nla iii  (New England Biolabs)


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    NlaIII
    Description:
    NlaIII 2 500 units
    Catalog Number:
    r0125l
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    282
    Size:
    2 500 units
    Category:
    Restriction Enzymes
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    New England Biolabs nla iii
    NlaIII
    NlaIII 2 500 units
    https://www.bioz.com/result/nla iii/product/New England Biolabs
    Average 99 stars, based on 168 article reviews
    Price from $9.99 to $1999.99
    nla iii - by Bioz Stars, 2020-09
    99/100 stars

    Images

    1) Product Images from "Type II restriction modification system in Ureaplasma parvum OMC-P162 strain"

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205328

    Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.
    Figure Legend Snippet: Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.

    Techniques Used: Activity Assay, Amplification, Polymerase Chain Reaction, Mutagenesis

    DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.
    Figure Legend Snippet: DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.

    Techniques Used: Plasmid Preparation

    Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.
    Figure Legend Snippet: Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.

    Techniques Used: Plasmid Preparation, Electrophoresis

    2) Product Images from "Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA"

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

    Journal: Genome Research

    doi: 10.1101/gr.306102

    Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.
    Figure Legend Snippet: Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.

    Techniques Used:

    3) Product Images from "Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle"

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.19-0095

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.
    Figure Legend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico

    4) Product Images from "Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle"

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.19-0095

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.
    Figure Legend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico

    5) Product Images from "Exploiting native forces to capture chromosome conformation in mammalian cell nuclei"

    Article Title: Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

    Journal: Molecular Systems Biology

    doi: 10.15252/msb.20167311

    Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.
    Figure Legend Snippet: Features of i3C performed in HUVEC s Overview of the i3C protocol. Living cells are harvested in a close‐to‐physiological buffer (PB; step 1); intact nuclei isolated by mild NP‐40 treatment (step 2); chromatin digested using Apo I or Nla III, nuclei spun to release unattached chromatin (step 3); and leave cut chromatin bound to the nuclear substructure (step 4). Then, ligation takes places in situ , and DNA is isolated (step 5). Percentage of total cell chromatin present at the different steps of the procedure (± SD; n = 2). Relative contribution of the different HUVEC ChromHMM features in each i3C fraction. i4C‐seq (blue shades) and conventional 4C (gray shades) were performed side by side in HUVECs, using Apo I and the SAMD4A TSS as a viewpoint (triangle); profiles from two replicates are overlaid. The browser view shows interactions in the ˜1 Mbp around SAMD4A . The zoom‐in shows interactions in the SAMD4A TAD (gray rectangle). Strong (red) and intermediate (brown) interactions called by fourSig , RefSeq gene models, and ENCODE ChIP‐seq data are shown below.

    Techniques Used: Isolation, Ligation, In Situ, Chromatin Immunoprecipitation

    Native interactions are confined by TAD boundaries and describe prelooping i4C‐seq was performed in HUVECs using Nla III and the TSSs of BMP4 , CDKN3 , CNIH , and SAMD4A as viewpoints (triangles). Interactions are shown aligned to TAD boundaries (gray rectangles; from Dixon et al , 2012 ) and HUVEC ENCODE ChIP‐seq data (below). Prelooping of the SAMD4A and BMP4 TNF‐responsive TSSs to enhancers is indicated (orange lines).
    Figure Legend Snippet: Native interactions are confined by TAD boundaries and describe prelooping i4C‐seq was performed in HUVECs using Nla III and the TSSs of BMP4 , CDKN3 , CNIH , and SAMD4A as viewpoints (triangles). Interactions are shown aligned to TAD boundaries (gray rectangles; from Dixon et al , 2012 ) and HUVEC ENCODE ChIP‐seq data (below). Prelooping of the SAMD4A and BMP4 TNF‐responsive TSSs to enhancers is indicated (orange lines).

    Techniques Used: Chromatin Immunoprecipitation

    6) Product Images from "Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle"

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.19-0095

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.
    Figure Legend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico

    7) Product Images from "Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle"

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.19-0095

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.
    Figure Legend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico

    8) Product Images from "Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum"

    Article Title: Gene expression analysis in cadmium-stressed roots of a low cadmium-accumulating solanaceous plant, Solanum torvum

    Journal: Journal of Experimental Botany

    doi: 10.1093/jxb/erp313

    A schematic diagram of the procedure for expression tag library preparation. Double-stranded cDNA was synthesized from mRNA using a biotinylated adaptor oligo(dT) primer. After a digestion with Nla III, the 3′ end cDNA fragments anchored to streptavidin magnetic beads were ligated to the adaptor 1_GT, the adaptor 1_CT, the adaptor 1_AC, and the adaptor 1_TC for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively. The adaptor 1-ligated cDNA fragments were digested with Eco P15I, and the released fragments were ligated to the adaptor 2. The adaptor 1-tag–adator 2 fragments were amplified using the GEX PCR primer set. The amplified fragments (∼104 bp) were separated by PAGE. Four independent library solutions (Cd0, Cd3h, Cd1d, and Cd3d) were mixed in an equal amount of DNA, and the blended library solution was subjected to high-throughput sequencing using an Illumina Genome Analyzer. Underlined characters indicate Nla III and Eco P15I recognition sites. Bold ‘XX’ indicates library coding nucleotides, and ‘GT’, ‘CT’, ‘AC’, and ‘TC’ were used for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively.
    Figure Legend Snippet: A schematic diagram of the procedure for expression tag library preparation. Double-stranded cDNA was synthesized from mRNA using a biotinylated adaptor oligo(dT) primer. After a digestion with Nla III, the 3′ end cDNA fragments anchored to streptavidin magnetic beads were ligated to the adaptor 1_GT, the adaptor 1_CT, the adaptor 1_AC, and the adaptor 1_TC for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively. The adaptor 1-ligated cDNA fragments were digested with Eco P15I, and the released fragments were ligated to the adaptor 2. The adaptor 1-tag–adator 2 fragments were amplified using the GEX PCR primer set. The amplified fragments (∼104 bp) were separated by PAGE. Four independent library solutions (Cd0, Cd3h, Cd1d, and Cd3d) were mixed in an equal amount of DNA, and the blended library solution was subjected to high-throughput sequencing using an Illumina Genome Analyzer. Underlined characters indicate Nla III and Eco P15I recognition sites. Bold ‘XX’ indicates library coding nucleotides, and ‘GT’, ‘CT’, ‘AC’, and ‘TC’ were used for Cd0, Cd3h, Cd1d, and Cd3d libraries, respectively.

    Techniques Used: Expressing, Synthesized, Magnetic Beads, Amplification, Polymerase Chain Reaction, Polyacrylamide Gel Electrophoresis, Next-Generation Sequencing

    9) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    10) Product Images from "Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients"

    Article Title: Stabilizing mutation of CTNNB1/beta-catenin and protein accumulation analyzed in a large series of parathyroid tumors of Swedish patients

    Journal: Molecular Cancer

    doi: 10.1186/1476-4598-7-53

    Analytical restriction enzyme cleavage analysis . (A), CTNNB1 exon 3 PCR fragments (176 bp) were digested with Xmn I or Nla III that cuts only wild-type or only S37A mutant sequences, respectively. Three out of the nine parathyroid adenomas with S37A mutation were identified in our previous study [10]. Nla III cuts also outside of codon 37, close to the fragment end (23 bp). U; uncleaved CTNNB1 exon 3 PCR fragment. (B), CTNNB1 exon 3 was PCR amplified from a 1:1 mixture of constitutional DNA and tumor DNA with the S37A mutation. The fragment was analyzed by restriction enzyme digestions as in (A).
    Figure Legend Snippet: Analytical restriction enzyme cleavage analysis . (A), CTNNB1 exon 3 PCR fragments (176 bp) were digested with Xmn I or Nla III that cuts only wild-type or only S37A mutant sequences, respectively. Three out of the nine parathyroid adenomas with S37A mutation were identified in our previous study [10]. Nla III cuts also outside of codon 37, close to the fragment end (23 bp). U; uncleaved CTNNB1 exon 3 PCR fragment. (B), CTNNB1 exon 3 was PCR amplified from a 1:1 mixture of constitutional DNA and tumor DNA with the S37A mutation. The fragment was analyzed by restriction enzyme digestions as in (A).

    Techniques Used: Polymerase Chain Reaction, Mutagenesis, Amplification

    11) Product Images from "Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle"

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    Journal: The American Journal of Tropical Medicine and Hygiene

    doi: 10.4269/ajtmh.19-0095

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.
    Figure Legend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Techniques Used: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico

    12) Product Images from "A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers"

    Article Title: A natural frameshift mutation in Campanula EIL2 correlates with ethylene insensitivity in flowers

    Journal: BMC Plant Biology

    doi: 10.1186/s12870-016-0786-4

    Detection of EIL2 in hybrids of C. formanekiana ( Cf ) and C. medium ( Cm ). The original PCR products ( EIL2/eil2 ) are 499 bp or 492 bp in Cf and Cm , respectively. Upon digestion with the restriction enzyme Nla III CfEIL2 produces DNA fragments of 362 bp, 122 bp and 16 bp ( a ). In contrast Cm containing the 7 bp deletion in eil2 produces DNA fragments of 476 bp and 16 bp ( b ). The 16 bp DNA fragments are not detected. Nla III restriction analysis of 5 heterozygote Cf × Cm hybrids shows that all hybrids contain both Cf and Cm specific DNA fragments ( c – g , hybrids A–E). Hybrids were produced according to [ 57 ]
    Figure Legend Snippet: Detection of EIL2 in hybrids of C. formanekiana ( Cf ) and C. medium ( Cm ). The original PCR products ( EIL2/eil2 ) are 499 bp or 492 bp in Cf and Cm , respectively. Upon digestion with the restriction enzyme Nla III CfEIL2 produces DNA fragments of 362 bp, 122 bp and 16 bp ( a ). In contrast Cm containing the 7 bp deletion in eil2 produces DNA fragments of 476 bp and 16 bp ( b ). The 16 bp DNA fragments are not detected. Nla III restriction analysis of 5 heterozygote Cf × Cm hybrids shows that all hybrids contain both Cf and Cm specific DNA fragments ( c – g , hybrids A–E). Hybrids were produced according to [ 57 ]

    Techniques Used: Polymerase Chain Reaction, Produced

    Alignment of genomic EIL2 DNA sequences. The aligned area presented is centered on the seven bp deletion in C. medium (represented with -). Nucleotides bordering the mutation are boxed. Conserved nucleotides are marked with asterisk. a EIL2 from six Campanula species, three C. medium cultivars, Edraianthus graminifolius , and Arabidopsis EIN3 . The restriction site of Nla III found in EIL2 Campanula sequences not having the mutation is marked with bold line b eil2 from three C. medium cultivars and two PKM breeding lines (line 1 (Sweet Mee®) and line 2)
    Figure Legend Snippet: Alignment of genomic EIL2 DNA sequences. The aligned area presented is centered on the seven bp deletion in C. medium (represented with -). Nucleotides bordering the mutation are boxed. Conserved nucleotides are marked with asterisk. a EIL2 from six Campanula species, three C. medium cultivars, Edraianthus graminifolius , and Arabidopsis EIN3 . The restriction site of Nla III found in EIL2 Campanula sequences not having the mutation is marked with bold line b eil2 from three C. medium cultivars and two PKM breeding lines (line 1 (Sweet Mee®) and line 2)

    Techniques Used: Mutagenesis

    13) Product Images from "DNA amplification method tolerant to sample degradation"

    Article Title: DNA amplification method tolerant to sample degradation

    Journal: Genome Research

    doi: 10.1101/gr.2813404

    ( A ) Outline of whole genome amplification of partially degraded FFPE samples via RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification). DNA damage sites (•) are indicated. The same approach may also be used to amplify intact or degraded genomic DNA or cDNA. ( B ) Comparison of RCA–RCA amplification products with MDA amplification products. MDA amplification products from H 2 O—no template present— (lane 1 ), 50 ng intact reference (lane 2 ), or FFPE #19 (lane 3 ) genomic DNA; RCA–RCA amplification products from H 2 O—no template present—(lane 4 ), 50 ng intact reference (lane 5 ) or FFPE #19 genomic DNA (lane 6 ). No Nla-III digestion has been applied at this stage following RCA–RCA. ( C ) Reference genomic DNA undigested (lane 1 ) or Nla-III-digested (lane 2 ) prior to RCA–RCA amplification.
    Figure Legend Snippet: ( A ) Outline of whole genome amplification of partially degraded FFPE samples via RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification). DNA damage sites (•) are indicated. The same approach may also be used to amplify intact or degraded genomic DNA or cDNA. ( B ) Comparison of RCA–RCA amplification products with MDA amplification products. MDA amplification products from H 2 O—no template present— (lane 1 ), 50 ng intact reference (lane 2 ), or FFPE #19 (lane 3 ) genomic DNA; RCA–RCA amplification products from H 2 O—no template present—(lane 4 ), 50 ng intact reference (lane 5 ) or FFPE #19 genomic DNA (lane 6 ). No Nla-III digestion has been applied at this stage following RCA–RCA. ( C ) Reference genomic DNA undigested (lane 1 ) or Nla-III-digested (lane 2 ) prior to RCA–RCA amplification.

    Techniques Used: Whole Genome Amplification, Formalin-fixed Paraffin-Embedded, Amplification, Multiple Displacement Amplification

    14) Product Images from "Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification"

    Article Title: Diagnosis of human respiratory syncytial virus infection using reverse transcription loop-mediated isothermal amplification

    Journal: Journal of Virological Methods

    doi: 10.1016/j.jviromet.2006.09.014

    Location of the RT-LAMP primer region. RT-LAMP primer sets were designed using PrimerExplorer online software. The position of six essential primers (F3, F2, F1, B1, B2, and B3) and the loop primer regions are shown by lines and bold font. Underlined sequences illustrate the recognition site of each restriction enzyme (CATG of Nla III and ACATGT of Xba I).
    Figure Legend Snippet: Location of the RT-LAMP primer region. RT-LAMP primer sets were designed using PrimerExplorer online software. The position of six essential primers (F3, F2, F1, B1, B2, and B3) and the loop primer regions are shown by lines and bold font. Underlined sequences illustrate the recognition site of each restriction enzyme (CATG of Nla III and ACATGT of Xba I).

    Techniques Used: Software

    15) Product Images from "Type II restriction modification system in Ureaplasma parvum OMC-P162 strain"

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0205328

    Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.
    Figure Legend Snippet: Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.

    Techniques Used: Activity Assay, Amplification, Polymerase Chain Reaction, Mutagenesis

    DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.
    Figure Legend Snippet: DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.

    Techniques Used: Plasmid Preparation

    Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.
    Figure Legend Snippet: Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.

    Techniques Used: Plasmid Preparation, Electrophoresis

    16) Product Images from "DNA amplification method tolerant to sample degradation"

    Article Title: DNA amplification method tolerant to sample degradation

    Journal: Genome Research

    doi: 10.1101/gr.2813404

    ( A ) Outline of whole genome amplification of partially degraded FFPE samples via RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification). DNA damage sites (•) are indicated. The same approach may also be used to amplify intact or degraded genomic DNA or cDNA. ( B ) Comparison of RCA–RCA amplification products with MDA amplification products. MDA amplification products from H 2 O—no template present— (lane 1 ), 50 ng intact reference (lane 2 ), or FFPE #19 (lane 3 ) genomic DNA; RCA–RCA amplification products from H 2 O—no template present—(lane 4 ), 50 ng intact reference (lane 5 ) or FFPE #19 genomic DNA (lane 6 ). No Nla-III digestion has been applied at this stage following RCA–RCA. ( C ) Reference genomic DNA undigested (lane 1 ) or Nla-III-digested (lane 2 ) prior to RCA–RCA amplification.
    Figure Legend Snippet: ( A ) Outline of whole genome amplification of partially degraded FFPE samples via RCA–RCA (Restriction and Circularization-Aided Rolling Circle Amplification). DNA damage sites (•) are indicated. The same approach may also be used to amplify intact or degraded genomic DNA or cDNA. ( B ) Comparison of RCA–RCA amplification products with MDA amplification products. MDA amplification products from H 2 O—no template present— (lane 1 ), 50 ng intact reference (lane 2 ), or FFPE #19 (lane 3 ) genomic DNA; RCA–RCA amplification products from H 2 O—no template present—(lane 4 ), 50 ng intact reference (lane 5 ) or FFPE #19 genomic DNA (lane 6 ). No Nla-III digestion has been applied at this stage following RCA–RCA. ( C ) Reference genomic DNA undigested (lane 1 ) or Nla-III-digested (lane 2 ) prior to RCA–RCA amplification.

    Techniques Used: Whole Genome Amplification, Formalin-fixed Paraffin-Embedded, Amplification, Multiple Displacement Amplification

    17) Product Images from "CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning"

    Article Title: CTCF-Binding Elements Mediate Accessibility of RAG Substrates During Chromatin Scanning

    Journal: Cell

    doi: 10.1016/j.cell.2018.04.035

    V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .
    Figure Legend Snippet: V H 81X-CBE Promotes Interactions of Its Flanking V H with the DJ H RC (A) Schematic representation of the 3C-HTGTS method for studying chromosomal looping interactions of a bait region of interest with the rest of Igh locus (see text and STAR Methods for details). (B) Schematic of the Nla III restriction fragment (indicated by a blue asterisk) and the relative positions of the biotinylated (cayenne arrow) and nested (blue arrow) PCR primers used for 3C-HTGTS from V H 81X bait in (C). (C) Top panel: schematic representation of chromosome interactions of V H 81X-CBE containing Nla III fragment with other Igh locales. Bottom two panels: 3C-HTGTS profiles of Rag2 −/− derivatives of control, V H 81X-CBE del , and V H 81X-CBE inv D H FL16.1J H 4 v-Abl lines using V H 81X-CBE locale as bait (blue asterisk). Owing to a D H FL16.1 to J H 4 rearrangement in the lines, the region spanning IGCR1, DJ H substrate and iEm appears as a broad interaction peak. As v-Abl lines lack locus contraction, we detected few substantial interactions with the upstream Igh locus beyond the most proximal V H ). Two independent datasets are shown from libraries normalized to 105,638 total junctions. .

    Techniques Used: Polymerase Chain Reaction

    18) Product Images from "The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination"

    Article Title: The Fundamental Role of Chromatin Loop Extrusion in Physiological V(D)J Recombination

    Journal: Nature

    doi: 10.1038/s41586-019-1547-y

    Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.
    Figure Legend Snippet: Working model for loop extrusion-mediated RAG downstream scanning. a-i, Model for cohesin-mediated loop extrusion of chromatin past nascent Igh RC in J H Δ v-Abl lines based on RAG2-deficient background analyses. For all examples, increased interactions of impediment sites with RC targets scanning activity in RAG-sufficient cells. a . Cohesin (red rings) are loaded at multiple sites in the RC-3'CBEs Igh sub-domain. Illustrations show cohesin loading at RC-downstream region. b. Cohesin-mediated extrusion promotes linear interaction of the nascent RC with downstream regions. c. Robust transcription (green arrow) across the Iγ2b/Sγ2b impedes loop extrusion. d. In a subset of cells, loop extrusion proceeds past Iγ2b/Sγ2b impediment to 3'CBEs loop anchor. e-i, Loop extrusion in J H Δ-dCas9-Sγ1-sgRNA lines is impeded, directly or indirectly, by the dCas9-bound Sγ1. As dCas9 impediment is not a complete block, loop extrusion in a subset of cells proceeds downstream, allowing dynamic sub-loop formation of RC with Iγ2b/Sγ2b or 3’CBEs. j-l, In RAG-sufficient cells, RC-bound RAG might enhance the dCas9-bound Sγ1 extrusion impediment. m-p, Elimination of Iγ2b-promoter-driven transcription permits unimpeded RAG-bound RC extrusion to 3’CBEs anchor, increasing RAG scanning activity there. q-r, 3C-HTGTS analysis of RC interactions with D H and flanking regions in J H Δ-dCas9 line ( q ) and D H -J H +/− line ( r ). DpnII ( n = 4, biological replicates) and NlaIII ( n = 3, biological replicates) digestions are shown for the J H Δ-dCas9 line. NlaIII digestion more clearly reveals interaction peak near D H 3-2 due to paucity of DpnII sites in that region. NlaIII digestion of D H -J H +/− line shows a similar RC interaction pattern to that of J H Δ-dCas9 line ( r, n = 2, technical repeats). Bar graphs show relative RC interaction of the 25kb intervening D H region (from D H 2-3 to D H 2-8) versus that of the same-size neighboring regions ( n as indicated above). Data represents mean ± s.d ( q ) or mean ( r ). P values calculated via two-tailed paired t -test.

    Techniques Used: Activity Assay, Blocking Assay, Two Tailed Test

    19) Product Images from "Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations"

    Article Title: Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.5.1617-1620.2002

    Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.
    Figure Legend Snippet: Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis

    PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.
    Figure Legend Snippet: PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced

    20) Product Images from "Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination"

    Article Title: Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkp814

    Visualization of DNA circles from high molecular weight telomere-enriched DNA from stn1-M1 . ( A–E ) Electron micrographs of DNA circles observed in the telomere-enriched fractions from stn1-M1 . Circle lengths for the molecules in A–E, respectively, are estimated at 15.7, 12.1, 3.1, 1.2 and 1.2, and 0.9 kb for fractions A–E, respectively. Note that there is also a ∼0.8 kb linear fragment in the lower right portion of panel A. Samples were prepared by coating the DNA in denatured cytochrome c and are shown in negative contrast. Bar is equivalent to 1.5 kb. ( F ) Relative DNA abundance and telomeric DNA content of eluted fractions from gel chromatography separation of Alu I, Hpa II and Nla III dirgest of stn1-M1 genomic DNA. Molecules shown in A–E are from fractions 26 to 28 (F). Size distribution of the measured circles from fractions 26 to 27 ( n = 29) and 28 ( n = 32).
    Figure Legend Snippet: Visualization of DNA circles from high molecular weight telomere-enriched DNA from stn1-M1 . ( A–E ) Electron micrographs of DNA circles observed in the telomere-enriched fractions from stn1-M1 . Circle lengths for the molecules in A–E, respectively, are estimated at 15.7, 12.1, 3.1, 1.2 and 1.2, and 0.9 kb for fractions A–E, respectively. Note that there is also a ∼0.8 kb linear fragment in the lower right portion of panel A. Samples were prepared by coating the DNA in denatured cytochrome c and are shown in negative contrast. Bar is equivalent to 1.5 kb. ( F ) Relative DNA abundance and telomeric DNA content of eluted fractions from gel chromatography separation of Alu I, Hpa II and Nla III dirgest of stn1-M1 genomic DNA. Molecules shown in A–E are from fractions 26 to 28 (F). Size distribution of the measured circles from fractions 26 to 27 ( n = 29) and 28 ( n = 32).

    Techniques Used: Molecular Weight, Chromatography

    21) Product Images from "A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment"

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-8-26

    (a) RFLP of the Bbs I-digested PCR fragments flanking the 1235T > C region for the family affected by LFSNHL and one normal control. The 672-bp PCR product obtained using the forward primer (5'-TTC CCA CGT ACC ATC TTT CC-3') and the reverse primer (5'-CAC ATC CAG GTT GGG CTC-3') contains a polymorphic restriction site at position 1235. Two fragments of 455 bp and 217 bp from genotype TT were unaffected by enzyme digestion, while three fragments of 672 bp (solid arrow), 455 bp, and 217 bp were observed from heterozygotes carrying genotype TC as I:2, II:1, II:3 and one normal control (C indicated by the open arrow). M: DNA marker. (b) RFLP of Nla III digested the fragment flanking the 2005T > C region of LFSNHL family members and normal controls. The 154-bp PCR product of the polymorphic restriction site at position 2005 was amplified by PCR using the forward primer (5'-GTC AAG CTC ATC CTG GTG TG-3') and reverse primer (5'-CCA TGT TGG TCT CCT TCC AG-3'). Genotype TT such as unaffected individuals and controls (C1, C2, C3 and C4) demonstrated two fragments of 78 and 75 bp, whereas the heterozygote TC such as I:2, II:1, and II:3 showed 4 fragments of 78, 75, 41 (solid arrow), and 37 bp (solid arrow). In all samples, the enzyme also generated a fragment of 1 bp, which was too small to be seen in the gel. U: undigested control, M: DNA marker. (c) Electrophoregrams of WFS1 gene sequences flanking position 2005 of the affected and unaffected family members. The affected family members are heterozygote (T and C) in position 2005 (labeled with a star), while the unaffected family members are homozygous as T in the same place.
    Figure Legend Snippet: (a) RFLP of the Bbs I-digested PCR fragments flanking the 1235T > C region for the family affected by LFSNHL and one normal control. The 672-bp PCR product obtained using the forward primer (5'-TTC CCA CGT ACC ATC TTT CC-3') and the reverse primer (5'-CAC ATC CAG GTT GGG CTC-3') contains a polymorphic restriction site at position 1235. Two fragments of 455 bp and 217 bp from genotype TT were unaffected by enzyme digestion, while three fragments of 672 bp (solid arrow), 455 bp, and 217 bp were observed from heterozygotes carrying genotype TC as I:2, II:1, II:3 and one normal control (C indicated by the open arrow). M: DNA marker. (b) RFLP of Nla III digested the fragment flanking the 2005T > C region of LFSNHL family members and normal controls. The 154-bp PCR product of the polymorphic restriction site at position 2005 was amplified by PCR using the forward primer (5'-GTC AAG CTC ATC CTG GTG TG-3') and reverse primer (5'-CCA TGT TGG TCT CCT TCC AG-3'). Genotype TT such as unaffected individuals and controls (C1, C2, C3 and C4) demonstrated two fragments of 78 and 75 bp, whereas the heterozygote TC such as I:2, II:1, and II:3 showed 4 fragments of 78, 75, 41 (solid arrow), and 37 bp (solid arrow). In all samples, the enzyme also generated a fragment of 1 bp, which was too small to be seen in the gel. U: undigested control, M: DNA marker. (c) Electrophoregrams of WFS1 gene sequences flanking position 2005 of the affected and unaffected family members. The affected family members are heterozygote (T and C) in position 2005 (labeled with a star), while the unaffected family members are homozygous as T in the same place.

    Techniques Used: Polymerase Chain Reaction, Marker, Amplification, CTG Assay, Generated, Labeling

    22) Product Images from "Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer"

    Article Title: Amplification of repeat-containing transcribed sequences (ARTS): a transcriptome fingerprinting strategy to detect functionally relevant microsatellite mutations in cancer

    Journal: Nucleic Acids Research

    doi:

    Description of the ARTS procedure and expected results. ( A ) Schematic flowchart representation of the procedure for obtaining ARTS products from a total transcriptome. As a first step, mRNAs are purified, reverse transcribed and converted into ds-cDNAs. The ds-cDNA population is then digested with a restriction endonuclease ( Nla III) that is expected to cleave most reverse transcripts at least once, generating 200–500 bp long ds-cDNA fragments. The fragments are subsequently ligated to an adapter (Ad), here indicated as ARTS-Ad. To specifically enrich for repeat-containing sequences, linear amplification is performed using a repeat-anchoring primer (R-An primer), designed as described in (B). Five linear amplification cycles are performed so that the anchoring (RAT) and repeat-specific (RSS) parts of the R-An primer are both required to achieve specific annealing. After the enrichment step, repeat-containing sequences are PCR amplified at high stringency using the same R-An primer and the ARTS-Ad primer, the latter specifically designed to avoid adapter–adapter amplification. ( B ) Design of the repeat-anchoring (R-An) primer. The R-An primer is composed of three modules, in 5′→3′ order: (i) a universal sequence (US), common to all R-An primers, which increases the efficiency of the final PCR amplification step; (ii) an arbitrary repeat-anchoring trinucleotide (RAT), which not only prevents slippage, but also defines the three bases immediately 5′ to the repeat being located at the 5′ end; (iii) a repeat-specific sequence (RSS), which contains the repeat to be explored. ( C ) Graphical representation of the expected ARTS products and their migration in PAGE, highlighting the differences between a PCR product containing a wild-type repeat or its longer and shorter versions, deriving from insertions and deletions, respectively. Due to the fact that the R-An primer is anchored 5′ of the repeat, the size of the ARTS product will be directly dependent on the repeat length.
    Figure Legend Snippet: Description of the ARTS procedure and expected results. ( A ) Schematic flowchart representation of the procedure for obtaining ARTS products from a total transcriptome. As a first step, mRNAs are purified, reverse transcribed and converted into ds-cDNAs. The ds-cDNA population is then digested with a restriction endonuclease ( Nla III) that is expected to cleave most reverse transcripts at least once, generating 200–500 bp long ds-cDNA fragments. The fragments are subsequently ligated to an adapter (Ad), here indicated as ARTS-Ad. To specifically enrich for repeat-containing sequences, linear amplification is performed using a repeat-anchoring primer (R-An primer), designed as described in (B). Five linear amplification cycles are performed so that the anchoring (RAT) and repeat-specific (RSS) parts of the R-An primer are both required to achieve specific annealing. After the enrichment step, repeat-containing sequences are PCR amplified at high stringency using the same R-An primer and the ARTS-Ad primer, the latter specifically designed to avoid adapter–adapter amplification. ( B ) Design of the repeat-anchoring (R-An) primer. The R-An primer is composed of three modules, in 5′→3′ order: (i) a universal sequence (US), common to all R-An primers, which increases the efficiency of the final PCR amplification step; (ii) an arbitrary repeat-anchoring trinucleotide (RAT), which not only prevents slippage, but also defines the three bases immediately 5′ to the repeat being located at the 5′ end; (iii) a repeat-specific sequence (RSS), which contains the repeat to be explored. ( C ) Graphical representation of the expected ARTS products and their migration in PAGE, highlighting the differences between a PCR product containing a wild-type repeat or its longer and shorter versions, deriving from insertions and deletions, respectively. Due to the fact that the R-An primer is anchored 5′ of the repeat, the size of the ARTS product will be directly dependent on the repeat length.

    Techniques Used: Purification, Amplification, Polymerase Chain Reaction, Sequencing, Migration, Polyacrylamide Gel Electrophoresis

    23) 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

    Nla III RLFP analysis of PCR-amplified crmB fragments from DNA of CPV isolates from human and animals. Nla III digest fragments after PCR amplification of OPV85 (human) (lane 1), OPV88/L (cat) (lane 2), OPV88/H (cat) (lane 3), OPV89/1 (cat)-M5 (lane 4), OPV89/2 (cat) (lane 5), OPV89/3 (cat) (lane 6), OPV89/4 (cat)-M6 (lane 7), OPV89/5 (cat)-M7 (lane 8), OPV90/1 (cat)-M8 (lane 9), OPV90/2 (human) (lane 10), OPV90/4 (dog) (lane 11), OPV90/5 (cat)-M9 (lane 12), OPV91/1 (cat) (lane 13), OPV91/2 (human) (lane 14), OPV91/3 (cow) (lane 15), CATPOX3 (lane 16), CATPOX5 (lane 17), RAT Moscow (rat) (lane 18), EP-1 (elephant) (lane 19), EP-2 (elephant)-M1 (lane 20), EP-3 (elephant) (lane 21), EP-4 (elephant) (lane 22), EP-5 (elephant) (lane 23), CPV BRT-Atlanta (lane 24), and CPV BRT-Munich (lane 25) are shown.
    Figure Legend Snippet: Nla III RLFP analysis of PCR-amplified crmB fragments from DNA of CPV isolates from human and animals. Nla III digest fragments after PCR amplification of OPV85 (human) (lane 1), OPV88/L (cat) (lane 2), OPV88/H (cat) (lane 3), OPV89/1 (cat)-M5 (lane 4), OPV89/2 (cat) (lane 5), OPV89/3 (cat) (lane 6), OPV89/4 (cat)-M6 (lane 7), OPV89/5 (cat)-M7 (lane 8), OPV90/1 (cat)-M8 (lane 9), OPV90/2 (human) (lane 10), OPV90/4 (dog) (lane 11), OPV90/5 (cat)-M9 (lane 12), OPV91/1 (cat) (lane 13), OPV91/2 (human) (lane 14), OPV91/3 (cow) (lane 15), CATPOX3 (lane 16), CATPOX5 (lane 17), RAT Moscow (rat) (lane 18), EP-1 (elephant) (lane 19), EP-2 (elephant)-M1 (lane 20), EP-3 (elephant) (lane 21), EP-4 (elephant) (lane 22), EP-5 (elephant) (lane 23), CPV BRT-Atlanta (lane 24), and CPV BRT-Munich (lane 25) are shown.

    Techniques Used: Polymerase Chain Reaction, Amplification

    Comparative Nla III RLFP analysis of PCR-amplified crmB fragments from intact genomes of OPV isolates. Nla III fragment sizes noted in parentheses were determined by direct sequence analysis of the crmB ) and agreed with sizes of fragments calculated by comparison to the 100-bp DNA ladder size marker (M). Lanes: 1, VAR major Bangladesh-1975; 2, VAR minor alastrim Brazil-Garcia-1966; 3, human MPV Congo-8; 4, human MPV 71-0082; 5, CML Somalia-1978; 6, CML (CP-5) Dubai-M4; 7, CPV Brighton; 8, CPV strain M1; 9, CPV strain M5; 10, CPV strain M6; 11, CPV strain M7; 12, CPV strain M8; 13, CPV strain M9; 14, CPV strain 58; 15, VAC Lister; 16, VAC Columbia; 17, BUF India-81-1985; 18, BUF India-3906; 19, RPV Utrecht; 20, TPV Dahomey-1971.
    Figure Legend Snippet: Comparative Nla III RLFP analysis of PCR-amplified crmB fragments from intact genomes of OPV isolates. Nla III fragment sizes noted in parentheses were determined by direct sequence analysis of the crmB ) and agreed with sizes of fragments calculated by comparison to the 100-bp DNA ladder size marker (M). Lanes: 1, VAR major Bangladesh-1975; 2, VAR minor alastrim Brazil-Garcia-1966; 3, human MPV Congo-8; 4, human MPV 71-0082; 5, CML Somalia-1978; 6, CML (CP-5) Dubai-M4; 7, CPV Brighton; 8, CPV strain M1; 9, CPV strain M5; 10, CPV strain M6; 11, CPV strain M7; 12, CPV strain M8; 13, CPV strain M9; 14, CPV strain 58; 15, VAC Lister; 16, VAC Columbia; 17, BUF India-81-1985; 18, BUF India-3906; 19, RPV Utrecht; 20, TPV Dahomey-1971.

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

    Nla III RLFP analysis of PCR-amplified crmB fragments from DNA of VAC variants. Nla III digest fragments of the PCR products of VAC COL (lane 1), VAC V (lane 2), VAC LIST (lane 3), VAC VNV (lane 4), VAC VCX (lane 5), VAC Wyeth (lane 6), VAC CV1-78 (lane 7), BUF 81 (lane 8), BUF 3906 (lane 9), RPV UTR (lane 10), Levaditi (lane 11), VAC M1 (lane 12), VAC COP wt (lane 13), VAC COP hr (lane 14), RPV Utrecht (lane 15), VAC Hagen (lane 16), VAC CVA (lane 17), VAC WR (lane 18), VAC Elstree (lane 19), and BUF BP-1 (lane 20) are shown.
    Figure Legend Snippet: Nla III RLFP analysis of PCR-amplified crmB fragments from DNA of VAC variants. Nla III digest fragments of the PCR products of VAC COL (lane 1), VAC V (lane 2), VAC LIST (lane 3), VAC VNV (lane 4), VAC VCX (lane 5), VAC Wyeth (lane 6), VAC CV1-78 (lane 7), BUF 81 (lane 8), BUF 3906 (lane 9), RPV UTR (lane 10), Levaditi (lane 11), VAC M1 (lane 12), VAC COP wt (lane 13), VAC COP hr (lane 14), RPV Utrecht (lane 15), VAC Hagen (lane 16), VAC CVA (lane 17), VAC WR (lane 18), VAC Elstree (lane 19), and BUF BP-1 (lane 20) are shown.

    Techniques Used: Polymerase Chain Reaction, Amplification

    RLFP analysis of PCR-amplified crmB fragments from genomes of CML and TPV isolates. Nla III digest fragments of the PCR products of CML Mauretania (lane 1), CML Somalia-1978 (lane 2), CML Niger (lane 3), CML (CP-1) Iran-M2 (lane 4), CML-Saudi-M3 (lane 5), and TPV Dahomey-1971 (lane 6) are shown in the left panel. Nci I digest fragments of the PCR products of TPV Dahomey-1971 (lane 7), CML Somalia-1978 (lane 8), CML Niger (lane 9), CML Mauretania (lane 10), and CML Iran-M2 (lane 11) are shown in the right panel. Size markers are provided by a 100-bp DNA ladder (lane M).
    Figure Legend Snippet: RLFP analysis of PCR-amplified crmB fragments from genomes of CML and TPV isolates. Nla III digest fragments of the PCR products of CML Mauretania (lane 1), CML Somalia-1978 (lane 2), CML Niger (lane 3), CML (CP-1) Iran-M2 (lane 4), CML-Saudi-M3 (lane 5), and TPV Dahomey-1971 (lane 6) are shown in the left panel. Nci I digest fragments of the PCR products of TPV Dahomey-1971 (lane 7), CML Somalia-1978 (lane 8), CML Niger (lane 9), CML Mauretania (lane 10), and CML Iran-M2 (lane 11) are shown in the right panel. Size markers are provided by a 100-bp DNA ladder (lane M).

    Techniques Used: Polymerase Chain Reaction, Amplification

    24) Product Images from "Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing"

    Article Title: Reference genome-independent assessment of mutation density using restriction enzyme-phased sequencing

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-13-72

    Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.
    Figure Legend Snippet: Size fractionation of digested DNA by affinity beads . A. Counts of restriction fragments by size after in silico digestion of the Oryza sativa Os6.1 genome with NlaIII . The Y-axis of the graph displays the count per 25 bp bins. The graph top axis displays the total count for in silico slices of 100 bp. The graph demonstrates how a size fraction from 100 to 200 bp would contain more than ten times the number of fragments found in the 600 to 700 bp fraction. B. Fractionation strategies with SPRI magnetic beads. On the left, a bottom-delimited size fraction of the digested input DNA can be taken in a single step (thicker arrows path), or a sliced size fraction in two steps (thinner arrows path). Slicing is demonstrated in a digital electrophoretogram on the right. In practice, bottom delimiting in a single step is the most practical solution since the larger size fragments contribute relatively less to the final library.

    Techniques Used: Fractionation, In Silico, Magnetic Beads

    25) Product Images from "Short interspersed elements (SINEs) are a major source of canine genomic diversity"

    Article Title: Short interspersed elements (SINEs) are a major source of canine genomic diversity

    Journal: Genome Research

    doi: 10.1101/gr.3765505

    Construction of libraries that are enriched for SINEC_Cf elements and flanking sequence. ( A ) Genomic DNA is cleaved with the frequently cutting restriction enzyme, NlaIII. ( B ) The cleaved fragments are self-ligated. ( C ) The circularized products are subjected to PCR using SINEC_Cf-specific primers. ( D ) The linear products are size-selected and cloned in a plasmid vector. ( E ) Inserts are sequenced with a vector-specific primer.
    Figure Legend Snippet: Construction of libraries that are enriched for SINEC_Cf elements and flanking sequence. ( A ) Genomic DNA is cleaved with the frequently cutting restriction enzyme, NlaIII. ( B ) The cleaved fragments are self-ligated. ( C ) The circularized products are subjected to PCR using SINEC_Cf-specific primers. ( D ) The linear products are size-selected and cloned in a plasmid vector. ( E ) Inserts are sequenced with a vector-specific primer.

    Techniques Used: Sequencing, Polymerase Chain Reaction, Clone Assay, Plasmid Preparation

    26) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    27) Product Images from "Somatic GNAS Mutation Causes Widespread and Diffuse Pituitary Disease in Acromegalic Patients with McCune-Albright Syndrome"

    Article Title: Somatic GNAS Mutation Causes Widespread and Diffuse Pituitary Disease in Acromegalic Patients with McCune-Albright Syndrome

    Journal: The Journal of Clinical Endocrinology and Metabolism

    doi: 10.1210/jc.2012-1274

    A, Tissue microdissection; representative examples of microdissected normal tissue, hyperplastic tissue, and neoplastic tissue are shown. Before genetic analysis, different areas of normal, hyperplastic, and neoplastic anterior pituitary gland tissue were microdissected from H E-stained slides; reticulin stains and immunohistochemical preparations from adjacent serial sections were used for identification of areas of interest. B, GNAS mutation analysis of pituitary gland tissue from three cases with MAS. DNA was extracted from microdissected tissue samples, amplified by PCR, and separately digested with Nla III and Pvu II, which can digest the PCR product in case the R201H mutation or the R201C of the GNAS gene is present, respectively. Case 1, Eleven foci of pituitary tissue were microdissected, mutation bands ( arrow ) are detected in different areas of hyperplasia and neoplasia, but not in three foci of normal-appearing pituitary gland (digestion with Nla III). Case 2, Fifteen foci of pituitary tissue were microdissected; mutation bands ( arrow ) are detected in different areas of hyperplasia and neoplasia, but not in five different foci of normal-appearing pituitary gland (digestion with Nla III). Case 3, Nine foci of pituitary gland tissue were microdissected; mutation bands ( arrow ) are detected in different areas of pituitary gland tissue with overrepresentation of somatotroph cells and areas of somatotroph hyperplasia (digestion with Pvu II).
    Figure Legend Snippet: A, Tissue microdissection; representative examples of microdissected normal tissue, hyperplastic tissue, and neoplastic tissue are shown. Before genetic analysis, different areas of normal, hyperplastic, and neoplastic anterior pituitary gland tissue were microdissected from H E-stained slides; reticulin stains and immunohistochemical preparations from adjacent serial sections were used for identification of areas of interest. B, GNAS mutation analysis of pituitary gland tissue from three cases with MAS. DNA was extracted from microdissected tissue samples, amplified by PCR, and separately digested with Nla III and Pvu II, which can digest the PCR product in case the R201H mutation or the R201C of the GNAS gene is present, respectively. Case 1, Eleven foci of pituitary tissue were microdissected, mutation bands ( arrow ) are detected in different areas of hyperplasia and neoplasia, but not in three foci of normal-appearing pituitary gland (digestion with Nla III). Case 2, Fifteen foci of pituitary tissue were microdissected; mutation bands ( arrow ) are detected in different areas of hyperplasia and neoplasia, but not in five different foci of normal-appearing pituitary gland (digestion with Nla III). Case 3, Nine foci of pituitary gland tissue were microdissected; mutation bands ( arrow ) are detected in different areas of pituitary gland tissue with overrepresentation of somatotroph cells and areas of somatotroph hyperplasia (digestion with Pvu II).

    Techniques Used: Laser Capture Microdissection, Staining, Immunohistochemistry, Mutagenesis, Amplification, Polymerase Chain Reaction

    28) Product Images from "The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families"

    Article Title: The p.M292T NDUFS2 mutation causes complex I-deficient Leigh syndrome in multiple families

    Journal: Brain

    doi: 10.1093/brain/awq232

    NDUFS1 homozygous mutation, c.1222C > T (p.R408C). ( A ) Family 5 pedigree, ( B ) sequence electropherograms of NDUFS1 gene with c.1222 position highlighted in patient and family members, ( C ) confirmation of homozygous c.1222C > T mutation by PCR–RFLP. U = uncut; C1 = Control 1; C2 = Control 2. RFLP products were separated through a 12% non-denaturing polyacrylamide gel. The wild-type PCR product contains a single Nla III site, which cuts the 218-bp amplicon into two fragments of 193 and 25 bp. In amplicons harbouring the c.1222C > T mutation, a second Nla III site is created that cuts the 193-bp fragment into two smaller fragments of 148 and 45 bp. Fragment sizes (bp) are shown on the left; ( D ) amino acid alignment of NDUFS1 orthologues. Alignments were generated with ClustalW using GenBank sequences from Mus musculus (NP_663493.1), Rattus norvegicus (NP_001005550.1), Homo sapiens (NP_004997.4), Pan troglodytes (XP_516047.2), Canis lupus familiaris (XP_859697.1), Bos taurus (NP_777245.1), Danio rerio (NP_001007766.1), Drosophila melanogaster (NP_727255.1), Arabidopsis thaliana (NP_568550.1), Oryza sativa (NP_001051072.1) and Caenorhabditis elegans (NP_503733.1). Position p.408 ( Homo sapiens ) is indicated. Conserved sites are indicated (asterisk).
    Figure Legend Snippet: NDUFS1 homozygous mutation, c.1222C > T (p.R408C). ( A ) Family 5 pedigree, ( B ) sequence electropherograms of NDUFS1 gene with c.1222 position highlighted in patient and family members, ( C ) confirmation of homozygous c.1222C > T mutation by PCR–RFLP. U = uncut; C1 = Control 1; C2 = Control 2. RFLP products were separated through a 12% non-denaturing polyacrylamide gel. The wild-type PCR product contains a single Nla III site, which cuts the 218-bp amplicon into two fragments of 193 and 25 bp. In amplicons harbouring the c.1222C > T mutation, a second Nla III site is created that cuts the 193-bp fragment into two smaller fragments of 148 and 45 bp. Fragment sizes (bp) are shown on the left; ( D ) amino acid alignment of NDUFS1 orthologues. Alignments were generated with ClustalW using GenBank sequences from Mus musculus (NP_663493.1), Rattus norvegicus (NP_001005550.1), Homo sapiens (NP_004997.4), Pan troglodytes (XP_516047.2), Canis lupus familiaris (XP_859697.1), Bos taurus (NP_777245.1), Danio rerio (NP_001007766.1), Drosophila melanogaster (NP_727255.1), Arabidopsis thaliana (NP_568550.1), Oryza sativa (NP_001051072.1) and Caenorhabditis elegans (NP_503733.1). Position p.408 ( Homo sapiens ) is indicated. Conserved sites are indicated (asterisk).

    Techniques Used: Mutagenesis, Sequencing, Polymerase Chain Reaction, Amplification, Generated

    29) Product Images from "Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA"

    Article Title: Enhancer-driven chromatin interactions during development promote escape from silencing by a long non-coding RNA

    Journal: Epigenetics & Chromatin

    doi: 10.1186/1756-8935-4-21

    (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.
    Figure Legend Snippet: (A) Developmental imprinting pattern of Kcnq1 . Allele-specific expression of Kcnq1 as assayed by reverse transcription (RT)-PCR and restriction digest with Nla III on E10.5, 11.5, 12.5, 13.5, 14.5, 16.5 and neonatal heart (nnH) from F1 hybrid B6(CAST7) × C57BL/6J crosses. Digestion products specific for B6(CAST7) (maternal) and C57BL/6J (paternal) alleles are indicated. Positive signs (+) denote addition of NlaIII to the RT-PCR product. (B) Quantification of relative paternal-specific and maternal-specific expression during development. (C) Kcnq1 RNA abundance during stages of development in which the imprinting pattern switches from monoallelic to biallelic, as assayed by real-time PCR. (D) Methylated DNA immunoprecipitation (MeDIP) analysis of the Kcnq1 and Kcnq1ot1 promoter regions in sperm and 7.5 days post coitum (dpc) embryos. 5meC lane = DNA precipitated by antibody against methylated cytosine; IgG = non-specific immunoprecipitation; Input = DNA before immunoprecipitation; - = no antibody control. Specific bands for Kcnq1 and Kcnq1ot1 are indicated; NS = non-specific amplification product. The Kcnq1ot1 promoter is methylated maternally in 7.5 dpc embryos and unmethylated in sperm, thus serving as a positive control for immunoprecipitation of methylated DNA in E7.5 DNA and a negative control in sperm DNA.

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Methylation, Immunoprecipitation, Methylated DNA Immunoprecipitation, Amplification, Positive Control, Negative Control

    30) Product Images from "Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations"

    Article Title: Detection of Ethambutol-Resistant Mycobacterium tuberculosis Strains by Multiplex Allele-Specific PCR Assay Targeting embB306 Mutations

    Journal: Journal of Clinical Microbiology

    doi: 10.1128/JCM.40.5.1617-1620.2002

    Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.
    Figure Legend Snippet: Schematic view of the embB gene fragment targeted by the MAS-PCR assay. Short arrows indicate the primers, long double-sided arrows indicate the allele-specific PCR fragments amplified in the absence of respective mutation. An X represents any base (A, T, C, or G). The embB codon 306 ATG is in boldface and in a shaded box. Nla III and Hae III restriction enzymes' sites related to embB 306 ATG are shown in the enlarged image.

    Techniques Used: Polymerase Chain Reaction, Amplification, Mutagenesis

    PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.
    Figure Legend Snippet: PCR-RFLP analysis of the amplified 118-bp embB306 fragment of M. tuberculosis strains with Nla III and Hae III. Lanes: 2 to 4, Nla III-RFLP profiles; 5 to 7, Hae III-RFLP profiles; 1, undigested PCR product (118 bp), 2 and 5, strains with embB 306 wild-type allele (ATG); 3 and 6, strains with embB codon 306 mutated in the first base (ATG→BTG); 4 and 7, strains with embB codon 306 mutated in the third base (ATG→ATH). Lane M, 50-bp DNA ladder (Amersham Pharmacia Biotech). Short triangular arrows indicate specific digests produced by Nla III (21/23, 30, and 44 bp in lane 2; 21/23 and 74 bp in lanes 3 and 4) and Hae III (50 and 68 bp in lanes 5 and 6). The 21- and 23-bp fragments present one weak band.

    Techniques Used: Polymerase Chain Reaction, Amplification, Produced

    31) Product Images from "The Glu727 Allele of Thyroid Stimulating Hormone Receptor Gene is Associated with Osteoporosis"

    Article Title: The Glu727 Allele of Thyroid Stimulating Hormone Receptor Gene is Associated with Osteoporosis

    Journal: North American Journal of Medical Sciences

    doi: 10.4103/1947-2714.98588

    Polymerase chain reaction (PCR) products at D727E site. PCR products were cut by NlaIII endonuclease and separated by agarose gel electrophoresis
    Figure Legend Snippet: Polymerase chain reaction (PCR) products at D727E site. PCR products were cut by NlaIII endonuclease and separated by agarose gel electrophoresis

    Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis

    32) Product Images from "Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases"

    Article Title: Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

    Journal: Journal of Clinical Microbiology

    doi:

    PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])
    Figure Legend Snippet: PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])

    Techniques Used: Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Software

    33) Product Images from "CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples"

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

    Journal: Nature Communications

    doi: 10.1038/s41467-019-12570-2

    CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file
    Figure Legend Snippet: CUTseq implementation and reproducibility. a CUTseq workflow. (1) RE, restriction enzyme. T7, T7 phage promoter. IVT, in vitro transcription. RA5, RA3, SP7, and SP9: Illumina’s sequencing adapters. b BT474 cells copy number profiles (100 kb resolution). ρ , Pearson’s correlation. c Pearson’s correlation ( ρ ) between the copy number profiles (100 kb resolution) of five cancer cell lines digested with HindIII (rows) or NlaIII (columns). d Chr17 copy number profiles (NlaIII, 100 kb resolution) in two HER2-positive (SKBR3 and BT474) and one HER2-negative cell line (MCF7). ERBB2/HER2 is highlighted in red. e Copy number profiles (NlaIII, 100 kb resolution) in five replicates (Rep) from FFPE tumor samples. COAD, colon adenocarcinoma. MELA, melanoma. ρ , Pearson’s correlation. f Pearson’s correlation ( ρ ) between the replicates shown in e at different resolutions. Each dot represents one pair of replicates. Error bars indicate the median and interquartile range. g Pearson’s correlation ( ρ ) between the fraction of the genome (100 kb resolution) either amplified or deleted in the replicates (Rep) shown in e . Each dot represents one pair of replicates. Dashed line: linear regression. h , i Length of amplified (AMP) or deleted (DEL) genomic segments in Rep1 ( h ) and Rep2 ( i ) samples shown in e , at various resolutions. j Zoom-in view on chr9 q-arm in sample TRN4 shown in e . Arrows indicate focal amplifications detected only at 10 kb resolution in both replicates. Red: centromeric region. The p-arm is not shown. k Copy number profiles (NlaIII, 100 kb resolution) determined using 120 pg of gDNA extracted from one FFPE breast cancer (BRCA) sample and three different numbers of PCR cycles. l Pearson’s correlation ( ρ ) between copy number profiles (100 kb resolution) determined using different amounts of gDNA extracted from the sample shown in k . In all the profiles, gray dots represent individual genomic windows, whereas black lines indicate segmented genomic intervals after circular binary segmentation 37 . The numbers below each box indicate chromosomes from chr1 (leftmost) to chr22 (rightmost). In all the cases, TRN refers to the ID of Turin samples, as shown in Supplementary Table 2 . All the source data for this figure are provided as a Source Data file

    Techniques Used: In Vitro, Sequencing, Formalin-fixed Paraffin-Embedded, Amplification, Polymerase Chain Reaction

    34) Product Images from "Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA"

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

    Journal: Genome Research

    doi: 10.1101/gr.306102

    Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.
    Figure Legend Snippet: Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.

    Techniques Used:

    35) Product Images from "DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells"

    Article Title: DNA Analysis by Restriction Enzyme (DARE) enables concurrent genomic and epigenomic characterization of single cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkz717

    Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.
    Figure Legend Snippet: Workflow of DNA Analysis by Restriction Enzyme (DARE) assay. ( A ) Workflow of DARE assay—cell lysis and protease treatment are followed by digestion of unmethylated CCGG sites with methylation sensitive HpaII enzyme. U-tag adapters are ligated and the remaining CCGG sites are digested by methylation insensitive MspI enzyme. NlaIII digestion is included to reduce the fragment length. This is followed by ligation with the respective adapters (M-tag and N-tag adapters). Thermolabile USER ® II enzyme is used to remove excess uracil-containing adapters after each ligation. ( B ) Adapter system: U-tag adapter consists of Read 1 primer sequence of Illumina adapter, unique molecular identifier (UMI), unmethylated site specific tag (U-tag), and CG overhang. M-tag adapter similarly consists of Read 1 primer sequence of Illumina adapter, UMI, methylated site specific tag (M-tag), and CG overhang. N-tag adapter consists of Read 2 primer sequence of Illumina adapter and CATG overhang.

    Techniques Used: Lysis, Methylation, Ligation, Sequencing

    36) Product Images from "Methylation Target Array for Rapid Analysis of CpG Island Hypermethylation in Multiple Tissue Genomes"

    Article Title: Methylation Target Array for Rapid Analysis of CpG Island Hypermethylation in Multiple Tissue Genomes

    Journal: The American Journal of Pathology

    doi:

    The content of repetitive sequences in different methylation targets. A: Serial dilution of different cutter-generated ( Mse I, Tsp509 I, Nla III, or Bfa I) methylation targets (15 to 500 ng) and an identical amount of human Cot-1 DNA were dotted on the nylon filter and hybridized with radiolabeled Cot-1 probe. B: The hybridization signals shown in A were quantified by a densitometer and subsequently standardized by its respective Cot-1 spots, as discussed in the text.
    Figure Legend Snippet: The content of repetitive sequences in different methylation targets. A: Serial dilution of different cutter-generated ( Mse I, Tsp509 I, Nla III, or Bfa I) methylation targets (15 to 500 ng) and an identical amount of human Cot-1 DNA were dotted on the nylon filter and hybridized with radiolabeled Cot-1 probe. B: The hybridization signals shown in A were quantified by a densitometer and subsequently standardized by its respective Cot-1 spots, as discussed in the text.

    Techniques Used: Methylation, Serial Dilution, Generated, Hybridization

    Map locations of restriction sites in the 10 genes analyzed by MTA. The shaded horizontal bar indicates that the first exon region and its position designation is relative to the transcription start site (+1). The short vertical bars indicate the flanking 4-base endonuclease recognition sites of Nla III (N), Tsp509 I (T), Bfa I (Bf), and Mse I (M), and the methylation-sensitive Hpa II (H) or Bst UI (B) sites in between. The horizontal line with flanking black circles represents the area analyzed by methylation-specific PCR. The thick horizontal line denotes the probe location for methylation target array analysis. Note that less GC-rich regions within the selected fragments were chosen as probes to prevent non-specific hybridization. The thin horizontal line under the schematics on right represents the distance of 100-bp.
    Figure Legend Snippet: Map locations of restriction sites in the 10 genes analyzed by MTA. The shaded horizontal bar indicates that the first exon region and its position designation is relative to the transcription start site (+1). The short vertical bars indicate the flanking 4-base endonuclease recognition sites of Nla III (N), Tsp509 I (T), Bfa I (Bf), and Mse I (M), and the methylation-sensitive Hpa II (H) or Bst UI (B) sites in between. The horizontal line with flanking black circles represents the area analyzed by methylation-specific PCR. The thick horizontal line denotes the probe location for methylation target array analysis. Note that less GC-rich regions within the selected fragments were chosen as probes to prevent non-specific hybridization. The thin horizontal line under the schematics on right represents the distance of 100-bp.

    Techniques Used: Methylation, Polymerase Chain Reaction, Hybridization

    37) Product Images from "A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment"

    Article Title: A novel mutation in the WFS1 gene identified in a Taiwanese family with low-frequency hearing impairment

    Journal: BMC Medical Genetics

    doi: 10.1186/1471-2350-8-26

    (a) RFLP of the Bbs I-digested PCR fragments flanking the 1235T > C region for the family affected by LFSNHL and one normal control. The 672-bp PCR product obtained using the forward primer (5'-TTC CCA CGT ACC ATC TTT CC-3') and the reverse primer (5'-CAC ATC CAG GTT GGG CTC-3') contains a polymorphic restriction site at position 1235. Two fragments of 455 bp and 217 bp from genotype TT were unaffected by enzyme digestion, while three fragments of 672 bp (solid arrow), 455 bp, and 217 bp were observed from heterozygotes carrying genotype TC as I:2, II:1, II:3 and one normal control (C indicated by the open arrow). M: DNA marker. (b) RFLP of Nla III digested the fragment flanking the 2005T > C region of LFSNHL family members and normal controls. The 154-bp PCR product of the polymorphic restriction site at position 2005 was amplified by PCR using the forward primer (5'-GTC AAG CTC ATC CTG GTG TG-3') and reverse primer (5'-CCA TGT TGG TCT CCT TCC AG-3'). Genotype TT such as unaffected individuals and controls (C1, C2, C3 and C4) demonstrated two fragments of 78 and 75 bp, whereas the heterozygote TC such as I:2, II:1, and II:3 showed 4 fragments of 78, 75, 41 (solid arrow), and 37 bp (solid arrow). In all samples, the enzyme also generated a fragment of 1 bp, which was too small to be seen in the gel. U: undigested control, M: DNA marker. (c) Electrophoregrams of WFS1 gene sequences flanking position 2005 of the affected and unaffected family members. The affected family members are heterozygote (T and C) in position 2005 (labeled with a star), while the unaffected family members are homozygous as T in the same place.
    Figure Legend Snippet: (a) RFLP of the Bbs I-digested PCR fragments flanking the 1235T > C region for the family affected by LFSNHL and one normal control. The 672-bp PCR product obtained using the forward primer (5'-TTC CCA CGT ACC ATC TTT CC-3') and the reverse primer (5'-CAC ATC CAG GTT GGG CTC-3') contains a polymorphic restriction site at position 1235. Two fragments of 455 bp and 217 bp from genotype TT were unaffected by enzyme digestion, while three fragments of 672 bp (solid arrow), 455 bp, and 217 bp were observed from heterozygotes carrying genotype TC as I:2, II:1, II:3 and one normal control (C indicated by the open arrow). M: DNA marker. (b) RFLP of Nla III digested the fragment flanking the 2005T > C region of LFSNHL family members and normal controls. The 154-bp PCR product of the polymorphic restriction site at position 2005 was amplified by PCR using the forward primer (5'-GTC AAG CTC ATC CTG GTG TG-3') and reverse primer (5'-CCA TGT TGG TCT CCT TCC AG-3'). Genotype TT such as unaffected individuals and controls (C1, C2, C3 and C4) demonstrated two fragments of 78 and 75 bp, whereas the heterozygote TC such as I:2, II:1, and II:3 showed 4 fragments of 78, 75, 41 (solid arrow), and 37 bp (solid arrow). In all samples, the enzyme also generated a fragment of 1 bp, which was too small to be seen in the gel. U: undigested control, M: DNA marker. (c) Electrophoregrams of WFS1 gene sequences flanking position 2005 of the affected and unaffected family members. The affected family members are heterozygote (T and C) in position 2005 (labeled with a star), while the unaffected family members are homozygous as T in the same place.

    Techniques Used: Polymerase Chain Reaction, Marker, Amplification, CTG Assay, Generated, Labeling

    38) Product Images from "Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases"

    Article Title: Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

    Journal: Journal of Clinical Microbiology

    doi:

    PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])
    Figure Legend Snippet: PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])

    Techniques Used: Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Software

    39) Product Images from "Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases"

    Article Title: Diversity of Helicobacter pylori vacA and cagA Genes and Relationship to VacA and CagA Protein Expression, Cytotoxin Production, and Associated Diseases

    Journal: Journal of Clinical Microbiology

    doi:

    PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])
    Figure Legend Snippet: PAGE after PCR amplification of H. pylori DNA using primers vac1F-vac1R and Nla III digestion. Only DNA amplicons obtained with vacA genotype s1 are cut by Nla III into fragments of 107 and 94 bp. Lanes: M, 100-bp DNA marker; 1, strain H. pylori 60190 ( vacA genotype s1); 2, Tx30a ( vacA genotype s2); 3 to 8, type s1 isolates; 9 to 11, type s2 isolates. (The photographs in this and subsequent figures were scanned with a Hewlett-Packard Scan Jet 4c using the PaperPort Software Version 3.0.1 for Windows [Visioneer Communications, Inc.])

    Techniques Used: Polyacrylamide Gel Electrophoresis, Polymerase Chain Reaction, Amplification, Marker, Software

    40) Product Images from "Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA"

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

    Journal: Genome Research

    doi: 10.1101/gr.306102

    Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.
    Figure Legend Snippet: Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.

    Techniques Used:

    Related Articles

    Polymerase Chain Reaction:

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle
    Article Snippet: .. All 24 clinical samples found positive by PCR amplification of the ND7 were further tested by digestion with Hpy CH4IV and Nla III. .. The ND7 PCR-RFLP was designed as a dual diagnostic assay, performing two separate digestions simultaneously, using different enzymes for each assay on a single clinical isolate.

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle
    Article Snippet: .. By ensuring that the same fingerprinting identification is reached from two separate digestions run concurrently, the restriction digest of ND7 -PCR amplicons with Nla III and Hpy CH4IV provide a robust, 2-fold diagnosis. ..

    Amplification:

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle
    Article Snippet: .. All 24 clinical samples found positive by PCR amplification of the ND7 were further tested by digestion with Hpy CH4IV and Nla III. .. The ND7 PCR-RFLP was designed as a dual diagnostic assay, performing two separate digestions simultaneously, using different enzymes for each assay on a single clinical isolate.

    other:

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA
    Article Snippet: The values in brackets are the position of the Nla III tagging site in the Y. pestis chromosome.

    Purification:

    Article Title: CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples
    Article Snippet: .. We digested purified gDNA with 20 U of HindIII (NEB, catalog number R3104) or NlaIII (NEB, catalog number R0125) enzyme in a final volume of 10 μl, by incubating for 14 h at 37 °C. .. Afterwards, we ligated HindIII or NlaIII cut sites with CUTseq adapters carrying the complementary staggered end, using 1000 U of T4 ligase (Thermo Fisher Scientific, catalog number EL0014) in a final volume of 30 μl, by incubating for 18 h at 16 °C.

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    New England Biolabs nla iii
    Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and <t>Nla</t> <t>III</t> for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.
    Nla Iii, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 45 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.

    Journal: PLoS ONE

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain

    doi: 10.1371/journal.pone.0205328

    Figure Lengend Snippet: Upa P162 activity on CATG motifs. (A) Schematic representation of amplified PCR products 1 to 8. CATG motifs are shown by red arrows and PCR product regions and the sizes, as well as mutation sites (black triangle), are indicated. (B) Digestion of products of 1 to 6 by UpaP 162 and Nla III for 1 h, respectively. (C) Digestion of PCR products 7 and 8 by Upa P162 and Nla III for 1 h, respectively.

    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Techniques: Activity Assay, Amplification, Polymerase Chain Reaction, Mutagenesis

    DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.

    Journal: PLoS ONE

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain

    doi: 10.1371/journal.pone.0205328

    Figure Lengend Snippet: DNA digestion by Upa P162 and Nla III. (A) The indicated Ureaplasma genomes were treated with either Upa P162 or Nla III for 1 h. (B) UPV229-treated and untreated pT7Blue plasmid was digested by either Upa P162 or Nla III for 1 h.

    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Techniques: Plasmid Preparation

    Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.

    Journal: PLoS ONE

    Article Title: Type II restriction modification system in Ureaplasma parvum OMC-P162 strain

    doi: 10.1371/journal.pone.0205328

    Figure Lengend Snippet: Digestion pattern of pT7Blue by Upa P162 and Nla III. The pT7Blue plasmid was either untreated or treated for 1 h with either Upa P162 or Nla III and products were separated by electrophoresis in 1% agarose.

    Article Snippet: Nla III was purchased from New England Biolabs (Tokyo, Japan), and Sph I was purchased from Takara Bio (Otsu, Shiga, Japan).

    Techniques: Plasmid Preparation, Electrophoresis

    Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.

    Journal: Genome Research

    Article Title: Genomic Signature Tags (GSTs): A System for Profiling Genomic DNA

    doi: 10.1101/gr.306102

    Figure Lengend Snippet: Schematic for GST preparation. In this method, DNA is first fragmented with a rare cutter such as Not I or a more frequent cutter such as Bam HI. Specific complementary biotinylated linkers are ligated to the free ends, and the DNA is then digested with Nla III. All subsequent steps in the protocol are identical.

    Article Snippet: The values in brackets are the position of the Nla III tagging site in the Y. pestis chromosome.

    Techniques:

    PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Journal: The American Journal of Tropical Medicine and Hygiene

    Article Title: Identification of Clinical Infections of Leishmania Imported into Australia: Revising Speciation with Polymerase Chain Reaction-RFLP of the Kinetoplast Maxicircle

    doi: 10.4269/ajtmh.19-0095

    Figure Lengend Snippet: PCR-RFLP analysis of Leishmania species with the restriction enzyme Nla III. Samples were run alongside a 50-bp molecular weight marker. Comparison of the band patterns of the DNA fragments generated from the Nla III digestion of in silico, culture, and clinical isolates. Digestion with Nla III can successfully differentiate between the extremely closely related Leishmania ( Viannia ) (L. [V.]) braziliensis and L. ( V. ) guyanensis / L. ( V. ) panamensis complex of the Viannia subgenus, which is extremely important in a clinical setting where treatment outcome can vary greatly between these two species.

    Article Snippet: All 24 clinical samples found positive by PCR amplification of the ND7 were further tested by digestion with Hpy CH4IV and Nla III.

    Techniques: Polymerase Chain Reaction, Molecular Weight, Marker, Generated, In Silico