aci i  (New England Biolabs)


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  • 96
    Name:
    AciI
    Description:
    AciI 1 000 units
    Catalog Number:
    r0551l
    Price:
    282
    Size:
    1 000 units
    Category:
    Restriction Enzymes
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    Structured Review

    New England Biolabs aci i
    AciI
    AciI 1 000 units
    https://www.bioz.com/result/aci i/product/New England Biolabs
    Average 96 stars, based on 68 article reviews
    Price from $9.99 to $1999.99
    aci i - by Bioz Stars, 2020-09
    96/100 stars

    Images

    1) Product Images from "Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats"

    Article Title: Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0148299

    PCR-RFLP of a gltA gene fragment using the restriction endonuclease Aci I. Lanes 1, 4 and 19 show 100 BP Ladder; Lane 2, Mt Lion L27-96; Lane 3, Mt Lion L42-94; Lane 5, Mt Lion L-39-97; Lane 6, Mt Lion FM98061; Lane 7, Bobcat L08-96; Lane 8, Bobcat L17-96; Lane 9, Bobcat DS08; Lane 10, Bobcat L10-97; Lane 11, Bobcat L11-97; Lane 12, Bobcat SC443; lane 13, Bobcat DS507; Lane 14, B . henselae Type I; Lane 15, B . henselae Type II; Lane 16, B . clarridgeiae ; Lane 17, B . koehlerae ; Lane18, B . bovis (“weissii” isolate).
    Figure Legend Snippet: PCR-RFLP of a gltA gene fragment using the restriction endonuclease Aci I. Lanes 1, 4 and 19 show 100 BP Ladder; Lane 2, Mt Lion L27-96; Lane 3, Mt Lion L42-94; Lane 5, Mt Lion L-39-97; Lane 6, Mt Lion FM98061; Lane 7, Bobcat L08-96; Lane 8, Bobcat L17-96; Lane 9, Bobcat DS08; Lane 10, Bobcat L10-97; Lane 11, Bobcat L11-97; Lane 12, Bobcat SC443; lane 13, Bobcat DS507; Lane 14, B . henselae Type I; Lane 15, B . henselae Type II; Lane 16, B . clarridgeiae ; Lane 17, B . koehlerae ; Lane18, B . bovis (“weissii” isolate).

    Techniques Used: Polymerase Chain Reaction

    2) Product Images from "Mutations causing slow-channel myasthenia reveal that a valine ring in the channel pore of muscle AChR is optimized for stabilizing channel gating"

    Article Title: Mutations causing slow-channel myasthenia reveal that a valine ring in the channel pore of muscle AChR is optimized for stabilizing channel gating

    Journal: Human mutation

    doi: 10.1002/humu.23043

    A. Pedigrees of patients 1, 2, and 3. The mother of patients 3 is mosaic for εV265A. Arrows point to the proband. B. Multiple alignment of the M2 of AChR subunits. βV266 and εV265 are conserved across all human AChR subunits and across AChR subunits of different species. C. Haplotype analysis of patients 2 and 3. SNPs on an allele carrying εV265A (star mark) are determined by analyzing the parents' DNA. Intron 6 to Intron 8 of CHRNE encoded on the reverse strand are drawn to scale. Note that haplotypes are not shared between patients 2 and 3. D. Sequencing chromatograms of the mother, and patient 3 of CHRNE exon 8. Note that the mother is mosaic for εV265A and the mutant peak in the mother is lower than that in patient 3. E. Scheme of SCCMS mutations at the M2. The M2 domains of five subunits (α1 × 2, β1, δ, and ε subunits) form α helices and constitute the ion channel pore that is opened by binding of two ACh molecules to the extracellular domains of these subunits. The smallest blue circle in the middle represents the narrowest leucine ring in the channel pore. The valine ring is 4 residues above the leucine ring and is indicated in red. The two large gray rings represent the amino acids margining the M2. The previously reported amino acid-substituting mutations in the M2 domains are indicated by closed symbols. βV266A and εV265A are shown in red. Mutations are indicated according to the legacy nomenclature, in which the first codon is the N-terminal end of a mature peptide. Add 20, 23, 21, and 20 codons for AChR α, β, δ, and ε subunits, respectively, to convert the legacy nomenclature to the HGVS nomenclature.
    Figure Legend Snippet: A. Pedigrees of patients 1, 2, and 3. The mother of patients 3 is mosaic for εV265A. Arrows point to the proband. B. Multiple alignment of the M2 of AChR subunits. βV266 and εV265 are conserved across all human AChR subunits and across AChR subunits of different species. C. Haplotype analysis of patients 2 and 3. SNPs on an allele carrying εV265A (star mark) are determined by analyzing the parents' DNA. Intron 6 to Intron 8 of CHRNE encoded on the reverse strand are drawn to scale. Note that haplotypes are not shared between patients 2 and 3. D. Sequencing chromatograms of the mother, and patient 3 of CHRNE exon 8. Note that the mother is mosaic for εV265A and the mutant peak in the mother is lower than that in patient 3. E. Scheme of SCCMS mutations at the M2. The M2 domains of five subunits (α1 × 2, β1, δ, and ε subunits) form α helices and constitute the ion channel pore that is opened by binding of two ACh molecules to the extracellular domains of these subunits. The smallest blue circle in the middle represents the narrowest leucine ring in the channel pore. The valine ring is 4 residues above the leucine ring and is indicated in red. The two large gray rings represent the amino acids margining the M2. The previously reported amino acid-substituting mutations in the M2 domains are indicated by closed symbols. βV266A and εV265A are shown in red. Mutations are indicated according to the legacy nomenclature, in which the first codon is the N-terminal end of a mature peptide. Add 20, 23, 21, and 20 codons for AChR α, β, δ, and ε subunits, respectively, to convert the legacy nomenclature to the HGVS nomenclature.

    Techniques Used: Sequencing, Mutagenesis, Binding Assay

    3) Product Images from "Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming"

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003636

    The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.
    Figure Legend Snippet: The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Techniques Used: Expressing, Ligation, Polymerase Chain Reaction, Amplification

    4) Product Images from "Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species"

    Article Title: Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

    Journal: Applied and Environmental Microbiology

    doi: 10.1128/AEM.71.10.5999-6007.2005

    Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.
    Figure Legend Snippet: Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.

    Techniques Used:

    5) Product Images from "Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia"

    Article Title: Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia

    Journal: Molecular Pathology

    doi:

    Restriction enzyme analysis and parallel direct sequencing of polymerase chain reaction (PCR) products. (A) Aci I and (B) Tau I endonuclease cleavage followed by 10% polyacrylamide gel analysis. M, molecular weight marker, 100 bp DNA ladder (Gibco BRL, Burlington, Ontario, Canada). (A) Lane 1, chronic lymphocytic leukaemia (CLL) sample 1, amplified BAX promoter segment before digestion; lanes 2–5, PCR products digested with Aci I; lane 2, CLL sample 2 with a heterozygous single nucleotide polymorphism (SNP); lanes 3 and 4, CLL samples 3 and 4 with no SNP; lane 5, RL cell line with homozygous SNP. (B) PCR products digested with Tau I; lane 2, CLL sample 2 with a heterozygous SNP; lane 3, CLL sample 3 with no SNP; lane 5, RL cell line with homozygous SNP. (C) Left hand column: direct sequence of the BAX promoter region. The control sample (I) shows no alteration, whereas a CLL sample (II) shows a heterozygous SNP, and the RL cell line (III) has a homozygous SNP. Middle and right hand columns: corresponding samples digested with the Aci I and Tau I enzymes, respectively
    Figure Legend Snippet: Restriction enzyme analysis and parallel direct sequencing of polymerase chain reaction (PCR) products. (A) Aci I and (B) Tau I endonuclease cleavage followed by 10% polyacrylamide gel analysis. M, molecular weight marker, 100 bp DNA ladder (Gibco BRL, Burlington, Ontario, Canada). (A) Lane 1, chronic lymphocytic leukaemia (CLL) sample 1, amplified BAX promoter segment before digestion; lanes 2–5, PCR products digested with Aci I; lane 2, CLL sample 2 with a heterozygous single nucleotide polymorphism (SNP); lanes 3 and 4, CLL samples 3 and 4 with no SNP; lane 5, RL cell line with homozygous SNP. (B) PCR products digested with Tau I; lane 2, CLL sample 2 with a heterozygous SNP; lane 3, CLL sample 3 with no SNP; lane 5, RL cell line with homozygous SNP. (C) Left hand column: direct sequence of the BAX promoter region. The control sample (I) shows no alteration, whereas a CLL sample (II) shows a heterozygous SNP, and the RL cell line (III) has a homozygous SNP. Middle and right hand columns: corresponding samples digested with the Aci I and Tau I enzymes, respectively

    Techniques Used: Sequencing, Polymerase Chain Reaction, Molecular Weight, Marker, Amplification

    (A) Aci I and (B) Tau I restriction enzyme maps for the BAX promoter region.
    Figure Legend Snippet: (A) Aci I and (B) Tau I restriction enzyme maps for the BAX promoter region.

    Techniques Used:

    Restriction enzyme analysis (with Aci I ) for the G(−248)A single nucleotide polymorphism (SNP) in the BAX gene. M, molecular weight marker (100 bp DNA ladder; Gibco BRL); N, control sample showing three distinct bands, 352 256 bp, and 96 bp; P1, positive control with heterozygous SNP showing the 352 bp (major) and 256 bp (minor) bands and an almost invisible 96 bp band; P2, positive control with a homozygous SNP, which abolishes a restriction enzyme site, resulting in a single 352 bp band; lanes 1–5, chronic lymphocytic leukaemia cases; lanes 1, 4, and 5, heterozygous SNP; lanes 2 and 3, no SNP; lane 6, RL showing the homozygous SNP; lanes 7–12, controls without the G(−248)A SNP.
    Figure Legend Snippet: Restriction enzyme analysis (with Aci I ) for the G(−248)A single nucleotide polymorphism (SNP) in the BAX gene. M, molecular weight marker (100 bp DNA ladder; Gibco BRL); N, control sample showing three distinct bands, 352 256 bp, and 96 bp; P1, positive control with heterozygous SNP showing the 352 bp (major) and 256 bp (minor) bands and an almost invisible 96 bp band; P2, positive control with a homozygous SNP, which abolishes a restriction enzyme site, resulting in a single 352 bp band; lanes 1–5, chronic lymphocytic leukaemia cases; lanes 1, 4, and 5, heterozygous SNP; lanes 2 and 3, no SNP; lane 6, RL showing the homozygous SNP; lanes 7–12, controls without the G(−248)A SNP.

    Techniques Used: Molecular Weight, Marker, Positive Control

    6) Product Images from "Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene"

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene

    Journal: The American Journal of Pathology

    doi:

    Alignment of the PTEN gene ( lower ) and pseudogene ( upper ). The region upstream of nucleotide (−841) represents a divergent sequence. MSP primer sets I, II, and III are shown. Set IV-A, -B primers were used for MSRA to amplify the PTEN gene and pseudogene, respectively. Aci I restriction sites are indicated with triangles. Differences between the two sequences are shown with the pseudogene sequence on top for MSRA are shown. Genomic position is defined by the location relative to the translational start site of PTEN (GenBank accession number AF143312).
    Figure Legend Snippet: Alignment of the PTEN gene ( lower ) and pseudogene ( upper ). The region upstream of nucleotide (−841) represents a divergent sequence. MSP primer sets I, II, and III are shown. Set IV-A, -B primers were used for MSRA to amplify the PTEN gene and pseudogene, respectively. Aci I restriction sites are indicated with triangles. Differences between the two sequences are shown with the pseudogene sequence on top for MSRA are shown. Genomic position is defined by the location relative to the translational start site of PTEN (GenBank accession number AF143312).

    Techniques Used: Sequencing

    Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.
    Figure Legend Snippet: Methylation-specific PCR using set III PTEN specific primers ( a ) and methylation-sensitive restriction analysis ( b, c, d ) using primer set IV-A ( PTEN ) respectively for cell lines. MSP analysis, as well as MSRA using primer set IV-A ( PTEN ), show lack of methylation in cell lines ( a, b ). Results for MSRA using set IV-B and -C primers ( c, d ) show methylation positive products in the same cell lines. NL treated with Sss I methyltransferase was used as a methylated positive control. Following Aci I digestion, equal amounts of cell line DNA were used for PCR analysis. U, unmethylated; M, methylated; N, undigested DNA; D, digested DNA; NL, normal lymphocyte DNA.

    Techniques Used: Methylation, Polymerase Chain Reaction, Positive Control

    7) Product Images from "APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure"

    Article Title: APOBEC3G cytosine deamination hotspots are defined by both sequence context and single-stranded DNA secondary structure

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkt246

    Experimental confirmation of ssDNA secondary structures. Two sets of oligonucleotides with restriction enzyme sites in the stem oligonucleotide were used (( A ) GCCG Set 2 Stem and GCCG Set 2 Open, and ( B ) GCCG Set 1 Stem and GCCG Set 1 Open). The stem bases in the structured oligonucleotides create an Aci I restriction site (A) or a Msp I restriction site (B). The oligonucleotides GCCG Set 2 Open and GCCG Set 1 Open did not fold to form the restriction enzyme sites and remained intact. The average and standard deviation from three independent experiments are shown.
    Figure Legend Snippet: Experimental confirmation of ssDNA secondary structures. Two sets of oligonucleotides with restriction enzyme sites in the stem oligonucleotide were used (( A ) GCCG Set 2 Stem and GCCG Set 2 Open, and ( B ) GCCG Set 1 Stem and GCCG Set 1 Open). The stem bases in the structured oligonucleotides create an Aci I restriction site (A) or a Msp I restriction site (B). The oligonucleotides GCCG Set 2 Open and GCCG Set 1 Open did not fold to form the restriction enzyme sites and remained intact. The average and standard deviation from three independent experiments are shown.

    Techniques Used: Standard Deviation

    8) Product Images from "The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive"

    Article Title: The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive

    Journal: BMC Plant Biology

    doi: 10.1186/1471-2229-14-174

    Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.
    Figure Legend Snippet: Analysis of DNA methylation. (a) Amplified methylation polymorphism (AMP) profiling. DNA from two biological replicates (lanes) was used for AMP before (on the left) or after (right) digestion with HpaII. Arrows point to the differences in methylation between 2HA and Jemalong. (b, c) Analysis of MtEIL1 methylation. (b) The MtEIL1 gene and promoter. Vertical bars depict positions of AciI cut sites, and bisulphate sequenced regions also indicated. After digestion of genomic DNA by AciI, qPCR was performed with primers F2/R2 and F4/R4 separately. (c) qPCR results show amount of undigested DNA due to methylation of digestion sites. Results are mean ± SE of 4 repeats.

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

    9) Product Images from "Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes"

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes

    Journal: Journal of Virology

    doi: 10.1128/JVI.00736-17

    EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P
    Figure Legend Snippet: EBV Rta expression increases DNA methylation and decreases CTCF binding in the promoter regions of MYC , CCND1 , and JUN . (A, left) Schematic diagrams of methylation-sensitive restriction enzyme sites, CTCF binding sites, and Rta binding sites in each target promoter region. These regions contain no EcoRI site, thus EcoRI served as an input control for AciI, HpaII, and HinP1I. The MYC gene body without Rta and CTCF binding sites served as a negative control (N.C.). Lengths of promoters are illustrated to scale. (Right) CpG methylation levels in the cellular promoters of 293TetLuc and 293TetER cells. Cellular DNAs of paired untreated and doxycycline (Dox)-treated (12 and 24 h) cells were extracted and subjected to restriction enzyme digestions. DNA fragments protected by each methylation-sensitive enzyme were quantified by real-time PCR. Fold changes of each restriction enzyme assessment denote the relative CpG methylation levels in the Dox-treated cells compared to their untreated counterparts. Error bars depict the means ± SD from four independent experiments. Student's t test was used to evaluate the significant difference between the indicated data set. ***, P

    Techniques Used: Expressing, DNA Methylation Assay, Binding Assay, Methylation, Negative Control, CpG Methylation Assay, Real-time Polymerase Chain Reaction

    10) Product Images from "How to Isolate a Plant's Hypomethylome in One Shot"

    Article Title: How to Isolate a Plant's Hypomethylome in One Shot

    Journal: BioMed Research International

    doi: 10.1155/2015/570568

    Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.
    Figure Legend Snippet: Complementary identification of genomic regions in rice due to restriction site locations. A detailed representation of the mapping results is shown for both enzymes, AciI and HpaII. The identified regions around the displayed gene differ due to the lack of recognition sites for the other enzyme. On the right, an example for overlapping but expanded regions is given.

    Techniques Used:

    Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.
    Figure Legend Snippet: Length distribution of genomic regions identified for AciI, HpaII, and the combined dataset in rice. The length distribution of hypomethylated regions identified with the three datasets up to the maximal length is shown as well as a closer view to the region between 0 and 2.000 bp, where an increase in length is visible for the combined dataset. Additionally, the amount of regions, the average and maximum length, and the average reads per region are given.

    Techniques Used:

    Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.
    Figure Legend Snippet: Genes and transposable elements identified in the rice genome with the methyl filtration technique. The regions comprised of at least five reads (left), and all regions (middle) show a clear depletion of transposable elements for AciI, Bsh1236I, and HpaII. On the right a representation of genes and transposable elements is given showing potential methylation sites within their gene space. All values are shown in percent based on the annotated 39.954 genes and 15.847 transposable elements.

    Techniques Used: Filtration, Methylation

    11) Product Images from "Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region"

    Article Title: Detection and Differentiation of Old World Orthopoxviruses: Restriction Fragment Length Polymorphism of the crmB Gene Region

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.39.1.94-100.2001

    RLFP analysis after Aci I digestion of PCR-amplified crmB fragments from genomes of VAR isolates. Lanes: 1, VAR major strain Bangladesh-1975; 2, VAR major strain Congo-1970; 3, VAR major Harvey-1944; 4, African VAR minor Somalia-1977; 5, “Whitepoxch9-2; 6, “Whitepox” ch9-4; 7, alastrim VAR minor strain Sierra Leone-1968; 8, alastrim VAR minor Garcia-1966.
    Figure Legend Snippet: RLFP analysis after Aci I digestion of PCR-amplified crmB fragments from genomes of VAR isolates. Lanes: 1, VAR major strain Bangladesh-1975; 2, VAR major strain Congo-1970; 3, VAR major Harvey-1944; 4, African VAR minor Somalia-1977; 5, “Whitepoxch9-2; 6, “Whitepox” ch9-4; 7, alastrim VAR minor strain Sierra Leone-1968; 8, alastrim VAR minor Garcia-1966.

    Techniques Used: Polymerase Chain Reaction, Amplification

    12) Product Images from "Mutations causing slow-channel myasthenia reveal that a valine ring in the channel pore of muscle AChR is optimized for stabilizing channel gating"

    Article Title: Mutations causing slow-channel myasthenia reveal that a valine ring in the channel pore of muscle AChR is optimized for stabilizing channel gating

    Journal: Human mutation

    doi: 10.1002/humu.23043

    A. Pedigrees of patients 1, 2, and 3. The mother of patients 3 is mosaic for εV265A. Arrows point to the proband. B. Multiple alignment of the M2 of AChR subunits. βV266 and εV265 are conserved across all human AChR subunits and across AChR subunits of different species. C. Haplotype analysis of patients 2 and 3. SNPs on an allele carrying εV265A (star mark) are determined by analyzing the parents' DNA. Intron 6 to Intron 8 of CHRNE encoded on the reverse strand are drawn to scale. Note that haplotypes are not shared between patients 2 and 3. D. Sequencing chromatograms of the mother, and patient 3 of CHRNE exon 8. Note that the mother is mosaic for εV265A and the mutant peak in the mother is lower than that in patient 3. E. Scheme of SCCMS mutations at the M2. The M2 domains of five subunits (α1 × 2, β1, δ, and ε subunits) form α helices and constitute the ion channel pore that is opened by binding of two ACh molecules to the extracellular domains of these subunits. The smallest blue circle in the middle represents the narrowest leucine ring in the channel pore. The valine ring is 4 residues above the leucine ring and is indicated in red. The two large gray rings represent the amino acids margining the M2. The previously reported amino acid-substituting mutations in the M2 domains are indicated by closed symbols. βV266A and εV265A are shown in red. Mutations are indicated according to the legacy nomenclature, in which the first codon is the N-terminal end of a mature peptide. Add 20, 23, 21, and 20 codons for AChR α, β, δ, and ε subunits, respectively, to convert the legacy nomenclature to the HGVS nomenclature.
    Figure Legend Snippet: A. Pedigrees of patients 1, 2, and 3. The mother of patients 3 is mosaic for εV265A. Arrows point to the proband. B. Multiple alignment of the M2 of AChR subunits. βV266 and εV265 are conserved across all human AChR subunits and across AChR subunits of different species. C. Haplotype analysis of patients 2 and 3. SNPs on an allele carrying εV265A (star mark) are determined by analyzing the parents' DNA. Intron 6 to Intron 8 of CHRNE encoded on the reverse strand are drawn to scale. Note that haplotypes are not shared between patients 2 and 3. D. Sequencing chromatograms of the mother, and patient 3 of CHRNE exon 8. Note that the mother is mosaic for εV265A and the mutant peak in the mother is lower than that in patient 3. E. Scheme of SCCMS mutations at the M2. The M2 domains of five subunits (α1 × 2, β1, δ, and ε subunits) form α helices and constitute the ion channel pore that is opened by binding of two ACh molecules to the extracellular domains of these subunits. The smallest blue circle in the middle represents the narrowest leucine ring in the channel pore. The valine ring is 4 residues above the leucine ring and is indicated in red. The two large gray rings represent the amino acids margining the M2. The previously reported amino acid-substituting mutations in the M2 domains are indicated by closed symbols. βV266A and εV265A are shown in red. Mutations are indicated according to the legacy nomenclature, in which the first codon is the N-terminal end of a mature peptide. Add 20, 23, 21, and 20 codons for AChR α, β, δ, and ε subunits, respectively, to convert the legacy nomenclature to the HGVS nomenclature.

    Techniques Used: Sequencing, Mutagenesis, Binding Assay

    13) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    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′ ).
    Figure Legend 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′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    14) Product Images from "Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications"

    Article Title: Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications

    Journal: bioRxiv

    doi: 10.1101/636936

    Data used to examine the effect of fragment-density test. (a) The gel was analyzed considering the portions delimited by the ROIs, using lane 1 (blue) as the reference ladder. ROIs 2–6 are tagmentation products of human genomic DNA; 7 and 8 are λ - Aci I digest samples (b) Summary plot of the fit for the gel in (a). Fragment-size mean and standard-deviation values for model distributions containing varying numbers of reference fragments (peaks). Symbols are colored according to the proportion of the original λ - Aci I basis fragments retained in the reference distribution; thus, 1:2 means that every other peak was eliminated from the fit, 2:3 every third peak, etc. Data are also reported in Table S4.
    Figure Legend Snippet: Data used to examine the effect of fragment-density test. (a) The gel was analyzed considering the portions delimited by the ROIs, using lane 1 (blue) as the reference ladder. ROIs 2–6 are tagmentation products of human genomic DNA; 7 and 8 are λ - Aci I digest samples (b) Summary plot of the fit for the gel in (a). Fragment-size mean and standard-deviation values for model distributions containing varying numbers of reference fragments (peaks). Symbols are colored according to the proportion of the original λ - Aci I basis fragments retained in the reference distribution; thus, 1:2 means that every other peak was eliminated from the fit, 2:3 every third peak, etc. Data are also reported in Table S4.

    Techniques Used: Standard Deviation

    Dependence of plug-in output on camera hardware. Estimates of average fragment size obtained for images of the low-resolution gel ( Figure 2c collected using four different camera systems, C1-C4 ( Table 1 ). The plug-in measurements of average fragment size were compared for the known λ - Aci I digest and the same λ -genome tagmented samples analyzed in Figure 2 . Error bars indicate the standard deviation ( i.e ., ) of the fitted distribution. Numerical data for this experiment are also provided in Table S2.
    Figure Legend Snippet: Dependence of plug-in output on camera hardware. Estimates of average fragment size obtained for images of the low-resolution gel ( Figure 2c collected using four different camera systems, C1-C4 ( Table 1 ). The plug-in measurements of average fragment size were compared for the known λ - Aci I digest and the same λ -genome tagmented samples analyzed in Figure 2 . Error bars indicate the standard deviation ( i.e ., ) of the fitted distribution. Numerical data for this experiment are also provided in Table S2.

    Techniques Used: Standard Deviation

    Tagmented libraries prepared from C. elegans genomic DNA. Gel images show the sections corresponding to a low-molecular-weight and b high-molecular-weight fractions. In both cases, lanes 2 and 3 are λ - Aci I digest samples; lanes 4-7 C. elegans genomic-DNA tagmentation reactions amplified by PCR under different conditions. Summary plot of average fragment size based on short- or long-exposure images, c low-molecular-weight, and d high-molecular-weight fractions (Table S3 in table form). Comparison of average fragment sizes obtained using the plug-in to those generated by TapeStation output for the same DNA fractions: e low molecular-weight, and f high molecular-weight (Table S6 in table form).
    Figure Legend Snippet: Tagmented libraries prepared from C. elegans genomic DNA. Gel images show the sections corresponding to a low-molecular-weight and b high-molecular-weight fractions. In both cases, lanes 2 and 3 are λ - Aci I digest samples; lanes 4-7 C. elegans genomic-DNA tagmentation reactions amplified by PCR under different conditions. Summary plot of average fragment size based on short- or long-exposure images, c low-molecular-weight, and d high-molecular-weight fractions (Table S3 in table form). Comparison of average fragment sizes obtained using the plug-in to those generated by TapeStation output for the same DNA fractions: e low molecular-weight, and f high molecular-weight (Table S6 in table form).

    Techniques Used: Molecular Weight, Amplification, Polymerase Chain Reaction, Generated

    15) Product Images from "Hypermethylation of miR-203 in endometrial carcinomas"

    Article Title: Hypermethylation of miR-203 in endometrial carcinomas

    Journal: Gynecologic oncology

    doi: 10.1016/j.ygyno.2014.02.009

    miR-203 is a novel hypermethylated marker in endometrial cancer. (A) The diagram of predicted miRNA binding sites on SOX4 3′-UTR. (B) Summary of the methylation status by COBRA of thirteen miRNA regions in endometrial cancer cell lines (from left to right: AN3CA, Ishikawa, HEC1A, KLE, RL95-2 and SK-UT-1B) and one normal (N) pooled sample derived from two noncancerous endometria as a negative control. m: methylated; and u: unmethylated. (C) Hypermethylation of miR-203 in endometrial cancer cell lines, as revealed by COBRA analysis. E6/E7: normal endometrial cell line; Sss I, methylated positive control; N: normal endometrium. +, Aci I restriction enzyme added; and −, without Aci I.
    Figure Legend Snippet: miR-203 is a novel hypermethylated marker in endometrial cancer. (A) The diagram of predicted miRNA binding sites on SOX4 3′-UTR. (B) Summary of the methylation status by COBRA of thirteen miRNA regions in endometrial cancer cell lines (from left to right: AN3CA, Ishikawa, HEC1A, KLE, RL95-2 and SK-UT-1B) and one normal (N) pooled sample derived from two noncancerous endometria as a negative control. m: methylated; and u: unmethylated. (C) Hypermethylation of miR-203 in endometrial cancer cell lines, as revealed by COBRA analysis. E6/E7: normal endometrial cell line; Sss I, methylated positive control; N: normal endometrium. +, Aci I restriction enzyme added; and −, without Aci I.

    Techniques Used: Marker, Binding Assay, Methylation, Combined Bisulfite Restriction Analysis Assay, Derivative Assay, Negative Control, Positive Control

    16) Product Images from "Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus"

    Article Title: Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0910955107

    Retrotransposon excision is associated with demethylation of the PvGlo locus. ( A ) Organization of wild-type, transposon, and revertant alleles of PvGlo showing location of upstream methylation-sensitive AciI restriction enzyme sites. The PvGlo -specific primers (F1, R1, R3) and transposon-specific PCR primers (R2, F2) are indicated. The retrotransposon insertion site is shown (-723); LTRs are shown in gray. ( B ) Methylation-sensitive PCR analysis of the wild-type, transposon, and revertant alleles of PvGlo using allele-specific primer combinations as indicated. Primer specificity is demonstrated on DNA from leaves of wild-type (wt) and homozygous Hose in Hose (HH) plants. DNA samples from whorl-1 tissue or combined whorl-2, -3, and -4 tissue from revertant (R), semirevertant (SR), and Hose in Hose (H) flowers from the same plant were either digested with Aci1 or uncut as indicated before PCR amplification using allele-specific primer combinations as shown before fractionation by agarose gel electrophoresis. The different alleles amplified by each primer combination are indicated and the excision allele-specific PCR product is highlighted (*). PCR products generated from uncut genomic DNA that are absent after predigestion with AciI reveal unmethylated sites.
    Figure Legend Snippet: Retrotransposon excision is associated with demethylation of the PvGlo locus. ( A ) Organization of wild-type, transposon, and revertant alleles of PvGlo showing location of upstream methylation-sensitive AciI restriction enzyme sites. The PvGlo -specific primers (F1, R1, R3) and transposon-specific PCR primers (R2, F2) are indicated. The retrotransposon insertion site is shown (-723); LTRs are shown in gray. ( B ) Methylation-sensitive PCR analysis of the wild-type, transposon, and revertant alleles of PvGlo using allele-specific primer combinations as indicated. Primer specificity is demonstrated on DNA from leaves of wild-type (wt) and homozygous Hose in Hose (HH) plants. DNA samples from whorl-1 tissue or combined whorl-2, -3, and -4 tissue from revertant (R), semirevertant (SR), and Hose in Hose (H) flowers from the same plant were either digested with Aci1 or uncut as indicated before PCR amplification using allele-specific primer combinations as shown before fractionation by agarose gel electrophoresis. The different alleles amplified by each primer combination are indicated and the excision allele-specific PCR product is highlighted (*). PCR products generated from uncut genomic DNA that are absent after predigestion with AciI reveal unmethylated sites.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Fractionation, Agarose Gel Electrophoresis, Generated

    17) Product Images from "Molecular Identification of Mucor and Lichtheimia Species in Pure Cultures of Zygomycetes"

    Article Title: Molecular Identification of Mucor and Lichtheimia Species in Pure Cultures of Zygomycetes

    Journal: Jundishapur Journal of Microbiology

    doi: 10.5812/jjm.35237

    Agarose Gel Electrophoresis of 18sS rRNA PCR Products of Different Mucorals After Restriction Digestion With XmnI and AcII Lanes 12, 11, 10, 9, 8, 2 and 13, M. circinelloides , M. racemosus , M. ramosissimus or M. plumbeus ; Lanes 3, 1, 7, 6, 5, 4, and 14, L. corymbifera or L. blakesleeana ; N, negative control; M, 100 bp molecular size marker.
    Figure Legend Snippet: Agarose Gel Electrophoresis of 18sS rRNA PCR Products of Different Mucorals After Restriction Digestion With XmnI and AcII Lanes 12, 11, 10, 9, 8, 2 and 13, M. circinelloides , M. racemosus , M. ramosissimus or M. plumbeus ; Lanes 3, 1, 7, 6, 5, 4, and 14, L. corymbifera or L. blakesleeana ; N, negative control; M, 100 bp molecular size marker.

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

    18) Product Images from "Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells"

    Article Title: Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells

    Journal: BMC Molecular Biology

    doi: 10.1186/1471-2199-15-24

    DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.
    Figure Legend Snippet: DNA methylation of GBGT1 promoter region. (A) Illustration of the organization of the CpG island encompassing the transcription start site of the GBGT1 gene. The horizontal black line represents the 217 bp amplicon generated using COBRA and bisulfite sequencing. Vertical lines represent the organization of individual CpG dinucleotides within the amplified region. Black triangles indicate the respective recognition sites of the restriction endonucleases ( Acil, Pvul and BsaAl ). Each of these enzymes digested the amplicon only when the respective CpG dinucleotide(s) within the enzyme recognition site was methylated in the original DNA prior to bisulfite conversion ( AciI -active 50 + 176 bp; PvuI -active 37 + 180 bp; BsaAI -active 192 + 25 bp). (B) COBRA with each of the three different endonucleases ( Acil, Pvul, BsaAl ) revealed considerable variation in the methylation status among the cell lines. The degree of methylation (which relates to the intensity of the lower digested bands as compared to the upper undigested band) was consistent between each restriction enzyme for each cell line. (C) Methylation profiles of individual CpG sites from single DNA strands derived from bisulfite sequencing in A2780, HOSE17-1, OVCAR3, and SKOV3. Columns represent individual CpG sites. Rows represent number of sequenced clones (n = 12). Methylated CpG (black), unmethylated (grey), unknown status (white). (D) Restoration of GBGT1 expression in A2780 induced by treatment with 2.5 μM 5-Aza. RT-qPCR shows a time-dependent increase in GBGT1 transcription. Left, data are presented as the number of PCR products in 5-Aza treated samples relative to the mock-treated control (y-axis) as a function of time (24 h, 48 h, 72 h) after treatment (x-axis). Right, RT-qPCR products of GBGT1 and normalization control YWHAZ . (E) Western blot (autoradiograph and corresponding quantitative analysis) showing 5-Aza -induced increase in GBGT1 protein expression as a function of time after treatment in A2780.

    Techniques Used: DNA Methylation Assay, Amplification, Generated, Combined Bisulfite Restriction Analysis Assay, Methylation Sequencing, Methylation, Derivative Assay, Clone Assay, Expressing, Quantitative RT-PCR, Polymerase Chain Reaction, Western Blot, Autoradiography

    19) Product Images from "Comparison between single and multi-locus approaches for specimen identification in Mytilus mussels"

    Article Title: Comparison between single and multi-locus approaches for specimen identification in Mytilus mussels

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-55855-8

    Restriction map of markers RFLP-PCR ( a ) Me15-16 Aci I, ( b ) ITS Hha I, ( c ) CO I Xba I and ( d ) 16S rRNA EcoR V, Nhe I and Spe I. *Is used to identify the new haplotypes found in this work. For clarity, we will conserve the name M. galloprovincialis to refer the former Northern Hemisphere haplotype and use M. chilensis for the former Southern Hemisphere haplotype.
    Figure Legend Snippet: Restriction map of markers RFLP-PCR ( a ) Me15-16 Aci I, ( b ) ITS Hha I, ( c ) CO I Xba I and ( d ) 16S rRNA EcoR V, Nhe I and Spe I. *Is used to identify the new haplotypes found in this work. For clarity, we will conserve the name M. galloprovincialis to refer the former Northern Hemisphere haplotype and use M. chilensis for the former Southern Hemisphere haplotype.

    Techniques Used: Polymerase Chain Reaction, Northern Blot

    Heatmaps indicating concordance (% of individuals) in species identification between PCR-RFLP Me15-16 Aci I and each of the other PCR-RFLP markers evaluated ( ITS, CO I and 16S rRNA ) and mac -1.
    Figure Legend Snippet: Heatmaps indicating concordance (% of individuals) in species identification between PCR-RFLP Me15-16 Aci I and each of the other PCR-RFLP markers evaluated ( ITS, CO I and 16S rRNA ) and mac -1.

    Techniques Used: Polymerase Chain Reaction

    Locations and codes for the six sampling sites. Codes for locations can be found in Table S5 . Color indicates species as determined using the PCR-RFLP Me15-16 Aci I assay: red for Mytilus chilensis , orange for Mytilus galloprovincialis , blue for Mytilus edulis and black for Mytilus trossulus . Background topographic map from GeoMapApp ( http://www.geomapapp.org ).
    Figure Legend Snippet: Locations and codes for the six sampling sites. Codes for locations can be found in Table S5 . Color indicates species as determined using the PCR-RFLP Me15-16 Aci I assay: red for Mytilus chilensis , orange for Mytilus galloprovincialis , blue for Mytilus edulis and black for Mytilus trossulus . Background topographic map from GeoMapApp ( http://www.geomapapp.org ).

    Techniques Used: Sampling, Polymerase Chain Reaction

    20) Product Images from "Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming"

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1003636

    The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.
    Figure Legend Snippet: The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Techniques Used: Expressing, Ligation, Polymerase Chain Reaction, Amplification

    21) Product Images from "Epigenetic Repression of microRNA-129-2 Leads to Overexpression of SOX4 Oncogene in Endometrial Cancer"

    Article Title: Epigenetic Repression of microRNA-129-2 Leads to Overexpression of SOX4 Oncogene in Endometrial Cancer

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-09-1499

    Reactivation of miR-129-2 in cancer cells by pharmacological induction of hyperacetylation and DNA demethylation lead to reduced SOX4 expression at both the mRNA and protein levels. A , genomic map of miR-129-2 CpG island and amplicon. Bar under line, CpG site; ↓, AciI cutting sites. B , COBRA analysis in endometrial cancer cell lines. u, unmethylated band; m, methylated bands; Sss I, 100% methylated control; Blood, a mix of 4 normal peripheral blood samples as negative control; +, Aci I restriction enzyme added; -, without Aci I. C , relative expression levels of miR-129-3p in endometrial cancer cell lines treated with DAC and/or TSA in relation to untreated controls was determined by RT-qPCR analysis. RNU48 was used as internal control gene. Error bar , SD; *, P
    Figure Legend Snippet: Reactivation of miR-129-2 in cancer cells by pharmacological induction of hyperacetylation and DNA demethylation lead to reduced SOX4 expression at both the mRNA and protein levels. A , genomic map of miR-129-2 CpG island and amplicon. Bar under line, CpG site; ↓, AciI cutting sites. B , COBRA analysis in endometrial cancer cell lines. u, unmethylated band; m, methylated bands; Sss I, 100% methylated control; Blood, a mix of 4 normal peripheral blood samples as negative control; +, Aci I restriction enzyme added; -, without Aci I. C , relative expression levels of miR-129-3p in endometrial cancer cell lines treated with DAC and/or TSA in relation to untreated controls was determined by RT-qPCR analysis. RNU48 was used as internal control gene. Error bar , SD; *, P

    Techniques Used: Expressing, Amplification, Combined Bisulfite Restriction Analysis Assay, Methylation, Negative Control, Quantitative RT-PCR

    22) Product Images from "Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology"

    Article Title: Germline genes hypomethylation and expression define a molecular signature in peripheral blood of ICF patients: implications for diagnosis and etiology

    Journal: Orphanet Journal of Rare Diseases

    doi: 10.1186/1750-1172-9-56

    Relative DNA methylation levels at germline gene promoters in whole blood and buccal swabs from ICF patients. Methylation analysis in whole blood (A) and buccal swabs (B) were assessed by Methylation-Sensitive Restriction Assay, followed by qRT-PCR amplification of the AciI digested product with primers flanking at least two AciI sites within the promoter CpG island. A non-cutter NcoI control digest served to normalize data that are presented as a percentage of methylation relative to the control digest. ICF subtypes 1, 2, and X are indicated and separated by dotted lines. For the X-linked gene TEX11, female patients are indicated as black bars. Raw data used to built this Figure can be found in Additional file 8 . WB and BS are control whole blood DNA and buccal swabs from healthy donor, respectively. Error bars represent standard error.
    Figure Legend Snippet: Relative DNA methylation levels at germline gene promoters in whole blood and buccal swabs from ICF patients. Methylation analysis in whole blood (A) and buccal swabs (B) were assessed by Methylation-Sensitive Restriction Assay, followed by qRT-PCR amplification of the AciI digested product with primers flanking at least two AciI sites within the promoter CpG island. A non-cutter NcoI control digest served to normalize data that are presented as a percentage of methylation relative to the control digest. ICF subtypes 1, 2, and X are indicated and separated by dotted lines. For the X-linked gene TEX11, female patients are indicated as black bars. Raw data used to built this Figure can be found in Additional file 8 . WB and BS are control whole blood DNA and buccal swabs from healthy donor, respectively. Error bars represent standard error.

    Techniques Used: DNA Methylation Assay, Methylation, Restriction Assay, Quantitative RT-PCR, Amplification, Western Blot

    23) Product Images from "Epigenetic regulation of matrix metalloproteinase expression in ameloblastoma"

    Article Title: Epigenetic regulation of matrix metalloproteinase expression in ameloblastoma

    Journal: BMC Clinical Pathology

    doi: 10.1186/1472-6890-12-11

    Representative figure of the methylation analysis. A : Methylation status of MMP-2 in ameloblastoma. M: PCR products when amplified by methylated primers (205 bp); U: PCR products when amplified by unmethylated primers (206 bp); +M: positive control for methylated reaction; +U: positive control for unmethylated reaction. -M and -U: negative controls without DNA. Lines 1 to 3 represent DNA from ameloblastoma samples. B : Methylation status of MMP-9 in ameloblastoma. DNA samples were digested by the AciI restriction enzyme followed by PCR, flanking the restriction sites. Absent band indicates unmethylated profile (U) due to DNA cleavage by the restriction enzyme. Presence of the PCR band represents methylated profile (M) of the MMP-9 gene. +M: methylated positive control; +U: unmethylated positive control; - : negative control without DNA.
    Figure Legend Snippet: Representative figure of the methylation analysis. A : Methylation status of MMP-2 in ameloblastoma. M: PCR products when amplified by methylated primers (205 bp); U: PCR products when amplified by unmethylated primers (206 bp); +M: positive control for methylated reaction; +U: positive control for unmethylated reaction. -M and -U: negative controls without DNA. Lines 1 to 3 represent DNA from ameloblastoma samples. B : Methylation status of MMP-9 in ameloblastoma. DNA samples were digested by the AciI restriction enzyme followed by PCR, flanking the restriction sites. Absent band indicates unmethylated profile (U) due to DNA cleavage by the restriction enzyme. Presence of the PCR band represents methylated profile (M) of the MMP-9 gene. +M: methylated positive control; +U: unmethylated positive control; - : negative control without DNA.

    Techniques Used: Methylation, Polymerase Chain Reaction, Amplification, Positive Control, Negative Control

    24) Product Images from "Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications"

    Article Title: Deconvolution of nucleic-acid length distributions: a gel electrophoresis analysis tool and applications

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz534

    Dependence of plug-in output on camera hardware. Estimates of average fragment size obtained for images of the low-resolution gel (Figure 2C collected using four different camera systems, C1–C4 (Table 1 ). The plug-in measurements of average fragment size were compared for the known λ- Aci I digest and the same λ-genome tagmented samples analyzed in Figure 2 . Error bars indicate the standard deviation (i.e. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\sqrt{\text{variance}}$\end{document} ) of the fitted distribution. Numerical data for this experiment are also provided in Supplementary Table S2 .
    Figure Legend Snippet: Dependence of plug-in output on camera hardware. Estimates of average fragment size obtained for images of the low-resolution gel (Figure 2C collected using four different camera systems, C1–C4 (Table 1 ). The plug-in measurements of average fragment size were compared for the known λ- Aci I digest and the same λ-genome tagmented samples analyzed in Figure 2 . Error bars indicate the standard deviation (i.e. \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{upgreek} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} }{}$\sqrt{\text{variance}}$\end{document} ) of the fitted distribution. Numerical data for this experiment are also provided in Supplementary Table S2 .

    Techniques Used: Standard Deviation

    Data used to examine the effect of fragment-density test. ( A ) The gel was analyzed considering the portions delimited by the ROIs, using lane 1 (blue) as the reference ladder. ROIs 2–6 are tagmentation products of human genomic DNA; 7 and 8 are λ- Aci I digest samples ( B ). Summary plot of the fit for the gel in panel (A). Fragment-size mean and standard-deviation values for model distributions containing varying numbers of reference fragments (peaks). Symbols are colored according to the proportion of the original λ- Aci I basis fragments retained in the reference distribution; thus, 1:2 means that every other peak was eliminated from the fit, 2:3 every third peak etc. Data are also reported in Supplementary Table S4 .
    Figure Legend Snippet: Data used to examine the effect of fragment-density test. ( A ) The gel was analyzed considering the portions delimited by the ROIs, using lane 1 (blue) as the reference ladder. ROIs 2–6 are tagmentation products of human genomic DNA; 7 and 8 are λ- Aci I digest samples ( B ). Summary plot of the fit for the gel in panel (A). Fragment-size mean and standard-deviation values for model distributions containing varying numbers of reference fragments (peaks). Symbols are colored according to the proportion of the original λ- Aci I basis fragments retained in the reference distribution; thus, 1:2 means that every other peak was eliminated from the fit, 2:3 every third peak etc. Data are also reported in Supplementary Table S4 .

    Techniques Used: Standard Deviation

    Tagmented libraries prepared from C. elegans genomic DNA. Gel images show the sections corresponding to panel ( A ) low-molecular-weight and panel ( B ) high-molecular-weight fractions. In both cases, lanes 2 and 3 are λ- Aci I digest samples; lanes 4–7 C. elegans genomic-DNA tagmentation reactions amplified by PCR under different conditions. Summary plot of average fragment size based on short- or long-exposure images, panel ( C ) low-molecular-weight and panel ( D ) high-molecular-weight fractions ( Supplementary Table S3 in table form). Comparison of average fragment sizes obtained using the plug-in to those generated by TapeStation output for the same DNA fractions: panel ( E ) low molecular weight and panel ( F ) high molecular weight ( Supplementary Table S6 in table form).
    Figure Legend Snippet: Tagmented libraries prepared from C. elegans genomic DNA. Gel images show the sections corresponding to panel ( A ) low-molecular-weight and panel ( B ) high-molecular-weight fractions. In both cases, lanes 2 and 3 are λ- Aci I digest samples; lanes 4–7 C. elegans genomic-DNA tagmentation reactions amplified by PCR under different conditions. Summary plot of average fragment size based on short- or long-exposure images, panel ( C ) low-molecular-weight and panel ( D ) high-molecular-weight fractions ( Supplementary Table S3 in table form). Comparison of average fragment sizes obtained using the plug-in to those generated by TapeStation output for the same DNA fractions: panel ( E ) low molecular weight and panel ( F ) high molecular weight ( Supplementary Table S6 in table form).

    Techniques Used: Molecular Weight, Amplification, Polymerase Chain Reaction, Generated

    25) Product Images from "Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0194036

    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′ ).
    Figure Legend 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′ ).

    Techniques Used: Methylation, DNA Methylation Assay, Microarray

    26) Product Images from "Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii"

    Article Title: Genetic and Physical Mapping of DNA Replication Origins in Haloferax volcanii

    Journal: PLoS Genetics

    doi: 10.1371/journal.pgen.0030077

    DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.
    Figure Legend Snippet: DNA Replication Origin on Contig 454 Is on Chromosomes pHV1 and pHV4 (A) Sequence features of the ARS element isolated from genomic libraries of WR340 DNA. Coordinates of plasmid inserts generated by HpaII and AciI digestion are shown (including insert in pTA194), in addition to the minimal ARS element determined by AciI digestion (in pTA250) or PCR amplification (in pCN12). Numbering refers to TIGR contig 454 (pHV1). (B) Sequence of repeats found at the intergenic region of the pHV1/4 replication origin (correspond to numbered arrows in Figure 1 A). Orientation is indicated by arrows (righthand side) and conserved sequences are shaded. Six of the 13 repeats feature a complete ORB element (boxed), while a core mini-ORB element is conserved in all repeats. The sequence motif found in repeats surrounding the DUE is indicated by the dashed box. (C) Southern blot of PFG of intact DNA from strains H53 and H230, probed with HpaII ARS insert from pTA194, or intergenic region replaced by trpA in pTA266 (see Figure 1 D). (D) Intergenic region of ori-pHV1/4 was replaced by trpA marker by using the deletion construct pTA266. H. volcanii H53 was transformed with pTA266 to generate H220, which was used to derive the pHV1/4 origin deletion strain H230. Predicted fragment sizes of StuI digest are indicated. (E) StuI digest of genomic DNA from strains H53, H220, and H230, probed with DNA flanking the intergenic region. The band indicated * represents episomal DNA carrying the pHV1/4 origin, resulting from excision of the integrated plasmid.

    Techniques Used: Sequencing, Isolation, Plasmid Preparation, Generated, Polymerase Chain Reaction, Amplification, Southern Blot, Marker, Construct, Transformation Assay

    DNA Replication Origin on the Main Chromosome: oriC1 (A) Sequence features of the ARS element isolated from genomic libraries of H230 DNA, including selected genes (see text for details). Coordinates of plasmid inserts generated by AciI digestion are shown (including insert in pTA313), in addition to the minimal ARS element in pTA441 and pCN11. See Figure 1 A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Above the line are sequences of repeats found at the intergenic region of the H. volcanii chromosomal replication origin (correspond to numbered arrows in Figure 2 A). Below the line are sequences of repeats found at other (presumed) archaeal origins. The species and relevant cdc6/orc1 genes are H. marismortui cdc6–4 (Hmar-1–2), Halobacterium sp. NRC-1 orc7 (NRC1-1-2), Natronomonas pharaonis cdc6–1 (Npha-1–2), S. solfataricus cdc6–1 (Sso-1–2), and P. abyssi cdc6 (Pab-1–4). The orientation is indicated by arrows and conserved positions are shaded. Among halophilic archaea, repeats surrounding the primary DUE feature a longer consensus sequence (Halo-ORB, boxed), which contains the core mini-ORB and “G-string” elements also found in other archaea, plus a halophile-specific “G-string.” (C) Southern blot of PFG of DNA from strain H53, probed with the AciI ARS insert from pTA313.
    Figure Legend Snippet: DNA Replication Origin on the Main Chromosome: oriC1 (A) Sequence features of the ARS element isolated from genomic libraries of H230 DNA, including selected genes (see text for details). Coordinates of plasmid inserts generated by AciI digestion are shown (including insert in pTA313), in addition to the minimal ARS element in pTA441 and pCN11. See Figure 1 A for key. Numbering refers to TIGR contig number 455. Main chromosome, Chr. (B) Above the line are sequences of repeats found at the intergenic region of the H. volcanii chromosomal replication origin (correspond to numbered arrows in Figure 2 A). Below the line are sequences of repeats found at other (presumed) archaeal origins. The species and relevant cdc6/orc1 genes are H. marismortui cdc6–4 (Hmar-1–2), Halobacterium sp. NRC-1 orc7 (NRC1-1-2), Natronomonas pharaonis cdc6–1 (Npha-1–2), S. solfataricus cdc6–1 (Sso-1–2), and P. abyssi cdc6 (Pab-1–4). The orientation is indicated by arrows and conserved positions are shaded. Among halophilic archaea, repeats surrounding the primary DUE feature a longer consensus sequence (Halo-ORB, boxed), which contains the core mini-ORB and “G-string” elements also found in other archaea, plus a halophile-specific “G-string.” (C) Southern blot of PFG of DNA from strain H53, probed with the AciI ARS insert from pTA313.

    Techniques Used: Sequencing, Isolation, Plasmid Preparation, Generated, Southern Blot

    27) Product Images from "Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System"

    Article Title: Small Fragment Homologous Replacement: Evaluation of Factors Influencing Modification Efficiency in an Eukaryotic Assay System

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0030851

    HpaII and AciI methylation analyses of integrated eGFP in C1 and D1 clones. A) Experimental design showing the amplicon regions and their length within eGFP locus integrated in genomic DNA. HpaII and AciI site are indicated. B) Densitometric analyses of parental C1 clone methylation pattern on eGFP + more positive, eGFP + less positive and eGFP − cells (see also Fig. S9B and Fig. S10B ). ANOVA test gave a statistical significance of p
    Figure Legend Snippet: HpaII and AciI methylation analyses of integrated eGFP in C1 and D1 clones. A) Experimental design showing the amplicon regions and their length within eGFP locus integrated in genomic DNA. HpaII and AciI site are indicated. B) Densitometric analyses of parental C1 clone methylation pattern on eGFP + more positive, eGFP + less positive and eGFP − cells (see also Fig. S9B and Fig. S10B ). ANOVA test gave a statistical significance of p

    Techniques Used: Methylation, Amplification

    28) Product Images from "Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing"

    Article Title: Staphylococcus aureus virulence genes identified by bursa aurealis mutagenesis and nematode killing

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.0404728101

    Plasmids used for transposon mutagenesis in S. aureus . ( A ) Bursa aurealis , a minimariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon ( rep ts ) and chloramphenicol resistance gene ( cat ) to generate pBursa; the 3.2-kb bursa aurealis encompasses the mariner terminal inverted repeats (TIR), the green fluorescent protein gene ( gfp ), the R6K replication origin ( oriV ), and the erythromycin-resistance determinant ( ermB ). The position of restriction enzyme recognition sites ( Aci I and Bam HI) is indicated. ( B ) Plasmid pFA545 encodes the mariner transposase ( tnp ), the origin of replication ( repC ), and the tetracycline- and ampicillin-resistance markers ( tetBD and bla , respectively). ( C ) The structure of the 5.3-kb transposon Tn 917 with the ermB erythromycin-resistance determinant, tnpR resolvase, and tnpA transposase is shown.
    Figure Legend Snippet: Plasmids used for transposon mutagenesis in S. aureus . ( A ) Bursa aurealis , a minimariner transposable element, was cloned into pTS2, with a temperature-sensitive plasmid replicon ( rep ts ) and chloramphenicol resistance gene ( cat ) to generate pBursa; the 3.2-kb bursa aurealis encompasses the mariner terminal inverted repeats (TIR), the green fluorescent protein gene ( gfp ), the R6K replication origin ( oriV ), and the erythromycin-resistance determinant ( ermB ). The position of restriction enzyme recognition sites ( Aci I and Bam HI) is indicated. ( B ) Plasmid pFA545 encodes the mariner transposase ( tnp ), the origin of replication ( repC ), and the tetracycline- and ampicillin-resistance markers ( tetBD and bla , respectively). ( C ) The structure of the 5.3-kb transposon Tn 917 with the ermB erythromycin-resistance determinant, tnpR resolvase, and tnpA transposase is shown.

    Techniques Used: Mutagenesis, Clone Assay, Plasmid Preparation

    Mapping insertion sites by inverse PCR. Fifteen bursa aurealis transposon mutants of S. aureus strain Newman were subjected to DNA purification, Aci I restriction, fragment ligation, inverse PCR, and agarose gel electrophoresis. M indicates the molecular weight marker (1-kb DNA ladder).
    Figure Legend Snippet: Mapping insertion sites by inverse PCR. Fifteen bursa aurealis transposon mutants of S. aureus strain Newman were subjected to DNA purification, Aci I restriction, fragment ligation, inverse PCR, and agarose gel electrophoresis. M indicates the molecular weight marker (1-kb DNA ladder).

    Techniques Used: Inverse PCR, DNA Purification, Ligation, Agarose Gel Electrophoresis, Molecular Weight, Marker

    29) Product Images from "Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells"

    Article Title: Bisulfite-independent analysis of CpG island methylation enables genome-scale stratification of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkx026

    Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.
    Figure Legend Snippet: Schematic representation of CGI-seq. TEST: single cell being studied. Methylation control (MC): cells that are used to define the detectable CGIs or any other accessible methylated regions (AMRs), sharing the same genome as the TEST. For CGI-seq analysis, the genomic DNA is classified into three classes of sequences: Me-CGIs (representing methylated CGIs, and more generally, Me-AMRs) indicated as a red line, Um-CGIs (unmethylated CGIs, and generally Um-AMRs) indicated as a blue line and undetectable regions indicated as a black line. Green lines are amplified sequences. Solid dot: Me-CpG sites. Hollow dot: Um-CpG sites. The intact gDNA is released from cell (step 1) and digested with RE1 (TEST) or no digestion (MC) (step 2), followed by MDA amplification (step 3). The amplification product is then digested by RE2 (step 4), in which the short fragments are converted t‘o a library and subjected to massive sequencing (step 5). The reads are aligned to the genome (step 6) and the methylation status of the AMRs (particularly CGIs) is determined (‘Materials and Methods’ section). The AMRs detected in both the TEST and the MC are called Me-CGIs (or Me-AMRs). The AMRs uniquely detected in the MC but not in the TEST are called Um-CGIs (or Um-AMRs) of the TEST. RE1 (four MREs in combination: Cfr42I (SacII), PdiI (NaeI), Eco52I (EagI) and PteI(BssHII), FastDigest enzyme from Thermal Scientific Inc.) distinguishes Me-CGIs (Me-AMRs) from Um-CGIs (Um-AMRs). MDA selectively depletes Um-CGIs (Um-AMRs) of the TEST and amplifies Me-CGIs (Me-AMRs). RE2 (either BstUI alone, or a combination of 3 separate REs (ABH: AciI, BstUI and Hinp1I from NEB)) generates fragments specifically enriching CGI sequences (and other CG-rich regions) from non-CGI sequences.

    Techniques Used: Methylation, Amplification, Multiple Displacement Amplification, Sequencing

    Related Articles

    Amplification:

    Article Title: Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats
    Article Snippet: .. The amplified product of the gltA gene was digested with Taq I (Promega, Madison, WI), Hha I (New England Biolabs, Beverly, M.A.), Mse I (New England Biolabs) and Aci I (New England Biolabs); the amplified product of the 16S rRNA gene was digested with Dde I (New England Biolabs); the amplified product of the ribC gene was digested with Taq I; and the amplified product of the 16S-23S ITS region was digested with Taq I and Hae III. ..

    Article Title: Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species
    Article Snippet: .. Three different restriction digests were performed on each sample by adding 7 μl of amplified product to 5 U of the following restriction enzymes in separate reactions: AciI (New England BioLabs, Beverly MA), HaeIII (Promega, Madison, WI), and MspI (Promega). ..

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
    Article Snippet: .. As a control for ligation products, genomic DNA regions covering restriction sites of interest were amplified by PCR, purified, mixed in equimolar quantities and then digested with Eco RI-HF, Hind III or Aci I (New England Biolabs) for 1.5 hours. .. Following heat inactivation at 65°C, the digested PCR products were incubated with 10 U T4 DNA-ligase (New England Biolabs) over a temperature range of 4 to 20°C overnight.

    Methylation:

    Article Title: The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive
    Article Snippet: .. To test for differential methylation within MtEIL1 in 2HA and Jemalong e.g. [ ], DNA (10 μg) was digested overnight by AciI (New England Biolabs, http://www.neb.com ) and cleaned through a PCR clean up column (Promega, http://www.promega.com ). ..

    Ligation:

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
    Article Snippet: .. As a control for ligation products, genomic DNA regions covering restriction sites of interest were amplified by PCR, purified, mixed in equimolar quantities and then digested with Eco RI-HF, Hind III or Aci I (New England Biolabs) for 1.5 hours. .. Following heat inactivation at 65°C, the digested PCR products were incubated with 10 U T4 DNA-ligase (New England Biolabs) over a temperature range of 4 to 20°C overnight.

    Purification:

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
    Article Snippet: .. As a control for ligation products, genomic DNA regions covering restriction sites of interest were amplified by PCR, purified, mixed in equimolar quantities and then digested with Eco RI-HF, Hind III or Aci I (New England Biolabs) for 1.5 hours. .. Following heat inactivation at 65°C, the digested PCR products were incubated with 10 U T4 DNA-ligase (New England Biolabs) over a temperature range of 4 to 20°C overnight.

    Incubation:

    Article Title: Considerations When Analyzing the Methylation Status of PTEN Tumor Suppressor Gene
    Article Snippet: .. Three hundred nanograms of genomic cell line DNA was incubated with 10 units of Aci I (New England Biolabs) at 37°C for 16 hours. .. Aci I digests only unmethylated DNA at its recognition sequence (5′- C↓ CGC- 3′), leaving methylated sites intact.

    Activity Assay:

    Article Title: Epstein-Barr Virus Rta-Mediated Accumulation of DNA Methylation Interferes with CTCF Binding in both Host and Viral Genomes
    Article Snippet: .. For each sample, 5 μg DNA was digested by AciI, HpaII, and HinP1I (NEB Inc.) at 37°C for 30 min, followed by heat inactivation to eliminate enzyme activity. .. EcoRI (NEB Inc.), which is insensitive to CpG methylation, was run in parallel to serve as a DNA input control.

    Polymerase Chain Reaction:

    Article Title: The 2HA line of Medicago truncatula has characteristics of an epigenetic mutant that is weakly ethylene insensitive
    Article Snippet: .. To test for differential methylation within MtEIL1 in 2HA and Jemalong e.g. [ ], DNA (10 μg) was digested overnight by AciI (New England Biolabs, http://www.neb.com ) and cleaned through a PCR clean up column (Promega, http://www.promega.com ). ..

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming
    Article Snippet: .. As a control for ligation products, genomic DNA regions covering restriction sites of interest were amplified by PCR, purified, mixed in equimolar quantities and then digested with Eco RI-HF, Hind III or Aci I (New England Biolabs) for 1.5 hours. .. Following heat inactivation at 65°C, the digested PCR products were incubated with 10 U T4 DNA-ligase (New England Biolabs) over a temperature range of 4 to 20°C overnight.

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    New England Biolabs aci i
    PCR-RFLP of a gltA gene fragment using the restriction endonuclease <t>Aci</t> I. Lanes 1, 4 and 19 show 100 BP Ladder; Lane 2, Mt Lion L27-96; Lane 3, Mt Lion L42-94; Lane 5, Mt Lion L-39-97; Lane 6, Mt Lion FM98061; Lane 7, Bobcat L08-96; Lane 8, Bobcat L17-96; Lane 9, Bobcat DS08; Lane 10, Bobcat L10-97; Lane 11, Bobcat L11-97; Lane 12, Bobcat SC443; lane 13, Bobcat DS507; Lane 14, B . henselae Type I; Lane 15, B . henselae Type II; Lane 16, B . clarridgeiae ; Lane 17, B . koehlerae ; Lane18, B . bovis (“weissii” isolate).
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    PCR-RFLP of a gltA gene fragment using the restriction endonuclease Aci I. Lanes 1, 4 and 19 show 100 BP Ladder; Lane 2, Mt Lion L27-96; Lane 3, Mt Lion L42-94; Lane 5, Mt Lion L-39-97; Lane 6, Mt Lion FM98061; Lane 7, Bobcat L08-96; Lane 8, Bobcat L17-96; Lane 9, Bobcat DS08; Lane 10, Bobcat L10-97; Lane 11, Bobcat L11-97; Lane 12, Bobcat SC443; lane 13, Bobcat DS507; Lane 14, B . henselae Type I; Lane 15, B . henselae Type II; Lane 16, B . clarridgeiae ; Lane 17, B . koehlerae ; Lane18, B . bovis (“weissii” isolate).

    Journal: PLoS ONE

    Article Title: Isolation of Bartonella henselae and Two New Bartonella Subspecies, Bartonellakoehlerae Subspecies boulouisii subsp. nov. and Bartonella koehlerae Subspecies bothieri subsp. nov. from Free-Ranging Californian Mountain Lions and Bobcats

    doi: 10.1371/journal.pone.0148299

    Figure Lengend Snippet: PCR-RFLP of a gltA gene fragment using the restriction endonuclease Aci I. Lanes 1, 4 and 19 show 100 BP Ladder; Lane 2, Mt Lion L27-96; Lane 3, Mt Lion L42-94; Lane 5, Mt Lion L-39-97; Lane 6, Mt Lion FM98061; Lane 7, Bobcat L08-96; Lane 8, Bobcat L17-96; Lane 9, Bobcat DS08; Lane 10, Bobcat L10-97; Lane 11, Bobcat L11-97; Lane 12, Bobcat SC443; lane 13, Bobcat DS507; Lane 14, B . henselae Type I; Lane 15, B . henselae Type II; Lane 16, B . clarridgeiae ; Lane 17, B . koehlerae ; Lane18, B . bovis (“weissii” isolate).

    Article Snippet: The amplified product of the gltA gene was digested with Taq I (Promega, Madison, WI), Hha I (New England Biolabs, Beverly, M.A.), Mse I (New England Biolabs) and Aci I (New England Biolabs); the amplified product of the 16S rRNA gene was digested with Dde I (New England Biolabs); the amplified product of the ribC gene was digested with Taq I; and the amplified product of the 16S-23S ITS region was digested with Taq I and Hae III.

    Techniques: Polymerase Chain Reaction

    The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Journal: PLoS Pathogens

    Article Title: Modulation of Enhancer Looping and Differential Gene Targeting by Epstein-Barr Virus Transcription Factors Directs Cellular Reprogramming

    doi: 10.1371/journal.ppat.1003636

    Figure Lengend Snippet: The influence of EBNA 3C on chromosome looping at the ADAM28/ADAMDEC1 locus. (A) Diagram (not to scale) showing the Hind III restriction fragments around the ADAM28 locus that encompass the promoter (P), the ADAM enhancer (E, located downstream of ADAM28 ) and two intervening control regions (con1 and con2). The arrow indicates the direction of transcription. (B) Chromosome conformation analysis of the ADAM28 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control regions. (C) Diagram (not to scale) showing the Aci I restriction fragments around the ADAMDEC1 locus that encompass the promoter (P), the ADAM enhancer (E, located upstream of ADAMDEC1 ) and an intervening control region (con). The arrow indicates the direction of transcription. (D) Chromosome conformation analysis of the ADAMDEC1 locus in the pz1 control BJAB cell line (−) and the E3C-3 stable EBNA 3C expressing cell line (+) using primer pairs that amplify across promoter-enhancer or promoter-control ligation junctions. Positive controls show PCR amplification from control digestion and ligation reactions carried out using PCR-amplified DNA fragments encompassing the promoter, enhancer and control region.

    Article Snippet: As a control for ligation products, genomic DNA regions covering restriction sites of interest were amplified by PCR, purified, mixed in equimolar quantities and then digested with Eco RI-HF, Hind III or Aci I (New England Biolabs) for 1.5 hours.

    Techniques: Expressing, Ligation, Polymerase Chain Reaction, Amplification

    Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.

    Journal: Applied and Environmental Microbiology

    Article Title: Comparison of Bacteroides-Prevotella 16S rRNA Genetic Markers for Fecal Samples from Different Animal Species

    doi: 10.1128/AEM.71.10.5999-6007.2005

    Figure Lengend Snippet: Examples of T-RFLP peaks resulting from cow and human fecal samples cut with AciI, MspI, and HaeIII.

    Article Snippet: Three different restriction digests were performed on each sample by adding 7 μl of amplified product to 5 U of the following restriction enzymes in separate reactions: AciI (New England BioLabs, Beverly MA), HaeIII (Promega, Madison, WI), and MspI (Promega).

    Techniques:

    Restriction enzyme analysis and parallel direct sequencing of polymerase chain reaction (PCR) products. (A) Aci I and (B) Tau I endonuclease cleavage followed by 10% polyacrylamide gel analysis. M, molecular weight marker, 100 bp DNA ladder (Gibco BRL, Burlington, Ontario, Canada). (A) Lane 1, chronic lymphocytic leukaemia (CLL) sample 1, amplified BAX promoter segment before digestion; lanes 2–5, PCR products digested with Aci I; lane 2, CLL sample 2 with a heterozygous single nucleotide polymorphism (SNP); lanes 3 and 4, CLL samples 3 and 4 with no SNP; lane 5, RL cell line with homozygous SNP. (B) PCR products digested with Tau I; lane 2, CLL sample 2 with a heterozygous SNP; lane 3, CLL sample 3 with no SNP; lane 5, RL cell line with homozygous SNP. (C) Left hand column: direct sequence of the BAX promoter region. The control sample (I) shows no alteration, whereas a CLL sample (II) shows a heterozygous SNP, and the RL cell line (III) has a homozygous SNP. Middle and right hand columns: corresponding samples digested with the Aci I and Tau I enzymes, respectively

    Journal: Molecular Pathology

    Article Title: Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia

    doi:

    Figure Lengend Snippet: Restriction enzyme analysis and parallel direct sequencing of polymerase chain reaction (PCR) products. (A) Aci I and (B) Tau I endonuclease cleavage followed by 10% polyacrylamide gel analysis. M, molecular weight marker, 100 bp DNA ladder (Gibco BRL, Burlington, Ontario, Canada). (A) Lane 1, chronic lymphocytic leukaemia (CLL) sample 1, amplified BAX promoter segment before digestion; lanes 2–5, PCR products digested with Aci I; lane 2, CLL sample 2 with a heterozygous single nucleotide polymorphism (SNP); lanes 3 and 4, CLL samples 3 and 4 with no SNP; lane 5, RL cell line with homozygous SNP. (B) PCR products digested with Tau I; lane 2, CLL sample 2 with a heterozygous SNP; lane 3, CLL sample 3 with no SNP; lane 5, RL cell line with homozygous SNP. (C) Left hand column: direct sequence of the BAX promoter region. The control sample (I) shows no alteration, whereas a CLL sample (II) shows a heterozygous SNP, and the RL cell line (III) has a homozygous SNP. Middle and right hand columns: corresponding samples digested with the Aci I and Tau I enzymes, respectively

    Article Snippet: Two restriction enzymes, Aci I (New England BioLabs Inc, Mississauga, Ontario, Canada) and Tau I (MBI Fermentas, Burlington, Ontario, Canada) were used to screen the samples for the SNP detection (fig 1 ).

    Techniques: Sequencing, Polymerase Chain Reaction, Molecular Weight, Marker, Amplification

    (A) Aci I and (B) Tau I restriction enzyme maps for the BAX promoter region.

    Journal: Molecular Pathology

    Article Title: Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia

    doi:

    Figure Lengend Snippet: (A) Aci I and (B) Tau I restriction enzyme maps for the BAX promoter region.

    Article Snippet: Two restriction enzymes, Aci I (New England BioLabs Inc, Mississauga, Ontario, Canada) and Tau I (MBI Fermentas, Burlington, Ontario, Canada) were used to screen the samples for the SNP detection (fig 1 ).

    Techniques:

    Restriction enzyme analysis (with Aci I ) for the G(−248)A single nucleotide polymorphism (SNP) in the BAX gene. M, molecular weight marker (100 bp DNA ladder; Gibco BRL); N, control sample showing three distinct bands, 352 256 bp, and 96 bp; P1, positive control with heterozygous SNP showing the 352 bp (major) and 256 bp (minor) bands and an almost invisible 96 bp band; P2, positive control with a homozygous SNP, which abolishes a restriction enzyme site, resulting in a single 352 bp band; lanes 1–5, chronic lymphocytic leukaemia cases; lanes 1, 4, and 5, heterozygous SNP; lanes 2 and 3, no SNP; lane 6, RL showing the homozygous SNP; lanes 7–12, controls without the G(−248)A SNP.

    Journal: Molecular Pathology

    Article Title: Molecular detection of the G(-248)A BAX promoter nucleotide change in B cell chronic lymphocytic leukaemia

    doi:

    Figure Lengend Snippet: Restriction enzyme analysis (with Aci I ) for the G(−248)A single nucleotide polymorphism (SNP) in the BAX gene. M, molecular weight marker (100 bp DNA ladder; Gibco BRL); N, control sample showing three distinct bands, 352 256 bp, and 96 bp; P1, positive control with heterozygous SNP showing the 352 bp (major) and 256 bp (minor) bands and an almost invisible 96 bp band; P2, positive control with a homozygous SNP, which abolishes a restriction enzyme site, resulting in a single 352 bp band; lanes 1–5, chronic lymphocytic leukaemia cases; lanes 1, 4, and 5, heterozygous SNP; lanes 2 and 3, no SNP; lane 6, RL showing the homozygous SNP; lanes 7–12, controls without the G(−248)A SNP.

    Article Snippet: Two restriction enzymes, Aci I (New England BioLabs Inc, Mississauga, Ontario, Canada) and Tau I (MBI Fermentas, Burlington, Ontario, Canada) were used to screen the samples for the SNP detection (fig 1 ).

    Techniques: Molecular Weight, Marker, Positive Control