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    MlyI 5 000 units
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    Restriction Enzymes
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    New England Biolabs mlyi
    MlyI
    MlyI 5 000 units
    https://www.bioz.com/result/mlyi/product/New England Biolabs
    Average 94 stars, based on 1594 article reviews
    Price from $9.99 to $1999.99
    mlyi - by Bioz Stars, 2020-09
    94/100 stars

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    1) Product Images from "A dual-tag microarray platform for high-performance nucleic acid and protein analyses"

    Article Title: A dual-tag microarray platform for high-performance nucleic acid and protein analyses

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn106

    Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.
    Figure Legend Snippet: Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.

    Techniques Used: Ligation, Sequencing, Amplification, Chromatin Immunoprecipitation, Hybridization, Microarray, Labeling

    2) Product Images from "A dual-tag microarray platform for high-performance nucleic acid and protein analyses"

    Article Title: A dual-tag microarray platform for high-performance nucleic acid and protein analyses

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn106

    Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.
    Figure Legend Snippet: Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.

    Techniques Used: Ligation, Sequencing, Amplification, Chromatin Immunoprecipitation, Hybridization, Microarray, Labeling

    3) Product Images from "High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides"

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkq677

    Block-based gene assembly. ( A ) Schematics of the process. Design: All genes in the set are combined into a virtual string and the resulting sequence is T m -normalized. For simplicity, a set of four color-coded genes is shown. Oligos: T m -normalized oligos are designed, synthesized and pooled into a single mixture. Blocks: a pool of oligos is converted into a mixture of partially overlapping blocks of uniform size. Blocks are assembled under tightly controlled, highly stringent conditions that prevent incorporation of improperly synthesized oligos. Assembled blocks are amplified to generate sufficient amount of material for the amplification of all individual genes designed to be built from the oligo pool. Genes: the amplified blocks are trimmed of their flanking regions and combined into a single sequence by several rounds of overlapping PCR. The assembled sequence is used as a template for amplification of all genes in the set, each with its own set of gene-specific primers. ( B ) Detailed diagram of block structure. Blocks are composed of unique gene-specific regions (GSR) built from GAOs and common flanking regions (UPR), which enables their co-amplification. UPRs carry Mly I recognition sites, which are used for UPR cleavage. Two distinct UPR sequences are used in alternation from block to block. Adjacent blocks share regions of overlap (BOR) enabling their covalent joining by overlapping PCR.
    Figure Legend Snippet: Block-based gene assembly. ( A ) Schematics of the process. Design: All genes in the set are combined into a virtual string and the resulting sequence is T m -normalized. For simplicity, a set of four color-coded genes is shown. Oligos: T m -normalized oligos are designed, synthesized and pooled into a single mixture. Blocks: a pool of oligos is converted into a mixture of partially overlapping blocks of uniform size. Blocks are assembled under tightly controlled, highly stringent conditions that prevent incorporation of improperly synthesized oligos. Assembled blocks are amplified to generate sufficient amount of material for the amplification of all individual genes designed to be built from the oligo pool. Genes: the amplified blocks are trimmed of their flanking regions and combined into a single sequence by several rounds of overlapping PCR. The assembled sequence is used as a template for amplification of all genes in the set, each with its own set of gene-specific primers. ( B ) Detailed diagram of block structure. Blocks are composed of unique gene-specific regions (GSR) built from GAOs and common flanking regions (UPR), which enables their co-amplification. UPRs carry Mly I recognition sites, which are used for UPR cleavage. Two distinct UPR sequences are used in alternation from block to block. Adjacent blocks share regions of overlap (BOR) enabling their covalent joining by overlapping PCR.

    Techniques Used: Blocking Assay, Sequencing, Synthesized, Amplification, Polymerase Chain Reaction

    Optimizing assembly of blocks into template for gene production. ( A ) Evaluating effect of annealing time. A 252-element mixture of Mly I cleaved blocks (20 fmol/block) assembled from a 2000-complex pool of microarray-synthesized oligos, was subjected to five cycles of PCA with increasing annealing times from 0 to 35 min. To evaluate robustness of the PCA-generated template 1 µl aliquots of the reactions were used for amplification of 2.1 and 1.3 kb genes with pairs of gene-specific primers. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Lanes 1–6—products amplified from the templates generated by the reactions that carried, respectively, 0, 2, 9, 18, 28 and 35-min long annealing steps; lane 7—100 bp DNA ladder (NEB); and lane 8–1 kb DNA ladder (NEB). ( B ) Evaluating effect of PCR cycle number. The block mixture described in ( A ) was subjected to an increasing number of PCA cycles performed with a 25-min annealing step. Robustness of the generated templates was evaluated as described in the ( A ). Lane 1—1 kb DNA ladder (NEB); and lanes 2–12—products amplified from the templates assembled using 0 to 10 PCA cycles, respectively.
    Figure Legend Snippet: Optimizing assembly of blocks into template for gene production. ( A ) Evaluating effect of annealing time. A 252-element mixture of Mly I cleaved blocks (20 fmol/block) assembled from a 2000-complex pool of microarray-synthesized oligos, was subjected to five cycles of PCA with increasing annealing times from 0 to 35 min. To evaluate robustness of the PCA-generated template 1 µl aliquots of the reactions were used for amplification of 2.1 and 1.3 kb genes with pairs of gene-specific primers. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Lanes 1–6—products amplified from the templates generated by the reactions that carried, respectively, 0, 2, 9, 18, 28 and 35-min long annealing steps; lane 7—100 bp DNA ladder (NEB); and lane 8–1 kb DNA ladder (NEB). ( B ) Evaluating effect of PCR cycle number. The block mixture described in ( A ) was subjected to an increasing number of PCA cycles performed with a 25-min annealing step. Robustness of the generated templates was evaluated as described in the ( A ). Lane 1—1 kb DNA ladder (NEB); and lanes 2–12—products amplified from the templates assembled using 0 to 10 PCA cycles, respectively.

    Techniques Used: Blocking Assay, Microarray, Synthesized, Generated, Amplification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction

    4) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.
    Figure Legend Snippet: Effect of read count on marker dataset size and imputation. Fraction of shared post-filter, pre-imputation genetic markers and fraction of post-imputation shared genome with original sample for subsamplings of A) RsaI F 2 -44 and B) HincII F 2 -23. C) Imputed genomes for each subsample in RsaI F 2 -44 displayed in concentric rings. Sample read count declines from the outermost ring to the innermost. D) Imputed genomes for each subsample in HincII F 2 -23 displayed in concentric circles. Sample read count declines from the outermost ring to the innermost.

    Techniques Used: Marker

    Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.
    Figure Legend Snippet: Trait mapping for yellowy ( y1 ) and sugary ( su1 ) in an F 2 admixture population. A green line in the plots annotates the locations of both genes. Pre-imputation markers are shown in black and grey. Markers, post-imputation and error correction are shown in color. A) RsaI GBS dataset, su1 map. B) RsaI GBS dataset, y1 map. C) HincII GBS dataset, su1 map. D) RsaI GBS dataset, y1 map.

    Techniques Used:

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    5) Product Images from "Flexible and scalable genotyping-by-sequencing strategies for population studies"

    Article Title: Flexible and scalable genotyping-by-sequencing strategies for population studies

    Journal: BMC Genomics

    doi: 10.1186/1471-2164-15-979

    Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.
    Figure Legend Snippet: Fraction of predicted sites covered in samples from a F 2 admixture population. Reads from each F 2 sample were aligned to predicted sites, then predicted sites were placed in 2 bp bins, with the fraction covered in each bin indicated by the heatmap. A) The RsaI dataset, aligned against total predicted sites. B) RsaI dataset, aligned against the subset of predicted sites with sequencing coverage in the original RsaI B73 GBS experiment. C) HincII dataset, aligned against total predicted sites. D) HincII dataset, aligned against predicted sites with at least one read coverage in the original HincII experiment. Sample order is given, left to right, in Additional file 5 : Table S1.

    Techniques Used: Sequencing

    6) Product Images from "Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification"

    Article Title: Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification

    Journal: Genome Biology

    doi: 10.1186/s13059-019-1691-6

    Illumina size standards allow measurement of sequencer-specific size biases. a Design of REcount-based Illumina size standard constructs. Each standard construct contains a normalization barcode, as well as a barcode associated with a variable size standard that can be liberated by Mly I digestion and directly sequenced. b Raw abundance data for all 30 size standards and normalization barcodes from a MiSeq run. c Run-to-run variability of multiple MiSeq runs ( n = 6 flow cells). d Size bias profiles of the iSeq ( n = 1 flow cell), MiSeq ( n = 6 flow cells), NextSeq ( n = 4 flow cells), and NovaSeq ( n = 4 flow cells, 4 lanes) sequencers. Note: Size bias data for other Illumina instruments is shown in Additional file 1 : Figure S5. e Size bias profiles of the same library either clustered on the MiSeq immediately after denaturation or clustered after freezing and thawing the denatured library. Error bars are ± s.e.m
    Figure Legend Snippet: Illumina size standards allow measurement of sequencer-specific size biases. a Design of REcount-based Illumina size standard constructs. Each standard construct contains a normalization barcode, as well as a barcode associated with a variable size standard that can be liberated by Mly I digestion and directly sequenced. b Raw abundance data for all 30 size standards and normalization barcodes from a MiSeq run. c Run-to-run variability of multiple MiSeq runs ( n = 6 flow cells). d Size bias profiles of the iSeq ( n = 1 flow cell), MiSeq ( n = 6 flow cells), NextSeq ( n = 4 flow cells), and NovaSeq ( n = 4 flow cells, 4 lanes) sequencers. Note: Size bias data for other Illumina instruments is shown in Additional file 1 : Figure S5. e Size bias profiles of the same library either clustered on the MiSeq immediately after denaturation or clustered after freezing and thawing the denatured library. Error bars are ± s.e.m

    Techniques Used: Construct

    7) Product Images from "A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis"

    Article Title: A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis

    Journal: BMC Biotechnology

    doi: 10.1186/1472-6750-7-49

    Solid support rolling circle DNA synthesis from amplified SF-WT90 oligonucleotide cleaved with Mly I . First row : The chemically synthesized padlock probe, WT90-66b, was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16-Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Second row : The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Third row : The SF-WT90oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Schematic representations indicate the expected outcome of the hybridization events. (+) and (-) indicate the polarity of the probes. The (+)-primer hybridizes to the (+)-probe and the (-)-primer hybridizes to the (-)-probe. Equimolar amounts of probe were applied in each reaction (0.1 μM). Scale bar, 100 μm. At the bottom of the figure is a schematic representation of the individual steps in the solid support assay.
    Figure Legend Snippet: Solid support rolling circle DNA synthesis from amplified SF-WT90 oligonucleotide cleaved with Mly I . First row : The chemically synthesized padlock probe, WT90-66b, was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16-Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Second row : The SF-WT90 oligonucleotide, was amplified one round by the method presented in figure 1 (from (+)-strand to (-)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Third row : The SF-WT90oligonucleotide was amplified two rounds by the method presented in figure 1 (from (+)-strand to (-)-strand and back to (+)-strand) and the suicide cassette was removed by cleavage with Mly I. Subsequently, the padlock probe generated was incubated with a covalently coupled primer (Amin-L16-Mly I (+) primer or Amin-L16- Mly I (-)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of either the ID 16 or the anti ID 16 detection oligonucleotide. Schematic representations indicate the expected outcome of the hybridization events. (+) and (-) indicate the polarity of the probes. The (+)-primer hybridizes to the (+)-probe and the (-)-primer hybridizes to the (-)-probe. Equimolar amounts of probe were applied in each reaction (0.1 μM). Scale bar, 100 μm. At the bottom of the figure is a schematic representation of the individual steps in the solid support assay.

    Techniques Used: DNA Synthesis, Amplification, Synthesized, Incubation, Sequencing, Hybridization, Generated

    Example of the sequence composition of a suicide cassette . The suicide cassette contains a binding site (green) and a cleavage site (indicated by a vertical arrow) for the nicking enzyme Nt. Alw I and a binding site (blue) and a cleavage site (indicated by two vertical arrows) for the restriction endonuclease Mly I. Furthermore, the cassette comprises a loop structure (yellow) and extra bases for improving the cleavage and ligation steps (red). Top : Linear sequence. Bottom : Base pairing in a hairpin structure.
    Figure Legend Snippet: Example of the sequence composition of a suicide cassette . The suicide cassette contains a binding site (green) and a cleavage site (indicated by a vertical arrow) for the nicking enzyme Nt. Alw I and a binding site (blue) and a cleavage site (indicated by two vertical arrows) for the restriction endonuclease Mly I. Furthermore, the cassette comprises a loop structure (yellow) and extra bases for improving the cleavage and ligation steps (red). Top : Linear sequence. Bottom : Base pairing in a hairpin structure.

    Techniques Used: Sequencing, Binding Assay, Ligation

    Comparison of the chemically synthesized oligonucleotide WT90-66b and the oligonucleotide contained within the suicide cassette in SF-WT90 following amplification in a solid support rolling circle DNA synthesis assay . The chemically synthesized padlock probe WT90-66b (left) and SF-WT90 (amplified two rounds (from (+)-strand to (-)-strand and back to (+)-strand) and cleaved with Mly I; the probe was purified by PAGE after each round) (right) were incubated with a covalently coupled primer (Amin-L16-Mly I (+)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of the ID 16 detection oligonucleotide. Equimolar amounts of probe were applied in each reaction (0.1 nM). Scale bar, 100 μm.
    Figure Legend Snippet: Comparison of the chemically synthesized oligonucleotide WT90-66b and the oligonucleotide contained within the suicide cassette in SF-WT90 following amplification in a solid support rolling circle DNA synthesis assay . The chemically synthesized padlock probe WT90-66b (left) and SF-WT90 (amplified two rounds (from (+)-strand to (-)-strand and back to (+)-strand) and cleaved with Mly I; the probe was purified by PAGE after each round) (right) were incubated with a covalently coupled primer (Amin-L16-Mly I (+)) in the presence of T4 DNA ligase. Only correctly hybridized padlock probes of the right sequence can be circularized. Ligated probes were subsequently amplified by rolling circle DNA synthesis and detected by hybridization to the rolling circle products of the ID 16 detection oligonucleotide. Equimolar amounts of probe were applied in each reaction (0.1 nM). Scale bar, 100 μm.

    Techniques Used: Synthesized, Amplification, DNA Synthesis, Purification, Polyacrylamide Gel Electrophoresis, Incubation, Sequencing, Hybridization

    Schematic overview of the amplification of an oligonucleotide contained within a suicide cassette . (A) A DNA sequence comprising an oligonucleotide to be amplified (red) and a suicide cassette (blue) is circularized by self-templated hybridization in the suicide cassette region. (B) The DNA circle is closed by self-templated ligation. (C) By the addition of a primer, complementary to part of the DNA sequence, dNTPs and a polymerase a rolling circle DNA synthesis can be initiated resulting in tandem repeats complementary to the templating circle. (D) The suicide cassette can be removed from the oligonucleotide by cleavage with the Mly I restriction enzyme. (E) The tandem repeats can be linearized without the loss of nucleotides using the Nt. Alw I nicking enzyme, resulting in monomers complementary to the initial DNA sequence. (F) Self-templated hybridization (see step A). (G) Self-templated ligation (see step B). (H) Rolling circle DNA synthesis (see step C). (I) Cleavage with Mly I (see step D). (J) Nicking, using Nt. Alw I, resulting in monomers of the same polarity as the initial DNA sequence. (+) and (-) indicate the polarity of the oligonucleotide.
    Figure Legend Snippet: Schematic overview of the amplification of an oligonucleotide contained within a suicide cassette . (A) A DNA sequence comprising an oligonucleotide to be amplified (red) and a suicide cassette (blue) is circularized by self-templated hybridization in the suicide cassette region. (B) The DNA circle is closed by self-templated ligation. (C) By the addition of a primer, complementary to part of the DNA sequence, dNTPs and a polymerase a rolling circle DNA synthesis can be initiated resulting in tandem repeats complementary to the templating circle. (D) The suicide cassette can be removed from the oligonucleotide by cleavage with the Mly I restriction enzyme. (E) The tandem repeats can be linearized without the loss of nucleotides using the Nt. Alw I nicking enzyme, resulting in monomers complementary to the initial DNA sequence. (F) Self-templated hybridization (see step A). (G) Self-templated ligation (see step B). (H) Rolling circle DNA synthesis (see step C). (I) Cleavage with Mly I (see step D). (J) Nicking, using Nt. Alw I, resulting in monomers of the same polarity as the initial DNA sequence. (+) and (-) indicate the polarity of the oligonucleotide.

    Techniques Used: Amplification, Sequencing, Hybridization, Ligation, DNA Synthesis

    Related Articles

    Amplification:

    Article Title: A Common Genetic Variant (97906C > A) of DAB2IP/AIP1 Is Associated with an Increased Risk and Early Onset of Lung Cancer in Chinese Males
    Article Snippet: .. The amplified fragments were digested with MlyI (New England BioLabs) overnight at 37°C, and the products were separated in 3.5% agarose gel. .. The −1420GG genotype produced 2 bands (112 and 20 bp), whereas the −1420TT genotype produced a single band (132 bp), and the heterozygotes displayed all 3 bands (132, 112 and 20 bp) ( ).

    Article Title: A new enzymatic route for production of long 5'-phosphorylated oligonucleotides using suicide cassettes and rolling circle DNA synthesis
    Article Snippet: .. To remove the suicide cassette, thereby terminating the amplification, cleavage of the rolling circle product was done by the addition of 1 volume of cleavage mixture containing 1× NEBuffer 4 (NEB), 0.2 μg/μl BSA (NEB) and 1 u/μl of Mly I (NEB). .. Cleavage reactions were incubated for 16 hours at 37°C and stopped by heat inactivation for 20 minutes at 65°C.

    Agarose Gel Electrophoresis:

    Article Title: A Common Genetic Variant (97906C > A) of DAB2IP/AIP1 Is Associated with an Increased Risk and Early Onset of Lung Cancer in Chinese Males
    Article Snippet: .. The amplified fragments were digested with MlyI (New England BioLabs) overnight at 37°C, and the products were separated in 3.5% agarose gel. .. The −1420GG genotype produced 2 bands (112 and 20 bp), whereas the −1420TT genotype produced a single band (132 bp), and the heterozygotes displayed all 3 bands (132, 112 and 20 bp) ( ).

    Blocking Assay:

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides
    Article Snippet: .. The resulting block mixture (600 ng) was digested in 50 µl of 1× NEBuffer 4 with 10 μ Mly I (NEB) at 37°C for 3 h and purified from the cleaved flanking regions by a DNA purification kit (Qiagen). .. Assembly of the gene amplification template and individual gene amplification The mixture of flank-trimmed blocks was assembled into a gene amplification template by an overlapping PCR.

    DNA Purification:

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides
    Article Snippet: .. The resulting block mixture (600 ng) was digested in 50 µl of 1× NEBuffer 4 with 10 μ Mly I (NEB) at 37°C for 3 h and purified from the cleaved flanking regions by a DNA purification kit (Qiagen). .. Assembly of the gene amplification template and individual gene amplification The mixture of flank-trimmed blocks was assembled into a gene amplification template by an overlapping PCR.

    Purification:

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides
    Article Snippet: .. The resulting block mixture (600 ng) was digested in 50 µl of 1× NEBuffer 4 with 10 μ Mly I (NEB) at 37°C for 3 h and purified from the cleaved flanking regions by a DNA purification kit (Qiagen). .. Assembly of the gene amplification template and individual gene amplification The mixture of flank-trimmed blocks was assembled into a gene amplification template by an overlapping PCR.

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    New England Biolabs mlyi
    Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), <t>MlyI</t> restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip <t>RCA</t> amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.
    Mlyi, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 94/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.

    Journal: Nucleic Acids Research

    Article Title: A dual-tag microarray platform for high-performance nucleic acid and protein analyses

    doi: 10.1093/nar/gkn106

    Figure Lengend Snippet: Schematic description of the dual-tag array procedure. ( a ) Probe design. Padlock probes are designed to comprise end sequences complementary to adjacent target sequences (black), MlyI restriction digestion cassettes (gray), and 3′ and 5′ tag sequences flanking a general detection cassette (blue, red and green, respectively). For proximity ligation each antibody is equipped with an oligonucleotide comprising a MlyI cassette, a tag sequence and half the detection sequence, which is also used for probe ligation. ( b ) The probes are ligated by target-mediated ligation. Solution phase amplification is performed and ( c ) restriction oligonucleotides are annealed to the amplification products directing the MlyI digestion, which creates reporter molecules with specific tag sequence elements at the 5′ and 3′-ends. ( d ) The reporter molecules are circularized by on-chip ligation after hybridization to microarray oligonucleotides, complementary to the tag sequences at either end of the reporter molecules. The immobilized oligonucleotides then prime on-chip RCA amplification detected by hybridizing a fluorophore-labeled detection oligonucleotide.

    Article Snippet: RCA products were cleaved with MlyI by addition of 5 μl S8 buffer with 0.1 μg/μl BSA, 10 pmol of each of the two restriction oligonucleotides 3′MC and 5′MC ( Supplementary Data Table 2 ), along with 5 U of MlyI (New England Biolabs, USA) for 1 h at 37°C.

    Techniques: Ligation, Sequencing, Amplification, Chromatin Immunoprecipitation, Hybridization, Microarray, Labeling

    Block-based gene assembly. ( A ) Schematics of the process. Design: All genes in the set are combined into a virtual string and the resulting sequence is T m -normalized. For simplicity, a set of four color-coded genes is shown. Oligos: T m -normalized oligos are designed, synthesized and pooled into a single mixture. Blocks: a pool of oligos is converted into a mixture of partially overlapping blocks of uniform size. Blocks are assembled under tightly controlled, highly stringent conditions that prevent incorporation of improperly synthesized oligos. Assembled blocks are amplified to generate sufficient amount of material for the amplification of all individual genes designed to be built from the oligo pool. Genes: the amplified blocks are trimmed of their flanking regions and combined into a single sequence by several rounds of overlapping PCR. The assembled sequence is used as a template for amplification of all genes in the set, each with its own set of gene-specific primers. ( B ) Detailed diagram of block structure. Blocks are composed of unique gene-specific regions (GSR) built from GAOs and common flanking regions (UPR), which enables their co-amplification. UPRs carry Mly I recognition sites, which are used for UPR cleavage. Two distinct UPR sequences are used in alternation from block to block. Adjacent blocks share regions of overlap (BOR) enabling their covalent joining by overlapping PCR.

    Journal: Nucleic Acids Research

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    doi: 10.1093/nar/gkq677

    Figure Lengend Snippet: Block-based gene assembly. ( A ) Schematics of the process. Design: All genes in the set are combined into a virtual string and the resulting sequence is T m -normalized. For simplicity, a set of four color-coded genes is shown. Oligos: T m -normalized oligos are designed, synthesized and pooled into a single mixture. Blocks: a pool of oligos is converted into a mixture of partially overlapping blocks of uniform size. Blocks are assembled under tightly controlled, highly stringent conditions that prevent incorporation of improperly synthesized oligos. Assembled blocks are amplified to generate sufficient amount of material for the amplification of all individual genes designed to be built from the oligo pool. Genes: the amplified blocks are trimmed of their flanking regions and combined into a single sequence by several rounds of overlapping PCR. The assembled sequence is used as a template for amplification of all genes in the set, each with its own set of gene-specific primers. ( B ) Detailed diagram of block structure. Blocks are composed of unique gene-specific regions (GSR) built from GAOs and common flanking regions (UPR), which enables their co-amplification. UPRs carry Mly I recognition sites, which are used for UPR cleavage. Two distinct UPR sequences are used in alternation from block to block. Adjacent blocks share regions of overlap (BOR) enabling their covalent joining by overlapping PCR.

    Article Snippet: The resulting block mixture (600 ng) was digested in 50 µl of 1× NEBuffer 4 with 10 μ Mly I (NEB) at 37°C for 3 h and purified from the cleaved flanking regions by a DNA purification kit (Qiagen).

    Techniques: Blocking Assay, Sequencing, Synthesized, Amplification, Polymerase Chain Reaction

    Optimizing assembly of blocks into template for gene production. ( A ) Evaluating effect of annealing time. A 252-element mixture of Mly I cleaved blocks (20 fmol/block) assembled from a 2000-complex pool of microarray-synthesized oligos, was subjected to five cycles of PCA with increasing annealing times from 0 to 35 min. To evaluate robustness of the PCA-generated template 1 µl aliquots of the reactions were used for amplification of 2.1 and 1.3 kb genes with pairs of gene-specific primers. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Lanes 1–6—products amplified from the templates generated by the reactions that carried, respectively, 0, 2, 9, 18, 28 and 35-min long annealing steps; lane 7—100 bp DNA ladder (NEB); and lane 8–1 kb DNA ladder (NEB). ( B ) Evaluating effect of PCR cycle number. The block mixture described in ( A ) was subjected to an increasing number of PCA cycles performed with a 25-min annealing step. Robustness of the generated templates was evaluated as described in the ( A ). Lane 1—1 kb DNA ladder (NEB); and lanes 2–12—products amplified from the templates assembled using 0 to 10 PCA cycles, respectively.

    Journal: Nucleic Acids Research

    Article Title: High-quality gene assembly directly from unpurified mixtures of microarray-synthesized oligonucleotides

    doi: 10.1093/nar/gkq677

    Figure Lengend Snippet: Optimizing assembly of blocks into template for gene production. ( A ) Evaluating effect of annealing time. A 252-element mixture of Mly I cleaved blocks (20 fmol/block) assembled from a 2000-complex pool of microarray-synthesized oligos, was subjected to five cycles of PCA with increasing annealing times from 0 to 35 min. To evaluate robustness of the PCA-generated template 1 µl aliquots of the reactions were used for amplification of 2.1 and 1.3 kb genes with pairs of gene-specific primers. The amplified products were separated by agarose gel electrophoresis and visualized by ethidium bromide staining. Lanes 1–6—products amplified from the templates generated by the reactions that carried, respectively, 0, 2, 9, 18, 28 and 35-min long annealing steps; lane 7—100 bp DNA ladder (NEB); and lane 8–1 kb DNA ladder (NEB). ( B ) Evaluating effect of PCR cycle number. The block mixture described in ( A ) was subjected to an increasing number of PCA cycles performed with a 25-min annealing step. Robustness of the generated templates was evaluated as described in the ( A ). Lane 1—1 kb DNA ladder (NEB); and lanes 2–12—products amplified from the templates assembled using 0 to 10 PCA cycles, respectively.

    Article Snippet: The resulting block mixture (600 ng) was digested in 50 µl of 1× NEBuffer 4 with 10 μ Mly I (NEB) at 37°C for 3 h and purified from the cleaved flanking regions by a DNA purification kit (Qiagen).

    Techniques: Blocking Assay, Microarray, Synthesized, Generated, Amplification, Agarose Gel Electrophoresis, Staining, Polymerase Chain Reaction

    Illumina size standards allow measurement of sequencer-specific size biases. a Design of REcount-based Illumina size standard constructs. Each standard construct contains a normalization barcode, as well as a barcode associated with a variable size standard that can be liberated by Mly I digestion and directly sequenced. b Raw abundance data for all 30 size standards and normalization barcodes from a MiSeq run. c Run-to-run variability of multiple MiSeq runs ( n = 6 flow cells). d Size bias profiles of the iSeq ( n = 1 flow cell), MiSeq ( n = 6 flow cells), NextSeq ( n = 4 flow cells), and NovaSeq ( n = 4 flow cells, 4 lanes) sequencers. Note: Size bias data for other Illumina instruments is shown in Additional file 1 : Figure S5. e Size bias profiles of the same library either clustered on the MiSeq immediately after denaturation or clustered after freezing and thawing the denatured library. Error bars are ± s.e.m

    Journal: Genome Biology

    Article Title: Measuring sequencer size bias using REcount: a novel method for highly accurate Illumina sequencing-based quantification

    doi: 10.1186/s13059-019-1691-6

    Figure Lengend Snippet: Illumina size standards allow measurement of sequencer-specific size biases. a Design of REcount-based Illumina size standard constructs. Each standard construct contains a normalization barcode, as well as a barcode associated with a variable size standard that can be liberated by Mly I digestion and directly sequenced. b Raw abundance data for all 30 size standards and normalization barcodes from a MiSeq run. c Run-to-run variability of multiple MiSeq runs ( n = 6 flow cells). d Size bias profiles of the iSeq ( n = 1 flow cell), MiSeq ( n = 6 flow cells), NextSeq ( n = 4 flow cells), and NovaSeq ( n = 4 flow cells, 4 lanes) sequencers. Note: Size bias data for other Illumina instruments is shown in Additional file 1 : Figure S5. e Size bias profiles of the same library either clustered on the MiSeq immediately after denaturation or clustered after freezing and thawing the denatured library. Error bars are ± s.e.m

    Article Snippet: Illumina size standards The following digest of the Illumina size standard pool was set up: 175 μl DNA (10 nM), 20 μl CutSmart buffer (NEB), 5 μl Mly I (NEB).

    Techniques: Construct