, 48, and 53 bp bands).

Article Snippet: For this purpose, 8 ml of the PCR product was digested with 10 units HhaI enzyme (Fermentas, Poland) for 4 hours.

Techniques: Polymerase Chain Reaction, Amplification

Journal: Microbial biotechnology

Article Title: Short-term changes in the composition of active marine bacterial assemblages in response to diesel oil pollution

doi: 10.1111/j.1751-7915.2010.00192.x

Figure Lengend Snippet: Variation in the relative abundance of selected T‐RFs, diagnostic for different bacterial groups, during the microcosm experiments. Square symbols with solid lines correspond to winter microcosms. Circle symbols and dashed lines correspond to summer microcosms. Closed symbols correspond to controls and open symbols to diesel treatments. A. SAR11 group (T‐RF AluI size 171 nt). B. Roseobacter group (sum of relative abundances of T‐RFs AluI sizes 245 and 248 nt). C. Oceanospirillaceae (sum of relative abundances of T‐RFs HhaI sizes 209, 361 and 369 nt). D. OMG group (sum of relative abundances of T‐RFs AluI 229 and HhaI 354 nt). E. SAR 86 group (sum of T‐RFs HhaI 365 and 366 nt). F. Synechococcus (sum of relative abundances of T‐RFs AluI sizes 125, 189 and 204 nt).

Article Snippet: Ten microlitres of the purified products was digested separately with 20 U of restriction endonucleases AluI and HhaI (Roche Applied Science and New England Biolabs respectively) at 37°C during 3.5 h. Restriction fragments were mixed with a ROX‐labelled size standard GeneScan‐500 (Applied Biosystems), denatured by heating at 96°C for 5 min in formamide and loaded in an ABI 3130 Genetic Analyzer (Applied Biosystems) for electrophoretic separation, using 36 cm length capillaries filled with POP‐7 polymer (Applied Biosystems).

Techniques: Diagnostic Assay